JPH0355464B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for separating amino acid enantiomers (left and right images) by chromatography.
Conventional methods for separating amino acid enantiomers using high-performance liquid chromatography include methods for converting amino acids into diastereomers (stereoisomers) prior to chromatography, and transfer using a chiral stationary phase or chiral reagent. There was a method using phase. The present invention belongs to the latter, but the former method, which is a conventional method, requires diastereomeric derivatives of amino acids prior to separation by chromatography, and this derivatization operation is complicated. , quantitative nature, etc. are often a problem. In most cases, separation must be performed by normal phase chromatography using an organic solvent as a mobile phase. In the latter case,
A conventional method uses a proline-copper complex as a mobile phase, but it is impossible to detect proline or hydroxyproline. In addition, in the conventional method using aspartyl cyclohexylimide and copper, the reagent itself has a primary amino group, so the ninhydrin method and o-
Highly sensitive detection methods such as the phthalaldehyde method could not be applied. Furthermore, as a conventional method, there is a method using N-(p-toluenesulfonyl)-L-phenylalanine, as disclosed in JP-A No. 57-176936, but this method has a poor ability to separate alanine in the DL form. It was something I didn't have. For example, the separation state shown in FIG. 3 of JP-A No. 57-176936 has the disadvantage that the separation of D-alanine and L-alanine is not always clear;
-Separation of serine and DL-glutamine was difficult. The present invention relates to a method for separating amino acid enantiomers by chromatography, which eliminates the above-mentioned drawbacks of conventional methods. - Phenylglycine and CuSO _{4.5H 2} O are included to separate D and L-amino acids, and N-(p-toluenesulfonyl)-D-phenylglycine is conventionally separated from free amino acids after separation. Ortho-phthalaldehyde, which had been used for trace detection, can be used as is as a detection reagent, making it possible to detect down to picomole (10 ^-12 mole) units. Furthermore, since this chiral reagent is negative for ninhydrin reactions, it is possible to detect amino acids with secondary amino groups such as proline and hydroxyproline by combining the ninhydrin method instead of the o-phthalaldehyde method. is also becoming possible. N-(p-toluenesulfonyl)-D-phenylglycine can separate racemic amino acids just by adding it to the mobile phase, and orthophthalaldehyde, which reacts with amino acids with primary amino groups, can be used as a detection reagent. . . In addition, in this use, the N-(P-toluenesulfonyl)-D-phenylglycine reagent is alkaline and causes precipitation of copper ions, but this can be prevented by adding disodium ethylenediaminetetraacetate to the reagent. It becomes possible. This method allows detection of amino acids down to picomole units. N-(p-toluenesulfonyl)-D-phenylglycine and CuSO ₄ .5H ₂ O ion in the mobile phase are a kind of binary complex, [(N-(p -Toluenesulfonyl)-D-phenylglycine) ₂ CuSO ₄ .
5H ₂ O]. Also free amino acids, N-(p-toluenesulfonyl)-D-phenylglycine and CuSO _{4.5H 2} O
is thought to form a ternary complex, with D-type and L-type
It is thought that complexes with different tertiary structures can be formed with these types of amino acids as shown below. In this case, the complex containing the D-amino acid is a trans form, which is considered to mean that it is more stable and more strongly retained in chromatography than the cis complex containing the L-amino acid. N-(p-toluenesulfonyl)-D-phenylglycine and CuSO _{4.5H 2} O-D amino acid N-(p-toluenesulfonyl)-D-phenylglycine and _CuSO4.5H2O - _L amino acids. This molecular model can be explained by the fact that the L-amino acid elutes faster than the D-isomer. Also, as explained later, there is a difference in the mobility of amino acids, and this difference is due to the n bonded on silica gel.
This appears to be due to the difference in hydrophobicity and steric interaction between the -octyl residue and the substituted alkyl group on the α-carbon of the amino acid. Therefore, amino acids with more hydrophobic alkyl substituents were retained on the column for longer periods of time than amino acids with less hydrophobic alkyl substituents. In the case of isomers having the same number of carbon atoms, they are amino acids with branched side chains. Valine and leucine elute faster than norvaline and norleucine, which have linear side chains. Furthermore, N-(p-toluenesulfonyl)-D-phenylglycine can be synthesized by synthesizing the following components according to the following procedure. D-phenylglycine 3.0wt% Water 71.3wt% Tetrahydrofuran 14.1wt% Triethylamine 5.9wt% Para-toluenesulfonyl chloride 5.7wt% First, 3.0wt% D-phenylglycine, 31.7wt% water, and 14.1wt% tetrahydrofuran are mixed. After that, cool it with cooling water. While this cooling continues, 5.9 wt% of triethylamine is added, then 5.7 wt% of para-toluenesulfonyl chloride is added little by little over 30 minutes, and stirring is continued for an additional 90 minutes. Next, after evaporating the tetrahydrofuran at room temperature until the odor is no longer noticeable, the remaining water is 39.6wt.
Add %. Next, wash with ether,
After extracting and removing excess para-toluenesulfonyl chloride and para-toluenesulfonic acid, dilute hydrochloric acid is added to adjust the pH to 3 to 4 to form crystals.
This crystal was mixed with 80wt% of methanol and 20wt% of water.
N-(p-toluenesulfonyl)-D-phenylglycine can be obtained by recrystallizing it in a liquid. As described above, the present invention provides N-
By using (p-toluenesulfonyl)-D-phenylglycine as the mobile phase, N-(p-
Toluenesulfonyl)-D-phenylglycine is
It was developed for the purpose of making the non-selective discrimination in the optical resolution of amino acids more efficient than the conventional method using N-(p-toluenesulfonyl)-L-phenylalanine. In the method of adding chiral ligands and metal ions, the ligand exchange during the chromatographic process is used to distinguish between the target optical enantiomers. The resolving power can be increased by taking measures to improve the stereoselectivity. N-
(p-Toluenesulfonyl)-D-phenylglycine has one less methylene group than N-(p-toluenesulfonyl)-D-phenylalanine, so it is formed as a result of ligand exchange. In the case of N-(p-toluenesulfonyl)-D-phenylglycine, the phenyl group is directly N- (p-toluenesulfonyl)
-D-phenylalanine is presumed to have a structure in which it is bonded via a methylene group.
Therefore, when N-(p-toluenesulfonyl)-D-phenylglycine is used, the degree of stereochemical freedom of the complex is lowered, and therefore the stereoselectivity during ligand exchange is higher. . In fact, when N-(p-toluenesulfonyl)-D-phenylglycine was used, when N-(p-toluenesulfonyl)-D-phenylalanine was used, the D,L- Glutamine was completely cleaved, and improvements in the cleavage efficiency of many other amino acids were observed. Specific examples of the present invention will be described below. First, regarding the reagents, the mobile phase contains N-(p-toluenesulfonyl)-D-
An acetonitrile-water system containing phenylglycine and CuSO ₄ .5H ₂ O (divalent copper ion) at a molecular ratio of 2:1 was used. The pH of the mobile phase was adjusted with an aqueous sodium carbonate solution. The orthophthalaldehyde reagent contains 2.5% disodium ethylenediaminetetraacetate.
It is included so that it becomes g/. Next, in the chromatographic system, the mobile phase and post-column reagent (ortho-phthalaldehyde reagent in this case) were flowed at a constant rate of 1.0 ml/min using a pump. As a filler, chemically bonded n-octylsilyl silica gel was used, with unreacted silanol groups masked with trimethylsilane. This slurry was packed into a 10 cm x 4.0 mm I.D stainless steel column. Chromatography was performed while keeping this packed column at 30°C or 80°C. The column eluate was mixed with an orthophthalaldehyde reagent using a T-shaped multi-tech tube, and reacted in a 50 cm x 0.5 mm I.D Teflon tube reaction coil. The fluorescence intensity of the eluate was measured using a spectrofluorescence monitor, with fluorescence excited at 340 nanometers and 455 nanometers. The results are as follows, and for convenience of explanation, the symbols used below indicate: K'=capacity ratio α=separation factor (relative retention power) R=separation power. These values vary depending on the pH of the mobile phase, the concentration of N-(p-toluenesulfonyl)-D-phenylglycine, CuSO ₄ .5H ₂ O complex, and acetonetrile in the mobile phase. Furthermore, K' and α become high in the PH5 to PH7 range, and most amino acids can be separated within this PH range. However, racemic forms of alanine, aspartic acid, glutamic acid, and asparagine can be separated at pH 6 or higher. Table 1 shows the capacity ratio K', the separation factor α, and the difference in free energy (△△G°=-
This shows the relationship between RTlnα) and acetonitrile concentration. The mobile phase contained 1mM N-(p-toluenesulfonyl)-D-phenylglycine and
An aqueous solution containing 0.5 mM CuSO ₄ .5H ₂ O at pH 6.0 to which acetonitrile was added at the indicated wt% was used.

【table】

【table】 [Brief explanation of drawings]

Figure 1A shows N-(p-toluenesulfonyl)-
D-phenylalanine and CuSO ₄ .5H ₂ O eluent, and _FIG .
This shows the separation state of DL-serine and DL glutamine using 5H ₂ O eluent, and A is the mobile phase.
1 mM of N-(p-toluenesulfonyl)-D-
Phenylalanine and _{0.5mM CuSO4.5H2O}
was used at pH 6.0, and each amino acid in the figure was used at 0.25 mM. B also contained 1mM of N-(p-toluenesulfonyl)-D-phenylglycine and 0.5mM of N-(p-toluenesulfonyl)-D-phenylglycine.
M CuSO ₄ .5H ₂ O was used at pH 6.0, and each amino acid in the figure was used at 0.25 mM. This result reveals the excellent separation ability of the present invention. Figure 2 shows N-(P
-Toluenesulfonyl)-D-phenylglycine and _CuSO4.5H2O eluent.The column was kept at 30 _℃ , and the mobile phase was 10% acetonitrile, 1mM N
-(p-Toluenesulfonyl)-D-phenylglycine and 0.5 mM CuSO ₄ .5H ₂ O were used at pH 6.0, and about 0.5 nmole of each amino acid shown in the figure was injected.
Figure 3 shows the separation of DL amino acids using N-(p-toluenesulfonyl)-D-phenylglycine and CuSO _{4.5H 2} O eluent. The column was kept at 80°C and the mobile phase to 0.0075% Na ₂ CO ₃ , 1
% acetonitrile, 1mM N-(P-toluenesulfonyl)-D-phenylglycine and 0.5mM
Using CuSO ₄ 5H ₂ O of M, each amino acid in the figure is
0.5 nmole was injected.

Claims (1)

[Claims] 1 Chromatography of amino acid enantiomers, characterized in that amino acid enantiomers are separated by reversed phase chromatography using N-(p-toluenesulfonyl)-D-phenylglycine and CuSO ₄ .5H ₂ O as a mobile phase. separation method.