WO2000059925A1 - A process for the production of glutamine derivatives and glutamine containing molecules - Google Patents

A process for the production of glutamine derivatives and glutamine containing molecules Download PDF

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Publication number
WO2000059925A1
WO2000059925A1 PCT/US2000/008178 US0008178W WO0059925A1 WO 2000059925 A1 WO2000059925 A1 WO 2000059925A1 US 0008178 W US0008178 W US 0008178W WO 0059925 A1 WO0059925 A1 WO 0059925A1
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Prior art keywords
glutamine
terminus
process according
unprotected
strong base
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PCT/US2000/008178
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French (fr)
Inventor
Timothy L. Macdonald
Thomas A. Miller
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The University Of Virginia
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Priority to AU39274/00A priority Critical patent/AU3927400A/en
Publication of WO2000059925A1 publication Critical patent/WO2000059925A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • 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
    • C07K1/066—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-amido functions
    • 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
    • C07K1/08—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 activating agents
    • C07K1/084—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 activating agents containing nitrogen
    • 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
    • C07K1/08—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 activating agents
    • C07K1/088—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 activating agents containing other elements, e.g. B, Si, As
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the preparation of amino acid derivatives particularly as related to the preparation of glutamine containing molecules and glutamine derivatives.
  • Glutamine is an important nutrient in culture media and in nutrient solutions for use with humans suffering from physiological or nutritional stress. Glutamine has proven difficult to use in such preparations due to its low thermal and aqueous stability.
  • glutamine-containing dipeptides exhibit good thermal and aqueous stability.
  • Many glutamine dipeptides are sufficiently thermally stable to withstand conditions of sterilization and significant ranges in pH.
  • glutamine-containing peptides can be quite difficult to prepare. The instability of the ⁇ -amido group of the glutamine residue leads to many difficulties including problems with various side reactions.
  • This process uses a weak base for the production of glutamine derivatives from glutamine and protected C-terminus activated amino acids.
  • the method of Kato and Kurauchi specifies the use of a weak base, since strong bases are associated with several competing reactions with glutamine.
  • the cyclization of glutamine to 5-oxoproline is known to be accelerated in the presence of a strong base.
  • racemization of the dipeptide product or either of the two amino acid precursors is known to be increased in the presence of a strong base.
  • a strong base improves several other aspects of glutamine derivative synthesis. These improvements include a reduction in the amount of base needed to stoichiomethric amounts, and increases in reaction rate and therefore greatly reduced reaction times. Additionally, the use of a strong base permits work-up procedures that do not involve gas evolution.
  • the present invention is directed to a method for preparing amino acid derivatives. More particularly the present invention is directed to preparation of glutamine containing molecules and glutamine derivatives.
  • glutamine containing molecules or glutamine derivatives are prepared by reacting glutamine in the presence of a strong base with a N-terminus protected activated ester of an amino acid or peptide to produce a N-terminus protected glutamine containing molecule or glutamine derivative that possesses glutamine at the C-terminus.
  • the N-terminus group may be removed yielding an unprotected or free glutamine containing molecule or glutamine derivative.
  • the present invention includes a process for producing a glutamine containing molecule or glutamine derivative where the process includes the steps of charging a reactor with a reaction solution.
  • the reaction solution includes a strong base, an unprotected glutamine, and an activated ester of an N-terminus protected acid where the N-terminus protected acid has a C-terminus.
  • the process also includes forming a bond between the C-terminus of the N-terminus protected acid and the unprotected glutamine by reacting the unprotected glutamine, in the presence of said strong base, with the activated ester of the N-terminus protected acid to form an N-terminus protected glutamine derivative.
  • the strong base may include, but is not limited to, sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, other metalooxides, metalofluorides and other materials capable of removing a proton from water.
  • the activated ester may include, but is not limited to N-hydroxysuccinimide, carbodiimide, hydroxy benzotriazole, nitrophenol, pentafluorophenol and other acylflourides.
  • the N-terminus protecting group of the N-terminus protected acid may include, but is not limited to, carbobenzoxy, benzyl, methoxybenzyl, t-butoxy carbonyl or similar N-terminous protecting group.
  • the present invention includes a process for producing a glutamine containing molecule or glutamine derivative.
  • the steps includes charging a reactor with a reaction solution that includes a strong base, an unprotected glutamine selected from the group consisting of L-glutamine, D-glutamine and D/L -glutamine, said glutamine having an N-terminus, and an activated ester of an N-terminus protected acid selected from the group consisting of an N-terminus protected amino acid and an N-terminus protected peptide, the N-terminus protected acid having a C- terminus.
  • the process further includes forming a bond between the C-terminus of the N-terminus protected acid and the unprotected glutamine by reacting the unprotected glutamine, in the presence of the strong base with the activated ester of the N- terminus protected acid to form an N-terminus protected glutamine derivative.
  • the process of the present invention may be performed with the temperature maintained between about -20°C and about 30°C. Additionally, the process may be performed in a water miscible solvent.
  • the invention may also include alanine as the N-terminus protected amino acid of the N-terminus protected acid.
  • the N-terminus protected acid may be an N-terminus protected peptide containing from about 2 to about 20 residues.
  • the process of the present invention may also include the additional step of crystallizing the N-terminus protected glutamine derivative.
  • the process may also include the step of forming an unprotected glutamine derivative by exposing the N-protected glutamine derivative to hydrogen in the presence of a catalyst.
  • the invention includes a process for producing an alanine derivative of glutamine, where the process includes charging a reactor with a reaction solution.
  • the reaction solution includes a strong base, an unprotected glutamine selected from the group consisting of L-glutamine, D-glutamine and D/L -glutamine, said glutamine having an N-terminus, and an activated ester of an N-terminus protected alanine said alanine having a C-terminus.
  • the process also includes forming a bond between the C-terminus of the N-terminus protected alanine and the unprotected glutamine by reacting the unprotected glutamine, in the presence of the strong base, with the activated ester of N- terminus protected alanine to form an N- terminus protected alanyl-glutamine derivative.
  • the present invention is a process for producing amino acid derivatives. More particularly the present invention is a process for producing glutamine containing molecules and glutamine derivatives. In a preferred embodiment, the process is directed to producing to alanyl-glutamine. This process couples N-terminus protected, activated esters of amino acids or peptides with glutamine in the presence of a strong base such as a metalhydroxide or a metalofluoride. The resulting product is a N-terminus protected amino acid or peptide conjugate possessing glutamine at the C-terminus. Typically, the N-terminus protecting group is subsequently removed to produce the unprotected or free glutamine containing molecule or glutamine derivative.
  • the strong base used with an embodiment of the present invention may be a metal hydroxide or metaloflouride.
  • Suitable metalhydroxides include, but are not limited to, sodium hydroxide, potassium hydroxide, lithium hydroxide or bases possessing comparable basicity.
  • Suitable metaloflourides include, but are not limited to, cesium fluoride and potassium fluoride.
  • Any naturally occurring or synthetic amino acid may be used in the process.
  • the stereochemistry of the amino acid may be L-, D-, or a mixtures thereof (D/L).
  • the process is amenable to peptides containing 2 or more residues, preferably between 2 and 20, and in a preferred embodiment, between 2 and 8 residues.
  • the stereochemistry of the amino acids that comprise the peptide may be D-, L-, or a mixture thereof (D/L).
  • the protection group for the N-terminus protected amino acid or peptide may include, but is not limited to benzyl, methoxybenzyl, carbobenzoxy, t-butoxy carbonyl or similar N-terminous protecting group.
  • One preferred protecting group is the carbobenzoxy protecting group because of its ease of removal.
  • the activated ester may include, but is not limited to, N-hydroxysuccinimide, carbodiimide, hydroxy benzotriazole, nitrophenol, pentafluorophenol and other acyl fluorides. It has been found that the use of N-hydroxysuccinimide produces a stable by-product.
  • the process is preferably performed at temperatures between about -20°C and about 30°C. In a preferred embodiment, the temperature is maintained between about 0°C and about 20°C. This may be accomplished by maintaining external cooling sources well known to those skilled in the art or by direct addition of ice to the reaction vessel. The process may be carried out between about 1 and about 48 hours, and preferably between about 1 and about 8 hours.
  • this reaction mixture is then acidified and upon standing crystalline carbobenzoxy-alanyl-glutamine is formed as a white powder.
  • carbobenzoxy-alanyl-glutamine can be further purified by dissolving it in a suitable solvent and then adjusting the pH to permit crystallization.
  • Suitable solvents for the process include any water miscible solvent, including, but not limited to, acetone, tetrahydrofuran, dimethoxyethane, and dioxane.
  • Water miscible etherial solvents such as tetrahydrofuran, dimothoxyethane or dioxane are preferred due to the low environmental impact and ease of removal.
  • the N-terminus protecting group may be removed by hydrogenolysis in an aqueous/alcoholic media in the presence of a catalyst.
  • a catalyst is Palladium on charcoal.
  • a solution of N-hydroxysuccinimide activated carbobenzoxy-analine-OH was prepared by adding dicyclohexylcarbodiimide (1.0 grams, 4.86 millimoles) in tetrahydrofuran (4 milliliters) dropwise to a tetrahydrofuran solution (10 milliliters), containing carbobenzoxy-alanine-OH (1.0 grams, 4.5 millimoles) and N- hydroxysuccinimide (0.61 grams, 5.3 millimoles) at about 20°C. The resultant solution was stirred for 3 to 6 hours.
  • the solution was filtered and then added to a solution of sodium glutamate which had been prepared by adding sodium hydroxide (0.4 grams, 10 millimoles) to an aqueous solution of glutamine (1.5 grams, 10.4 millimoles in 7 milliliters of water). After one hour, the reaction mixture was concentrated to approximately 7 milliliters and diluted with brine (saturated aqueous sodiumchloride, 10 milliliters). The solution was then filtered to remove insoluble material (primarily excess glutamine) and acidified to a pH of approximately 3.5. Upon standing at 0°C for about two hours the solution yielded crystalline carbobenzoxy-alanyl-glutamine-OH (1.3 grams, 3.7 millimoles, 82%) as a white powder. Carbobenzoxy- Alanyl-glutamine is then crystallized by dissolving in sodium bicarbonate (saturated) solution and careful acidification to a pH between 3 and 4.

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  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
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Abstract

The present invention is a process for preparing glutamine derivates including alanyl-glutamine. Glutamine containing molecules or glutamine derivatives are prepared in the presence of a strong base by reacting an activated ester of an N-terminus protected amino acid or peptide with unprotected glutamine to produce an N-terminus protected derivative with a C-terminal glutamine. The N-terminus protecting group may then be eliminated to yield an unprotected or free glutamine containing molecule or glutamine derivative.

Description

A PROCESS FOR THE PRODUCTION OF GLUTAMINE DERIVATIVES AND
GLUTAMINE CONTAINING MOLECULES
CROSS REFERENCE The present utility patent application is based on and claims the benefit under 35 U.S.C. §119(e) of United States Provisional Patent Application Serial No. 60/127,315, which was filed on April 1, 1999.
BACKGROUND OF THE INVENTION The present invention relates to the preparation of amino acid derivatives particularly as related to the preparation of glutamine containing molecules and glutamine derivatives. Glutamine is an important nutrient in culture media and in nutrient solutions for use with humans suffering from physiological or nutritional stress. Glutamine has proven difficult to use in such preparations due to its low thermal and aqueous stability. However, glutamine-containing dipeptides exhibit good thermal and aqueous stability. Many glutamine dipeptides are sufficiently thermally stable to withstand conditions of sterilization and significant ranges in pH. Unfortunately, glutamine-containing peptides can be quite difficult to prepare. The instability of the β-amido group of the glutamine residue leads to many difficulties including problems with various side reactions. Several publications including: Y. Shimonishi, S. Sakakibara and S. Akabori, Bull. Chem. Soc. Japan, 35, 1966-1970; Y. Shimonishi, Bull. Chem. Soc. Jap., 37, 200-203; and S. Akabori, S. Sakakibara and Y. Shimonishi, Bull. Chem. Soc. Jap., 34, 739 describe the synthesis of glutamine-containing peptides by preparation methods which can include many difficulties, low yields and/or impure product. Another approach to preparing the glutamine containing dipeptides is described in U.S. Patent No. 5,032,675 and EPO Patent No. 0311057 to T. Kato and M. Kurauchi. This process uses a weak base for the production of glutamine derivatives from glutamine and protected C-terminus activated amino acids. The method of Kato and Kurauchi specifies the use of a weak base, since strong bases are associated with several competing reactions with glutamine. The cyclization of glutamine to 5-oxoproline is known to be accelerated in the presence of a strong base. Additionally racemization of the dipeptide product or either of the two amino acid precursors is known to be increased in the presence of a strong base.' However, we have found that with the judicious use of a strong base in the production of glutamine derivatives as described herein, these complications do not interfere and, in fact, yields greater then those reported by Kato and that Kurauchi are obtained. Additionally the use of a strong base improves several other aspects of glutamine derivative synthesis. These improvements include a reduction in the amount of base needed to stoichiomethric amounts, and increases in reaction rate and therefore greatly reduced reaction times. Additionally, the use of a strong base permits work-up procedures that do not involve gas evolution.
SUMMARY OF THE INVENTION The present invention is directed to a method for preparing amino acid derivatives. More particularly the present invention is directed to preparation of glutamine containing molecules and glutamine derivatives. In the process of this invention, glutamine containing molecules or glutamine derivatives are prepared by reacting glutamine in the presence of a strong base with a N-terminus protected activated ester of an amino acid or peptide to produce a N-terminus protected glutamine containing molecule or glutamine derivative that possesses glutamine at the C-terminus. The N-terminus group may be removed yielding an unprotected or free glutamine containing molecule or glutamine derivative.
The present invention includes a process for producing a glutamine containing molecule or glutamine derivative where the process includes the steps of charging a reactor with a reaction solution. The reaction solution includes a strong base, an unprotected glutamine, and an activated ester of an N-terminus protected acid where the N-terminus protected acid has a C-terminus. The process also includes forming a bond between the C-terminus of the N-terminus protected acid and the unprotected glutamine by reacting the unprotected glutamine, in the presence of said strong base, with the activated ester of the N-terminus protected acid to form an N-terminus protected glutamine derivative.
The strong base may include, but is not limited to, sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, other metalooxides, metalofluorides and other materials capable of removing a proton from water.
Further, the activated ester may include, but is not limited to N-hydroxysuccinimide, carbodiimide, hydroxy benzotriazole, nitrophenol, pentafluorophenol and other acylflourides. The N-terminus protecting group of the N-terminus protected acid may include, but is not limited to, carbobenzoxy, benzyl, methoxybenzyl, t-butoxy carbonyl or similar N-terminous protecting group.
Still further, the present invention includes a process for producing a glutamine containing molecule or glutamine derivative. The steps includes charging a reactor with a reaction solution that includes a strong base, an unprotected glutamine selected from the group consisting of L-glutamine, D-glutamine and D/L -glutamine, said glutamine having an N-terminus, and an activated ester of an N-terminus protected acid selected from the group consisting of an N-terminus protected amino acid and an N-terminus protected peptide, the N-terminus protected acid having a C- terminus. The process further includes forming a bond between the C-terminus of the N-terminus protected acid and the unprotected glutamine by reacting the unprotected glutamine, in the presence of the strong base with the activated ester of the N- terminus protected acid to form an N-terminus protected glutamine derivative. Further, the process of the present invention may be performed with the temperature maintained between about -20°C and about 30°C. Additionally, the process may be performed in a water miscible solvent.
The invention may also include alanine as the N-terminus protected amino acid of the N-terminus protected acid. Further, the N-terminus protected acid may be an N-terminus protected peptide containing from about 2 to about 20 residues. Still further, the process of the present invention may also include the additional step of crystallizing the N-terminus protected glutamine derivative. The process may also include the step of forming an unprotected glutamine derivative by exposing the N-protected glutamine derivative to hydrogen in the presence of a catalyst.
Still further, the invention includes a process for producing an alanine derivative of glutamine, where the process includes charging a reactor with a reaction solution. The reaction solution includes a strong base, an unprotected glutamine selected from the group consisting of L-glutamine, D-glutamine and D/L -glutamine, said glutamine having an N-terminus, and an activated ester of an N-terminus protected alanine said alanine having a C-terminus. The process also includes forming a bond between the C-terminus of the N-terminus protected alanine and the unprotected glutamine by reacting the unprotected glutamine, in the presence of the strong base, with the activated ester of N- terminus protected alanine to form an N- terminus protected alanyl-glutamine derivative.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a process for producing amino acid derivatives. More particularly the present invention is a process for producing glutamine containing molecules and glutamine derivatives. In a preferred embodiment, the process is directed to producing to alanyl-glutamine. This process couples N-terminus protected, activated esters of amino acids or peptides with glutamine in the presence of a strong base such as a metalhydroxide or a metalofluoride. The resulting product is a N-terminus protected amino acid or peptide conjugate possessing glutamine at the C-terminus. Typically, the N-terminus protecting group is subsequently removed to produce the unprotected or free glutamine containing molecule or glutamine derivative.
The strong base used with an embodiment of the present invention may be a metal hydroxide or metaloflouride. Suitable metalhydroxides include, but are not limited to, sodium hydroxide, potassium hydroxide, lithium hydroxide or bases possessing comparable basicity. Suitable metaloflourides include, but are not limited to, cesium fluoride and potassium fluoride.
Any naturally occurring or synthetic amino acid may be used in the process. The stereochemistry of the amino acid may be L-, D-, or a mixtures thereof (D/L). For peptides the process is amenable to peptides containing 2 or more residues, preferably between 2 and 20, and in a preferred embodiment, between 2 and 8 residues. The stereochemistry of the amino acids that comprise the peptide may be D-, L-, or a mixture thereof (D/L).
The protection group for the N-terminus protected amino acid or peptide may include, but is not limited to benzyl, methoxybenzyl, carbobenzoxy, t-butoxy carbonyl or similar N-terminous protecting group. One preferred protecting group is the carbobenzoxy protecting group because of its ease of removal.
The activated ester may include, but is not limited to, N-hydroxysuccinimide, carbodiimide, hydroxy benzotriazole, nitrophenol, pentafluorophenol and other acyl fluorides. It has been found that the use of N-hydroxysuccinimide produces a stable by-product.
The process is preferably performed at temperatures between about -20°C and about 30°C. In a preferred embodiment, the temperature is maintained between about 0°C and about 20°C. This may be accomplished by maintaining external cooling sources well known to those skilled in the art or by direct addition of ice to the reaction vessel. The process may be carried out between about 1 and about 48 hours, and preferably between about 1 and about 8 hours.
In a preferred embodiment this reaction mixture is then acidified and upon standing crystalline carbobenzoxy-alanyl-glutamine is formed as a white powder. Additionally, in a preferred embodiment the carbobenzoxy-alanyl-glutamine can be further purified by dissolving it in a suitable solvent and then adjusting the pH to permit crystallization.
Suitable solvents for the process include any water miscible solvent, including, but not limited to, acetone, tetrahydrofuran, dimethoxyethane, and dioxane. Water miscible etherial solvents such as tetrahydrofuran, dimothoxyethane or dioxane are preferred due to the low environmental impact and ease of removal.
The N-terminus protecting group may be removed by hydrogenolysis in an aqueous/alcoholic media in the presence of a catalyst. One preferred catalyst is Palladium on charcoal.
The above is a description of the general parameters of the process of the present invention. Specific examples of the invention as it pertains to alanyl- glutamine are given below. It will be appreciated that these examples are representative and are not intended to limit the invention in any respect. The examples are provided for a better understanding of the methods of practice for this invention.
EXAMPLE 1 Preparation of Protected Alanyl-Glutamine from N-hydroxysuccinimide Activated
Carbobenzoxy- Alanine-OH
A solution of N-hydroxysuccinimide activated carbobenzoxy-analine-OH was prepared by adding dicyclohexylcarbodiimide (1.0 grams, 4.86 millimoles) in tetrahydrofuran (4 milliliters) dropwise to a tetrahydrofuran solution (10 milliliters), containing carbobenzoxy-alanine-OH (1.0 grams, 4.5 millimoles) and N- hydroxysuccinimide (0.61 grams, 5.3 millimoles) at about 20°C. The resultant solution was stirred for 3 to 6 hours. The solution was filtered and then added to a solution of sodium glutamate which had been prepared by adding sodium hydroxide (0.4 grams, 10 millimoles) to an aqueous solution of glutamine (1.5 grams, 10.4 millimoles in 7 milliliters of water). After one hour, the reaction mixture was concentrated to approximately 7 milliliters and diluted with brine (saturated aqueous sodiumchloride, 10 milliliters). The solution was then filtered to remove insoluble material (primarily excess glutamine) and acidified to a pH of approximately 3.5. Upon standing at 0°C for about two hours the solution yielded crystalline carbobenzoxy-alanyl-glutamine-OH (1.3 grams, 3.7 millimoles, 82%) as a white powder. Carbobenzoxy- Alanyl-glutamine is then crystallized by dissolving in sodium bicarbonate (saturated) solution and careful acidification to a pH between 3 and 4.
EXAMPLE 2 Preparation of Free Alanyl-Glutamine from Protected Alanyl-Glutamine
Hydrogeno lysis of protected carbobenzoxy- Alanyl-glutamine-OH to free H-alanyl- glutamine-OH was performed by dissolution of carbobenzoxy-alanyl-glutamine-OH (1.3 grams, 3.7 millimoles) in methanol (7 milliliters) and water (2 milliliters) with magnetic stirring followed by the addition of 10% palladium on carbon (0.1 grams) and exposure to hydrogen at 1 atmosphere. After approximately 3 hours, the solution was filtered to remove the catalyst and concentrated to 4 milliliters by rotary evaporation. Isopropanol (15 milliliters) was added to the solution and this mixture was stirred in an open vessel for several minutes. Upon vacuum drying, alanyl- glutamine product (0.7 grams, 3.3 millimoles, 90%) was obtained. This alanyl- glutamine product elicited the spectral and physical properties of commercially available Alanyl-glutamine.
It will be readily understood by those persons skilled in the art that the present invention is susceptible to broad utility and application. Many embodiments and adaptations of the present invention other than those herein described, as well as many variations, modifications and equivalent arrangements will be apparent from or reasonably suggested by the present invention and the foregoing description without departing from the substance or scope of the present invention.
Accordingly, while the present invention has been described in detail in relation to its preferred embodiment, it is to be understood that this disclosure is only illustrative and exemplary of the present invention. The foregoing disclosure is not intended to be construed to limit the present invention or otherwise exclude any other embodiments, adaptations, variations, modifications or equivalent arrangements, the present invention being limited only by the claims and the equivalents thereof.

Claims

CLAIMS What is claimed is:
1. A process for producing a glutamine containing molecule or glutamine derivative, said process comprising the steps of: charging a reactor with a reaction solution comprising: a strong base, an unprotected glutamine, an activated ester of an N-terminus protected acid, said N-terminus protected acid having a C-terminus; and forming a bond between said C-terminus of said N-terminus protected acid and said unprotected glutamine by reacting said unprotected glutamine, in the presence of said strong base with said activated ester of said N- terminus protected acid to form an N-terminus protected glutamine derivative.
2. The process according to claim 1 wherein said strong base is selected from the group consisting of a metalooxide and a metalofluoride.
3. The process according to claim 1 wherein said strong base is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide and cesium hydroxide.
4. The process according to claim 1 wherein said strong base is sodium hydroxide.
5. The process according to claim 1 wherein said activated ester is formed from a compound selected from the group consisting of N-hydroxysuccinimide, carbodiimide, hydroxy benzotriazole, nitrophenol and pentafluorophenol.
6. The process according to claim 1 wherein said activated ester is formed from N-hydroxysuccinimide.
7. The process according to claim 1 wherein a N-terminus protecting group of said N-terminus protected acid is selected from the group consisting' of carbobenzoxy, benzyl, methoxybenzyl and t-butoxy carbonyl.
8. The process according to claim 1 wherein said N-terminus protecting group of said n-terminus protected acid is carbobenzoxy.
9. A process for producing a glutamine containing molecule or glutamine derivative, said process comprising the steps of: charging a reactor with a reaction solution comprising: a strong base, an unprotected glutamine selected from the group consisting of L- glutamine, D-glutamine and D/L -glutamine, said glutamine having an N-terminus, and an activated ester of an N-terminus protected acid selected from the group consisting of an N-terminus protected amino acid and an N-terminus protected peptide, said N-terminus protected acid having a C-terminus; and forming a bond between said C-terminus of said N-terminus protected acid and said unprotected glutamine by reacting said unprotected glutamine, in the presence of said strong base with said activated ester of said N- terminus protected acid to form an N-terminus protected glutamine derivative.
10. The process according to claim 9 wherein said strong base is selected from the group consisting of metalooxide and metalofluoride.
11. The process according to claim 9 wherein said strong base is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide and cesium hydroxide.
12. The process according to claim 9 wherein said strong base is sodium hydroxide.
13. The process according to claim 9 wherein said activated ester is formed from a compound selected from the group consisting of N-hydroxysuccinimide, carbodiimide, hydroxy benzotriazole, nitrophenol and pentafluorophenol.
14. The process according to claim 9 wherein said activated ester is formed from N-hydroxysuccinimide.
15. The process according to claim 9 wherein a N-terminus protecting group of said N-terminus protected acid is selected from the group consisting of carbobenzoxy, benzyl, methoxybenzyl, and t-butoxy carbonyl.
16. The process according to claim 9 wherein said N-terminus protecting group of said n-terminus protected acid is carbobenzoxy.
17. The process according to claim 9 wherein the process is performed with the temperature maintained between about -20°C and about 30°C.
18. The process according to claim 9 wherein the process is performed in a water miscible solvent.
19. The process according to claim 9 wherein said N-terminus protected amino acid of N-terminus protected acid is alanine.
20. The process according to claim 9 wherein said N-terminus protected acid is said N-terminus protected peptide containing about 2 to about 20 residues.
21. The process according to claim 9 comprising the additional step of crystallizing said N-terminus protected glutamine derivative.
22. The process of claim 9 further comprising the step of forming an unprotected glutamine derivative by exposing said N-protected glutamine derivative to hydrogen in the presence of a catalyst.
23. A process for producing an alanine derivative of glutamine, said process comprising the steps of: charging a reactor with a reaction solution comprising, a strong base, an unprotected glutamine from the group consisting of L-glutamine, D- glutamine and D/L -glutamine, said glutamine having an N- terminus, and an activated ester of an N-terminus protected alanine said alanine having a C-terminus; and forming a bond between said C-terminus of said N-terminus protected alanine and said unprotected glutamine by reacting said unprotected glutamine, in the presence of said strong base, with said activated ester of N- terminus protected alanine to form an N-terminus protected alanyl- glutamine derivative.
24. The process according to claim 23 wherein said strong base is selected from the group consisting of metalooxide and metalofluoride.
25. The process according to claim 23 wherein said strong base is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, and cesium hydroxide.
26. The process according to claim 23 wherein said strong base is sodium hydroxide.
27. The process according to claim 23 wherein said activated ester is formed from a compound selected from the group consisting of N-hydroxysuccinimide, carbodiimide, hydroxy benzotriazole, nitrophenol, and pentafluorophenol.
28. The process according to claim 23 wherein said activated ester is formed from the compound N-hydroxysuccinimide.
29. The process according to claim 23 wherein the N-terminus protecting group of said alanine is selected from the group consisting of carbobenzoxy, benzyl, methoxybenzyl, and t-butoxy carbonyl.
30. The process according to claim 23 wherein the N-terminus protecting group of said N-terminus protected alanine is carbobenzoxy.
31. The process according to claim 23 wherein the process is performed with the temperature maintained between about -20°C and about 30°C.
32. The process according to claim 23 wherein the process is performed in a water miscible solvent.
33. The process according to claim 23 comprising the additional step of crystallizing said N-terminus protected alanyl-glutamine derivative.
34. The process according to claim 33 further comprising the step of forming an unprotected alanyl-glutamine by exposing said N-terminus protected alanyl- glutamine derivative to hydrogen in the presence of a catalyst.
35. The process according to claim 23 wherein the molar ratio of said activated ester of N-terminus protected alanine to said unprotected glutamine is approximately about 1 to about 2.
36. The process according to claim 23 further comprising the step of forming an unprotected alanyl-glutamine by exposing said N-terminus protected alanyl- glutamine derivative to hydrogen in the presence of a catalyst.
37. The process according to claim 36 wherein the catalyst is palladium on charcoal.
38. A process for producing a glutamine containing molecule or glutamine derivative, said process comprising the steps of: providing a strong base; providing an unprotected L-,D or D-/L-glutamine in the presence of said strong base, said unprotected L-,D- or D- or D-/L-glutamine having an N- terminus; providing an activated ester of an N-terminus protected amino acid or peptide, said N-terminus protected amino acid or peptide having a C-terminus; forming a bond between said C-terminus of said N-terminus protected amino acid or peptide and said N-terminus of said unprotected L-,D- or D-/L- glutamine; said forming step comprising reacting said unprotected L-,D- or D-/L- glutamine, in the presence of said strong base, as a reactant with said activated ester of said N-terminus protected amino acid or peptide.
PCT/US2000/008178 1999-04-01 2000-03-28 A process for the production of glutamine derivatives and glutamine containing molecules WO2000059925A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10392821B4 (en) * 2002-06-17 2011-12-15 Xiamen University Syntetic method for alanylglutamine
CN103360462A (en) * 2013-07-09 2013-10-23 山东齐都药业有限公司 Refining method of alanyl-glutamine crude drug

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5032675A (en) * 1987-07-10 1991-07-16 Ajinomoto Co., Inc. Process for the production of glutamine derivatives
US5550283A (en) * 1992-10-29 1996-08-27 Kyowa Hakko Kogyo Co., Ltd. Process for producing alanylglutamine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5032675A (en) * 1987-07-10 1991-07-16 Ajinomoto Co., Inc. Process for the production of glutamine derivatives
US5550283A (en) * 1992-10-29 1996-08-27 Kyowa Hakko Kogyo Co., Ltd. Process for producing alanylglutamine

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10392821B4 (en) * 2002-06-17 2011-12-15 Xiamen University Syntetic method for alanylglutamine
CN103360462A (en) * 2013-07-09 2013-10-23 山东齐都药业有限公司 Refining method of alanyl-glutamine crude drug

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