Compositions – Compositions containing a single chemical reactant or plural...
Reexamination Certificate
2000-04-14
2004-06-01
Toomer, Cephia D. (Department: 1714)
Compositions
Compositions containing a single chemical reactant or plural...
C562S434000, C562S435000, C562S452000, C562S453000, C562S457000
Reexamination Certificate
active
06743373
ABSTRACT:
The invention relates to a process for the separation of enantiomers and to reagents based on an enantiopure amino acid which can be used in the separation of enantiomers.
The separation of enantiomers is a matter of great, importance in the pharmaceutical, chemical and biotechnology industries. This is because the two enantiomers of a chemical substance with an identical composition can have radically different biological activities. It is thus desirable to have available separation reagents and techniques which make it possible to separate the enantiomers and to analyse the enantiomeric purity of pharmaceutical, chemical and biotechnology products.
An article by Marfey, P., (Carlsberg Res. Comm., 49, 1984, 591-596) describes a process for the separation of enantiomers by RP-HPLC. According to this known process, 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide is used as reagent for the derivatization of amino acids. Other similar processes are also known. However, the process and reagent which are described by Marfey and the other processes and reagents exhibit numerous disadvantages.
The derivative obtained in the reaction of the amino acid with the reagent has to be isolated by successive neutralization, drying, redissolution and filtration operations. These operations take a great deal of time and are therefore not very advantageous in an industrial application. Furthermore, there is a risk, in cases of analytical applications, of errors in the analytical results caused by differing solubility of the diastereomeric derivatives in the redissolution solvent. When quantitative analyses are carried out using UV spectrometry, difficulties due to differences in absorption coefficient of the diastereomeric derivatives are encountered in the known process. Finally, the high cost of the reagent renders it desirable to find alternatives.
The invention is targeted at overcoming these problems.
The invention consequently relates to a process for the separation of enantiomers comprising at least one free functional group, in which
(a) a mixture comprising the enantiomers is reacted in basic medium with a reagent based on an enantiopure amino acid, in which reagent at least one amino group of the amino acid carries an activating group, in order to form an active precursor of an isocyanate group, and in which reagent at least one carboxyl group of the amino acid is substituted, and
(b) the mixture of diastereomers obtained is subjected to a separation operation.
It has been found, surprisingly, that the process according to the invention makes it possible to obtain good results with regard to the separation of enantiomers comprising at least one free functional group, in particular in quantitative analytical applications. The process according to the invention makes possible rapid derivatization and rapid separation of enantiomers under flexible and economical conditions.
The invention also relates to a reagent based on an enantiopure amino acid in which at least one amino group of the amino acid carries an activating group in order to form an active precursor of an, isocyanate group and in which at least one carboxyl group of the amino acid is substituted.
The term “amino acid” is understood to denote, for the purposes of the present invention, any compound comprising at least one NH
2
group and at least one carboxyl group. The amino acids used in the present invention are chiral amino acids comprising at least one asymmetric carbon. Use may be made of any chiral amino acid well known in itself of natural or synthetic origin.
Examples of reagents according to the invention are based, for example, on the following natural amino acids: alanine, valine, norvaline, leucine, norleucine, isoleucine, serine, isoserine, homoserine, threonine, allothreonine, methionine, ethionine, glutamic acid, aspartic acid, asparagine, cysteine, cystine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, ornithine, glutamine and citrulline.
Unnatural enantiomers can also be used.
Examples of amino acids of synthetic origin which can bemused as basis for the reagent according to the invention comprise, for example, the following amino acids: (1-naphthyl)alanine, (2-naphthyl)alanine, homophenylalanine, (4-chlorophenyl)alanine, (4-fluoro-phenyl)alanine, (3-pyridyl)alanine, phenylglycine, diaminopimelic acid (2,6-diaminoheptane-1,7-dioic acid), 2-aminobutyric acid, 2-aminotetralin-2-carboxylic acid, erythro-&bgr;-methylphenylalanine, threo-&bgr;-methylphenylalanine, (2-methoxyphenyl)alanine, 1-amino-5-hydroxyindan-2-carboxylic acid, 2-amino-heptane-1,7-dioic acid, (2,6-dimethyl-4-hydroxyphenyl)-alanine, erythro-&bgr;-methyltyrosine or threo-&bgr;-methyl-tyrosine.
The term “enantiopure amino acid” is understood to denote a chiral amino acid composed essentially of one enantiomer. The enantiomeric excess (ee) is defined: ee (%)=100(x
1
−x
2
)/(x
1
+x
2
) with x
1
>x
2
; x
1
and x
2
represent the content of enantiomer 1 or 2 respectively in the mixture.
Use is generally made of an enantiopure amino acid with an enantiomeric excess of greater than or equal to 99%. Preference is given to an enantiopure amino acid with an enantiomeric excess of greater than or equal to 99.5%. In a particularly preferred way, use is made of an enantiopure amino acid with an enantiomeric excess of greater than or equal to 99.9%.
Any enantiopure amino acid can be used as basis for the reagent according to the invention. The enantiopure amino acid is preferably selected from the abovenamed amino acids of natural or synthetic origin. Amino acids comprising at least one aromatic nucleus, such as, for example, phenylalanine or its derivatives, are particularly well suited as enantiopure amino acid. In a particularly preferred way, the enantiopure amino acid is selected from phenylalanine, (1-naphthyl)-alanine, (2-naphthyl)alanine or &agr;- or &bgr;-tryptophan ((2-indolyl)alanine or (3-indolyl)alanine), which are optionally substituted.
In the reagent according to the invention, at least one amino group of the enantiopure amino acid carries an activating group in order to form an active precursor of an isocyanate group.
The term “active precursor of an isocyanate group” is understood to denote any precursor which, when it is employed in a solvent which can be used in the process according to the invention with 1 equivalent of phenylalanine in the presence of 1 equivalent of base, reacts at a temperature of less than or equal to 35° C. essentially completely in a period of time of less than or equal to 30 min to form the corresponding urea. The reactive precursor preferably releases the isocyanate group at a temperature of less than or equal to 30° C. in a period of time of less than or equal to 15 min. In a very particular preferred way, the reactive precursor releases the isocyanate group at room temperature in a period of time of less than or equal to 10 min. Test conditions which can be used to determine the active precursor are described, for example, in Example 3 below.
The activating group is generally composed of a carbonyl derivative bonded to ant electronegative substituent. Use may: be made, for example, as activating group, of an aryloxycarbonyl, heteroaryloxycarbonyl, 1,3-imidazolyl-N-carbonyl or 1,2,4-triazolyl-N-carbonyl group. The aryloxycarbonyl groups which are well suited include those which carry at least one −I, −M substituent on an aromatic nucleus. An −I, −M substituent is a group which has a negative inductive effect and negative resonance effect as defined in J. March, Advanced Organic Chemistry, 4th Ed., 1992, p. 17-19, 273-275. The −I, −M substituents include, for example, —NO
2
, —SO
2
R, —SO
2
OR, —NR
3
+
and SR
2
+
. The substituents are preferably found at at least one of the 2, 4 or 6 positions of the aromatic nucleus or at positions analogous to the 2 or 4 positions in condensed aromatic systems. It is preferable to use an aryloxycarbonyl activating group which carries at least one nitro substituent on the aromatic nucleus. The (4
Callens Roland
Delplanche Thierry
Connolly Bove & Lodge & Hutz LLP
Solvay ( Societe Anonyme)
Toomer Cephia D.
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