Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2002-08-30
2004-12-14
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
active
06830904
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for producing single diastereomers of isoleucine in high stereochemical purity.
BACKGROUND
Unnatural or non-proteinogenic amino acids, which are structural analogs of the naturally-occurring amino acids that are the constituents of proteins, have important applications as pharmaceutical intermediates. For example, the anti-hypertensives ramipril, enalapril, benazapril, and prinivil are all based on L-homophenylalanine; certain second generation pril analogs are synthesized from p-substituted-L-homophenylalanine. Various &bgr;-lactam antibiotics use substituted D-phenylglycine side chains, and newer generation antibiotics are based on aminoadipic acid and other UAAs. The unnatural amino acid L-tert-leucine has been used as a precursor in the synthesis of a number of different developmental drugs.
Unnatural amino acids are used almost exclusively as single stereoisomers. Since unnatural amino acids are not natural metabolites, traditional production methods for amino acids based on fermentation cannot generally be used since no metabolic pathways exist for their synthesis. Given the growing importance of unnatural amino acids as pharmaceutical intermediates, various methods have been developed for their enantiomerically pure preparation. Commonly employed methods include resolutions by diastereomeric crystallization, enzymatic resolution of derivatives, and separation by simulated moving bed (SMB) chiral chromatography. These methods can be used to separate racemic mixtures, but the maximum theoretical yield is only 50%.
The amino acid isoleucine poses special problems due to the presence of a second chiral center. Four distinct diastereomers exist for the constitutional carbon skeleton of isoleucine, consisting of two enantiomeric pairs: L-isoleucine, D-isoleucine, L-allo-isoleucine, and D-allo-isoleucine, having the (2S,3S), (2R,3R), (2S,3R), and (2R,3S) absolute configurations, respectively. The naturally-occurring L-isoleucine can be produced by fermentation, taking advantage of the existing metabolic pathway to introduce both chiral centers. Production of the other isoleucine diastereomers is considerably more difficult, however, because metabolic pathways for their production do not exist. Separation of an equimolar mixture of the four diastereomers, which is extremely difficult and costly due to the chemical similarity of the compounds, can produce only a maximum theoretical yield of 25% of any single diastereomer, and in practice it is always much lower. Synthesis of a racemate in which the relative stereochemistry of the two chiral centers is controlled will still only permit a maximum theoretical yield of 50% when the enantiomers are separated.
D-isoleucine, or (2R,3R)-2-amino-3-methylpentanoic acid, D-allo-isoleucine, or (2R,3S)-2-amino-3-methylpentanoic acid, L-allo-isoleucine, or (2S,3R)-2-amino-3-methylpentanoic acid, and L-isoleucine, or (2S,3S)-2-amino-3-methylpentanoic acid all have applications as pharmaceutical intermediates and as chemicals for medical and biochemical research. Various derivatives are also required for the synthesis of peptides and peptide analogs. Thus, an efficient method for preparation of a single diastereomer of D-isoleucine or D- or L-allo-isoleucine in high stereochemical purity would be highly desirable. The present invention is directed toward a method for the preparation of any of the four diastereomers of the isoleucine carbon skeleton in high stereochemical purity.
SUMMARY OF THE INVENTION
The invention is directed to methods for the preparation of any of the four diastereomers of the isoleucine carbon skeleton in high stereochemical purity. In one embodiment as shown in Scheme 1, the invention is directed to a method for producing D-isoleucine comprising converting (R)-2-methylbutyraldehyde to a diastereomeric mixture of D-isoleucine hydantoin (5R-[(R)-1-methylpropyl]hydantoin) and L-allo-isoleucine hydantoin (5S-[(R)-1-methylpropyl]hydantoin) under conditions whereby no significant racemization of the chiral center in (R)-2-methylbutyraldehyde occurs, followed by contacting said diastereomeric hydantoin mixture with a D-hydantoinase to stereoselectively hydrolyze any D-isoleucine hydantoin in the mixture to the corresponding N-carbamoyl-D-isoleucine. Preferably in the claimed method the contacting of the diastereomeric hydantoin mixture with a D-hydantoinase is carried out under conditions permitting the simultaneous epimerization of the chiral center at C-5 of the hydantoin. As discussed further below, the simultaneous epimerization permits the reaction to be carried out to substantial completion so that the diastereomeric hydantoin mixture is converted to N-carbamoyl-D-isoleucine in high yield. The N-carbamoyl-D-isoleucine is then decarbamoylated to produce D-isoleucine.
In another embodiment, as shown in Scheme 2, the invention is directed to a method for producing L-allo-isoleucine comprising converting (R)-2-methylbutyraldehyde to a diastereomeric mixture of D-isoleucine hydantoin and L-allo-isoleucine hydantoin under conditions whereby no significant racemization of the chiral center in (R)-2-methylbutyraldehyde occurs, followed by contacting said diastereomeric hydantoin mixture with an L-hydantoinase to stereoselectively hydrolyze any L-allo-isoleucine hydantoin in the mixture to the corresponding N-carbamoyl-L-allo-isoleucine. Preferably in the claimed method the contacting of the diastereomeric hydantoin mixture with an L-hydantoinase is carried out under conditions permitting the simultaneous epimerization of the chiral center at C-5 of the hydantoin. As discussed further below, the simultaneous epimerization permits the reaction to be carried out to substantial completion so that the diastereomeric hydantoin mixture is converted to N-carbamoyl-L-allo-isoleucine in high yield. The N-carbamoyl-L-allo-isoleucine is then decarbamoylated to produce L-allo-isoleucine.
In another embodiment, as shown in Scheme 3, the invention is directed to a method for producing D-allo-isoleucine comprising converting (S)-2-methylbutyraldehyde to a diastereomeric mixture of D-allo-isoleucine hydantoin (5R-[(S)-1-methylpropyl]hydantoin) and L-isoleucine hydantoin (5S-[(S)-1-methylpropyl]hydantoin) under conditions whereby no significant racemization of the chiral center in (S)-2-methylbutyraldehyde occurs, followed by contacting said diastereomeric hydantoin mixture with a D-hydantoinase to stereoselectively hydrolyze any D-allo-isoleucine hydantoin in the mixture to the corresponding N-carbamoyl-D-allo-isoleucine. Preferably in the claimed method the contacting of the diastereomeric hydantoin mixture with a D-hydantoinase is carried out under conditions permitting the simultaneous epimerization of the chiral center at C-5 of the hydantoin. As discussed further below, the simultaneous epimerization permits the reaction to be carried out to substantial completion so that the diastereomeric hydantoin mixture is converted to N-carbamoyl-D-allo-isoleucine in high yield. The N-carbamoyl-D-allo-isoleucine is then decarbamoylated to produce D-allo-isoleucine.
In another embodiment, as shown in Scheme 4, the invention is directed to a method for producing L-isoleucine comprising converting (S)-2-methylbutyraldehyde to a diastereomeric mixture of D-allo-isoleucine hydantoin and L-isoleucine hydantoin under conditions whereby no significant racemization of the chiral center in (S)-2-methylbutyraldehyde occurs, followed by contacting said diastereomeric hydantoin mixture with an L-hydantoinase to stereoselectively hydrolyze any L-isoleucine hydantoin in the mixture to the corresponding N-carbamoyl-L-isoleucine. Preferably in the claimed method the contacting of the diastereomeric hydantoin mixture with an L-hydantoinase is carried out under conditions permitting the simultaneous epimerization of the chiral center at C-5 of the hydantoin. As discussed further below, the simultaneous epimerization permits the reaction to be carried out to substantial co
Paulson Basil P.
Rozzell, Jr. J. David
BioCatalytics, Inc.
Christie Parker & Hale LLP
Lilling Herbert J.
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