KR950008080B1 - Hplc chiral column for optical resolution of racemic amino acids - Google Patents

Abstract

A HPLC chiral column for optical resolution of racemic aminoacid is manufactured by dissolving N-carboxymethyl N-alkylaminoalcohol mono sodium salt or mono potassium salt in a mixt. solvent of water and an organic solvent; and circulating the obtd. soln. in C8 or C18 reversed phase column for HPLC. The N-carboxymethyl N-alkylaminoalcohol mono sodium salt or mono potassium salt is a deriv. of optically active aminoalcohol; and is prepd. by a reduction of an aminoacid.

Description

HPLC Chiral Column for Optical Fractionation of Racemic Amino Acids

The present invention relates to the preparation of a chiral column for HPLC that can be used to optically split racemic compounds that can act as multidentate ligands, such as racemic amino acids or racemic hydroxy carboxylic acids.

The material constituting the HPLC chiral column is a commercial C ₈ or C ₁₈ HPLC column with a chiral selector. The chiral selector having a long alkyl group is dissolved in a mixed solvent of water and an organic solvent, and then purified through a commercial C ₈ or C ₁₈ HPLC column. By pouring, chiral columns for HPLC can be prepared.

At this time, as the chiral selector, optically active amino acid derivatives, chiral crown ethers or (IS, 2R) -norephedrine derivatives have been used, but in the present invention, amino alcohol derivatives obtained by reduction of amino acids are used as chiral selectors. This extends the range of chiral selectivity and the magnitude of chiral selectivity.

Referring to the manufacturing method and process of the chiral selector used herein are as follows.

1. Manufacturing method of chiral selector

Amino alcohols (alanial, leucinol, valinol, phenyglycnol, etc.) are reacted with lauroyl chloride in the presence of triethylamine, for example, to obtain N-lauroylamino alcohol.

N-lauroylamino alcohol is reacted with LiAH₄ in THF solution to obtain N-dodecyl amino alcohol. The synthesized N-dodecylamino alcohol is dissolved in dichloromethane and reacted with ethylbromoacetate in the presence of propyleneoxide to obtain N-ethoxycarbonylmethy N-dodeylamino alcohol.

Finally, N-ethoxycarbonylmethyl N-dodecylamino alcohol is dissolved in methanol and treated with IN NaOH (or INKOH) solution to hydrolyze to obtain N-carboxymethyl N-dodecylamino alcohol mono sodium salt.

After dissolving about 2 g of the synthesized salt in 50 ml of a mol-methanol mixed solvent (1: 1, v / v), circulating through a commercial C HPLC column for 3 hours and flowing it with pure water after 3 hours, racemics such as racemic amino acids HPLC chiral columns are made that are applicable to the optical splitting of compounds.

When the racemic amino acid is optically divided by the HPLC chiral column, the structure of the ternary complex, which plays an important role in the optical division, that is, the ternary complex between the chiral selector Cu (II) and the amino acid having a hydrophobic bond to the C ₁₈ alkyl chain The structure of is expected to be as shown in Figure 1. A chiral selector was synthesized using (R) -Alaninol as a starting material, and the chiral selector was circulated for 3 hours through a C ₁₈ HPLC column to prepare a chiral column, and then racemic amino acid and racemic carboxylic acid were obtained using this chiral column. Table 1 shows the results of optical splitting when optical splitting is performed. Below is an example of the detailed preparation of the chiral selector and chiral column using (R) -Alaninol as starting material.

(1) Synthesis of N-Lauroyl- (R) -alaninol.

CH2Cl₂ (50ml), triethylamine (8.9ml, 64mM) and (R) -Alaninol (5ml, 64mM) were added to a 250ml round bottom flask, and slowly added Lauroyl chloride (15ml, 64mM) dissolved in 20ml CH₂Cl₂. Stir at room temperature for about 2 hours.

After stirring for 2 hours, the reaction mixture was washed three times with saturated aqueous NaHCO₃ solution, dried over anhydrous MgSO₄, and concentrated under reduced pressure to obtain a solid material. After dissolving this solid material in acetone, recrystallization by adding hexane gradually gave white crystals. (13.5g, 80% yield)

m.p. 70-72 ℃

H NMR (CDCl₃) δ0.8-1.5 (m, 24H), 2.1 (t, 2H), 3.5 (s, 2H), 3.9 (m, 2H), 6.0 (s, 1H)

IR (KBr) cm ^-1 3300, 2925, 1640, 1548, 1462, 1387

(2) Synthesis of N-Dodecyl- (R) -alaninol

N-Lauroyl- (R) -alaninol (10 g, 38 mM) and LiAIH₄ (6 g) were added to a 250 ml two-necked flask, and a small amount of THF (50 ml) dried under nitrogen stream at 0 ° C. was added. After reaching THF, reflux for about 24 hours. After refluxing for 24 hours, the reaction mixture is cooled to room temperature, water is added under ice bath conditions to terminate the reaction, and concentrated under reduced pressure to remove THF. CH₂Cl₂ (70ml) was added thereto, washed three times with water, dried over anhydrous MgSO₄ and concentrated under reduced pressure to obtain a solid material. This solid material was dissolved in CH₂Cl₂ and recrystallized with a small amount of nucleic acid to give white crystals (7.6g, 83% yield).

m.p. 63-65 ℃

H NMR (CDCl₃) δ1.5 (m, 26H), 2.5 (m, 3H), 3.4 (m, 2H)

IR (KBr) cm 3320, 3110, 2950, 1550, 1462, 1337.

(3) Synthesis of N-Ethoxycarbonylmethyl N-Dodecyl- (R) -alaninol

In a 250 ml round bottom flask, N-Dodecyl- (R) -alaninol (6 g, 24.8 mM) synthesized above was added, dissolved in 50 ml of CH₂Cl₂, and then Ethoxybromoacetate (25 mM) and Propyleneoxide (1 ml) were added and the mixture was stirred at room temperature.

Stir for about 7 hours, you can see that a white solid is produced. After 24 hours, stop the reaction, filter the white solid with filter paper, and wash the filtrate three times with water. The organic solution washed with water was dried over anhydrous MgSO₄, and then separated by column chromatography to obtain white crystals (2.2g, 27% yield).

m.p. 78-80 ℃

H NMR (CDCl₃) δ1.2 (m, 29H), 2.9 (m, 2H), 3.25 (s, 2H), 3.4 (s, 2H), 4.2 (t, 2H)

IR (KBr) cm ^-1 2900, 1750, 1420, 1380.

(4) Synthesis of N-Carboxymethyl N- (R) -alaninol Monosodium Salt

In a 50 ml round bottom flask, N-ethoxycarbonymethyl N-Dodecyl- (R) -alaninol (2.0 g, 6.08 mM) synthesized above was added and dissolved in 20 ml of methanol.

Add NaOH (methanol solution) of IN slowly through the dropping funnel. Stir at room temperature for about 5 hours and concentrate under reduced pressure to give an oily product (1.8 g).

IR (KBr) cm ^-1 2900, 1620, 1480, 1410

2. Manufacturing method of chiral column

The compound synthesized above, N-carboxymethl N-dodecyl- (R) -alaninol monosodium salt (1.8 g), is dissolved in 50 ml of water-methanol (1: 1) mixed solvent and circulated through a C ₁₈ reversed phase column for HPLC. At this time, the flow rate of HPLC is maintained at 0.8 ml / min.

After observing that the absorption of UV is kept constant by the UV detector, the HPLC detector, the water-methanol (1: 1) mixed solvent is continuously flowed.

At this time, the HPLC flow rate was maintained at 0.3 ml / min. After 7 hours, the mobile phase was changed to pure water and kept flowing for 5 hours to obtain a chiral column that can be used for optical division of racemic amino acids.

3. Optical division of racemic amino acids by chiral column.

Dissolve an appropriate amount of CuSO 메탄 in a suitable mixed solvent of water-methanol or water-acetonitrile (the composition of the organic solvent in the mixed solvent should not exceed 20% to keep the chiral selector on the column) ( Considering the solubility of CuSO ₄ below 0.3mM) mobile phase is made and flowed through the prepared HPLC chiral column. When the racemic amino acid or hydroxy carboxylic acid to be optically injected is injected, the two optical isomers are separated.

Figure 1.Structure of a tertiary complex formed between a chiral selector immobilized on a C ₁₈ alkyl chain and Cu (II) and an amino acid.

The (D) -amino acid stays longer in the chiral column because of the hydrophobic interaction between the C ₁₈ alkyl chain and the R group of (D) -amino acid.

Table 1.Optical fractionation of racemic amino acids and racemic hydroxycarboxylic acids on chiral stationary phases based on (R) -Alaniol derivatives.

TABLE 1

a: mobile phase 1: pure water (0.25 mM CuSO ₄ ), 2: methanol-water (10:90, 0.25 mM CuSO ₄ )

As described above, as a method for preparing chiral stationary phase for HPLC, which is useful for the synthesis of chiral selectors used in the chiral column of the present invention and the optical separation of racemic amino acids using chiral selectors, racemic amino acids and racemic HPLC chiral columns with high stereoselectivity and relatively easy production are prepared for transition metal ions such as hydroxycarboxylic acids and racemic compounds that can be coordinated at various positions. It is big.

Claims (1)

N-carboxymethyl N-alkiylaminoalcohol mono sodium mono potassium salt or N-carboxymethyl N-alkiylaminoalcohol mono sodium satl or mono potassium salt, a derivative of optically active aminoalcohol obtained by reduction of amino acids, is dissolved in a mixed solvent of water and organic solvent HPLC chiral column for optical separation of racemic amino acids, characterized in that the circulating through a C ₈ or C ₁₈ reverse phase column to prepare an HPLC chiral column usable for optical division of racemic amino acids.