JPH0440661B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a chromatographic packing material grafted with a novel optically active organosilane and -OCNH- group, -OH group, -
OCO- group, -OCONH- group or

This invention relates to a method for separating and analyzing a mixture of enantiomers of a compound having a group represented by the formula: by liquid chromatography. As a filler grafted with an optically active compound for directly separating and analyzing an enantiomeric mixture of a compound having an asymmetric carbon by liquid chromatography, for example, Davankov et al. grafted optically active proline. A method using a ligand exchange using a filler, a method using a charge transfer complex using a filler grafted with an optically active compound lacking π electrons by Gil-Av, etc., a method using a charge transfer complex using a filler grafted with an optically active N-acylated amino acid by Hara et al. Separation of N-acylated amino acid esters and N-acylated dipeptide esters using packing materials or Pirkle
3,5-dinitrobenzoylated amino acids using optically active 1-(9-anthryl)trifluoroethanol-grafted fillers by et al.
Separation of amines, oxyacids, sulfoxides, etc. and separation of aromatic alcohols using fillers grafted with 3,5-dinitrobenzoylated optically active phenylglycine have been reported. However, these methods are limited to a narrow range of compounds that can be separated, the degree of separation is small, and it is difficult to produce grafted fillers, making it difficult to produce fillers with reproducible performance. It is difficult to obtain fillers and it is difficult to call them practical fillers. Under such circumstances, the present inventors have continued to conduct intensive studies with the aim of developing a grafted filler that is applicable to a wide range of compounds that can be analyzed, is relatively easy to manufacture, and is chemically stable and practical. As a result, a chromatographic packing material in which a compound formed by bonding an optically active isocyanate derivative and an aminoalkylsilane to an inorganic carrier having hydroxyl groups on its surface was grafted was bonded to the asymmetric carbon.
CONH- group, -OH group, -OCO- group, -OCONH-
It not only shows an excellent effect in separating enantiomeric mixtures of compounds having -N-CONH- or -N-CONH- groups, but also is extremely useful because it can be easily produced by ordinary chemical reactions and is chemically stable. It was discovered that it is a filler, leading to the present invention. That is, the present invention provides a chromatographic packing material in which a compound formed by bonding an optically active isocyanate derivative and an aminoalkylsilane is grafted onto an inorganic carrier having a hydroxyl group on its surface, and a chromatographic packing material that is used in liquid chromatography. -CONH- group, -OH group, bonded to asymmetric carbon used in the stationary phase,
-OCO- group, -OCONH- group or

Provided are methods for separating and analyzing enantiomeric mixtures of compounds having the group ##STR1##. In the present invention, examples of the grafted compound include the general formula [] [In the formula, R ₁ , R ₂ and R ₃ are the same or different and represent an alkyl group, an alkoxyl group, a hydroxyl group or a halogen atom, and at least one of R ₁ , R ₂ and R ₃ is an alkoxyl group or It is a halogen atom. X is

【formula】

【formula】

[expression] or

[Formula] represents. Here, R ₅ represents an aryl group, an alkyl group or an aralkyl group. R ₄ represents an alkyl group or an aralkyl group, and Ar represents a phenyl group or a naphthyl group which may have a substituent. However, when Ar represents a naphthyl group, R ₄
represents a methyl group. n represents an integer from 2 to 4, and * represents an asymmetric carbon. ] Organosilanes shown in the following can be mentioned,
More specifically, in the compound represented by the above general formula [], the optically active isocyanate as a raw material is a lower alkyl isocyanate substituted with an aryl group at the α position or an aralkyl isocyanate substituted with an aryl group at the α position. is preferable, and examples thereof include 1-phenylethyl isocyanate, 1-(α-naphthyl)ethyl isocyanate, and 1-phenyl-2-(4-tolyl)ethyl isocyanate. In addition, the X part, for example, -NHCO-CH(C ₆ H ₅ )-NH- or -N
-NHCO-CH( _C6H5 )-O- or -NHCO _- CH( _C6H5 )-O- in _which a derivative _produced by reacting mandelic acid with an isocyanate is bonded to an aminoalkylsilane. -N
Examples include _H3.OCO -CH _{( C6H5} )-O-. In addition, as an aminoalkylsilane component, ω-
Aminoalkylalkoxysilanes or ω-aminoalkylhalogenosilanes are preferred, e.g.
Examples include ω-aminopropyltriethoxysilane and ω-aminopropyltrichlorosilane. In the present invention, the inorganic carrier having a hydroxyl group on its surface is preferably a silica-containing carrier such as silica gel, and the shape of the carrier is spherical,
Although the particles may be in any shape, including crushed ones, fine particles with as uniform a particle size as possible are preferred in order to obtain a highly efficient chromatographic column. Various grafting methods can be employed to prepare the novel chromatographic packing material of the present invention, including the following methods. An aminoalkylsilane is reacted with an inorganic carrier having a hydroxyl group on its surface to introduce an aminoalkylsilyl residue onto the surface of the inorganic carrier. Next, this is reacted with an N-carbamoylated amino acid or an O-carbamoylated oxyacid using an optically active isocyanate,
A method of dehydration condensation or ionic bonding. Specifically, an inorganic carrier having a hydroxyl group on its surface has the general formula [] [In the formula, R ₁ , R ₂ , R ₃ and n have the same meanings as above. ] The aminoalkylsilane represented by the formula [] is reacted by a known method to introduce an aminoalkylsilyl residue onto the surface of the inorganic carrier, and then the general formula [] [In the formula, R ₄ , Ar and * have the same meanings as above. ] An optically active isocyanate represented by the formula is reacted with an amino acid or an oxyacid to form the general formula [ ] [In the formula, R ₄ , Ar and * have the same meanings as above, R ₆ represents an aryl group, an alkyl group or an aralkyl group, and Y represents an -NH- group or an oxygen atom. ] The desired filler can be obtained by converting the derivative of the isocyanate represented by the formula below into a dehydration condensation or ionic bonding with an inorganic carrier into which the above-mentioned aminoalkyl residue has been introduced. The optically active isocyanate represented by the above general formula [] can be prepared by commonly used methods such as 1-phenylethylamine, 1-(α-
naphthyl)ethylamine or 1-phenyl-
The isocyanate derivatives obtained by reacting 2-(4-tolyl)ethylamine with phosgene and represented by the above general formula [] can also be prepared by commonly used methods such as 1-phenylethylisocyanate, 1-(α −naphthyl)
Ethyl isocyanate or 1-phenyl-2
-(4-tolyl)ethyl isocyanate, D
-obtained by reacting with a sodium salt of an amino acid such as phenylglyne in aqueous solution, or by reacting with a triethylamine salt of an oxyacid such as (S)-mandelic acid in dehydrated tetrahydrofuran. A method in which an organosilane obtained by reacting an aminoalkylsilane with an N-carbamoylated amino acid or an O-carbamoylated oxyacid using an optically active isocyanate is grafted onto an inorganic carrier having a hydroxyl group on its surface. Specifically, an organosilane represented by the general formula [] obtained by reacting the isocyanate represented by the above general formula [] with an aminoalkylsilane represented by the general formula [] is grafted onto an inorganic carrier such as silica gel. By doing so, the desired filler can be obtained. The chromatographic packing material obtained according to the present invention is packed into a chromatographic column according to a conventional method and used as a stationary phase in liquid chromatography. In the case of the above-mentioned grafting, an inorganic carrier such as silica gel is reacted with an aminoalkylsilane represented by the general formula Therefore, a stationary phase for similar liquid chromatography can also be prepared by filling a chromatography column and grafting the isocyanate derivative represented by the general formula [] into the column. By selecting appropriate elution conditions in liquid chromatography using this stationary phase, especially the commonly used normal phase distribution or reversed phase distribution conditions,
-CONH- group, -OH group, - bonded to asymmetric carbon
OCO− group, −OCONH− group, or

Separation and analysis of a mixture of enantiomers of a compound having the group [Formula] can be performed with good resolution and in a short time. Example 1 Silica gel (average particle size 10 μm, average pore size 60 Å,
After drying 10 g (surface area: 500 m ² /g) under reduced pressure at 130°C for 4 hours, 20 g of 3-aminopropyltriethoxysilane was dissolved in 200 ml of dehydrated toluene, and the mixture was stirred at 60°C for 6 hours. Filter the reaction;
The residue was washed with 100 ml of acetone and dried to obtain 3-aminopropylsilylated silica gel (hereinafter abbreviated as APS). The elemental analysis value of this item is N: 1.20
%, C: 3.40%, which corresponds to about 0.90 mmol of 3-aminopropyl group grafted to 1 g of this product. Separately, add 145 g of trichloromethyl chloroformate to 50 ml of toluene, shake gently, add 1 g of granular activated carbon, leave overnight to gently generate phosgene, and filter the activated carbon to remove the liquid. Add a solution of 31 g of R(+)-1-(α-naphthyl)ethylamine dissolved in 50 ml of toluene,
Stir. Next, after heating and refluxing this mixture for 4 hours,
The mixture was allowed to cool, and excess phosgene and the solvent toluene were distilled off under reduced pressure to obtain 35.3 g of R(-)-1-(α-naphthyl)ethyl isocyanate. This is a viscous yellow-brown liquid at room temperature. Optical rotation; [α] ²⁰ _D = -54.6° (c = 0.83%, toluene) Elemental analysis (as C ₁₃ H ₁₁ NO) Carbon (%) Hydrogen (%) Nitrogen (%) Calculated value 79.17 5.62 7.10 Actual value 78.98 5.67 6.97 Dissolve 3.0 g of D-phenyglycine in 20 ml of 1N aqueous sodium hydroxide solution, and add 4 ml of tetrahydrofuran.
and while stirring, the above R(-)-1-(α-
Add 4.0 g of ethyl isocyanate and continue stirring at room temperature overnight. After adding 10 ml of 1N aqueous sodium hydroxide solution and 30 ml of water to the reaction solution, it was washed twice with 50 ml of ethyl acetate, acidified with 6N hydrochloric acid,
The resulting white solid substance was diluted with 100 ml of ethyl acetate.
Extract times. The extract was washed twice with 100 ml of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then recrystallized from a mixture of ethyl acetate and n-hexane.
{(R)-1-(α-naphthyl)ethylcarbamoyl}
3.1 g of -D-phenylglycine was obtained as white crystals. Foam decomposition point: 201-202℃ Optical rotation: [α] ²⁰ _D = -126° (c = 0.87%, methanol) Elemental analysis (as C ₂₁ H ₂₀ N ₂ O ₃ ) Carbon (%) Hydrogen (%) Nitrogen (%) Calculated value 72.40 5.79 8.04 Actual value 72.26 5.77 7.93 Next, take 1.74 g of this compound, add 20 ml of methanol-tetrahydrofuran (1:1) mixture to dissolve it, and add this to 2.5 g of APS obtained in Example 1. After adding and suspending the mixture and thoroughly deaerating it under reduced pressure, the mixture was gently stirred at room temperature for one day and night. The reaction mixture was diluted four times with 30 ml of tetrahydrofuran, then twice with 30 ml of methanol.
The desired filler (hereinafter NEC-PHG- (abbreviated as Si) was obtained. The elemental analysis values of this are N: 2.10%, C:
12.8%, which means that 1g of this substance contains N-
{(R)-1-(α-naphthyl)ethylcarbamoyl}
It shows that about 0.45 mmol of -D-phenylglycine was grafted. The thus obtained packing material was slurried packed into a stainless steel column with an inner diameter of 4 mm and a length of 25 cm.
O-(3,5-dinitrophenylcarbamoyl)-(±)-1-phenylethyl alcohol was analyzed under the following conditions, and the chromatogram shown in Figure 1 was obtained. Temperature: Room temperature Mobile phase: Hexane/dichloroethane/ethanol (15:4:1) Flow rate: 1.0ml/min Detector: Ultraviolet absorption meter (wavelength 254nm) In Figure 1, peak number (1) is O-( 3,5-dinitrophenylcarbamoyl)-(-)-1-phenylethyl alcohol, (2) is each O-(3,5-dinitrophenylcarbamoyl)-(+)-1-phenylethyl alcohol It is the peak. The time required for peak (2) to elute is approximately 15 minutes. The separation factor is
1.41, and the area ratio of peaks (1) and (2) was 50:50. Example 2 According to the method for producing R(-)-1-(α-naphthyl)-ethyl isocyanate in Example 1, S(+)-1-
By reacting phenyl-2-(4-tolyl)ethylamine with phosgene, S(+)-1-phenyl-2
-(4-tolyl)ethyl isocyanate was obtained.
This is a pale yellow liquid at room temperature. Optical rotation; [α] ²⁰ _D = -14.0° (c = 1.74%, toluene) Elemental analysis (as C ₁₆ H ₁₅ NO) Carbon (%) Hydrogen (%) Nitrogen (%) Calculated value 80.99 6.37 5.90 Actual value 80.72 6.43 5.78 Separately, 3.78 g of D-phenylglycine was dissolved in 25 ml of 1N aqueous sodium hydroxide solution, 5 ml of tetrahydrofuran was added, and while stirring, the above-mentioned S(+)-1-phenyl-2-(4-tolyl) was added. ) Add 5.22 g of ethyl isocyanate and continue stirring at room temperature overnight. 1N sodium hydroxide aqueous solution in the reaction solution
After adding 10ml, wash twice with 40ml of ethyl acetate,
Acidify with 6N hydrochloric acid and extract the resulting white solid substance three times with 100 ml of ethyl acetate. The extract was washed twice with 100 ml of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then recrystallized from a mixture of ethyl acetate and n-hexane to obtain N{(S)-1-
5.7 g of phenyl-2-(4-tolyl)ethylcarbamoyl}-D-phenylglycine was obtained as white crystals. Flash decomposition point; 173°C to 175°C Optical rotation; [α] ²⁰ _D = -83° (c = 0.39%, methanol) Elemental analysis (as C ₂₄ H ₂₄ N ₂ O ₃ ) Carbon (%) Hydrogen (%) Nitrogen (%) Calculated value 74.20 6.23 7.21 Actual value 74.23 6.20 7.15 Next, take 2.0 g of this compound, add 100 ml of tetrahydrofuran and dissolve it, and separately add the APS obtained in Example 1 to a stainless steel plate with an inner diameter of 4 mm and a length of 25 cm. Approximately 2 ml/ml of slurry is filled in the column.
N-{(S)-1-phenyl-2-(4-tolyl)ethylcarbamoyl} by circulating at a flow rate of min for 2 hours.
After grafting -D-phenylglycine, the column is conditioned by sequentially flowing tetrahydrofuran, methanol and chloroform (hereinafter this column will be abbreviated as PTC-PHG-Si). Using this column, N-(3,5-dinitrobenzoyl)-D,L-valine methyl ester was analyzed under the following conditions, and the chromatogram shown in Figure 2 was obtained. Temperature: Room temperature Mobile phase: n-hexane/dichloromethane/ethanol (15:4:1) Flow rate: 1.0ml/min Detector: Ultraviolet absorption meter (wavelength 254nm) In Figure 2, peak number (1) is N-( 3,5-dinitrobenzoyl)-D-valine methyl ester,
(2) is each peak of N-(3,5-dinitrobenzoyl)-L-valine methyl ester. The time required for peak (2) to elute was approximately 8 minutes, the separation factor was 1.98, and the area ratio of peaks (1) and (2) was 50:50. Example 3 After dissolving 3.35 g of S(+)-mandelic acid in 30 ml of dehydrated tetrahydrofuran and adding 3.0 g of triethylamine, R(-)-1-(α
- Add 4.33 g of naphthyl)ethisocyanate,
Heat to reflux on a boiling water bath for 5 hours. After cooling, the solvent was concentrated under reduced pressure and ethyl acetate-hexane (1:
1) After dissolving in 100 ml of the mixture, extract twice with 100 ml of 5% aqueous sodium bicarbonate solution, wash the combined extract once with 50 ml of ethyl acetate-hexane (1:1) mixture, acidify with 6N hydrochloric acid, The resulting pale yellow oil is extracted twice with 100 ml of ethyl acetate. The extract was washed twice with 100 ml of water, dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure, and recrystallized from a mixture of ethyl acetate and n-hexane.
{(R)-1-(α-naphthyl)ethylcarbamoyl}
2.9 g of -(S)-mandelic acid was obtained as white crystals. Flash decomposition point: 178-180℃ Optical rotation: [α] ²⁰ _D = 67.5゜ (c = 0.90%, methanol) Elemental analysis (as C ₂₁ H ₁₈ NO ₄ ) Carbon (%) Hydrogen (%) Nitrogen (%) Calculated value 72.19 5.48 4.01 Actual value 72.31 5.67 3.89 Next, take 2.0 g of this compound and dissolve it in 20 ml of dehydrated tetrahydrofuran. To this, 2.0 g of N-methoxycarbonyl-2-ethoxy-1,2-dihydroquinoline was added and dissolved, and the mixture was stirred at room temperature for 15 minutes. To this, 2.5 g of APS obtained in Example 1 is added and suspended, and after sufficient deaeration under reduced pressure, the mixture is gently stirred at room temperature overnight. The reaction mixture was diluted four times with 30 ml of tetrahydrofuran, then twice with 30 ml of methanol.
Wash twice with 30 ml of ethyl ether and dry to obtain the desired filler (hereinafter NEC-MNC −Si) was obtained.
The elemental analysis values of this item are N: 1.68%, C: 13.6%
This means that for 1 g of this material, O-{(R)-
This shows that about 0.47 mmol of (α-naphthyl)ethylcarbamoyl}-(S)-mandelic acid was grafted. The filler thus obtained had an inner diameter of 4 mm,
The slurry was packed into a stainless steel column with a length of 25 cm, and N-(3,5-dinitrobenzoyl)-(±)-1-phenyl-2-(4-tolyl)ethylamine was analyzed under the following conditions. A chromatogram was obtained. Temperature: Room temperature Mobile phase: Hexane/dichloromethane/ethanol (15:4:1) Flow rate: 1.0ml/min Detector: Ultraviolet absorption meter (wavelength 254nm) In Figure 3, peak number (1) is N-(3, 5-dinitrobenzoyl)-(+)-1-phenyl-2-(4
-tolyl)ethylamine, (2) is N-(3,5-dinitrobenzoyl)-(-)-1-phenyl-2-
These are the peaks of (4-tolyl)ethylamine.
The time required for peak (2) to elute is approximately 25
, the separation factor is 1.66, and the area ratio of peaks (1) and (2) is
It was 50:50. Example 4 NEC-PHG-Si obtained in Example 1 and NEC-MNC-Si obtained in Example 3 were slurried into stainless steel columns with an inner diameter of 4 mm and a length of 25 cm. PTC-PHC-Si column obtained in Example 2, and further Example 1
N-{(R)-1-phenylethylcarbamoyl}-D-phenylglycine prepared in a similar manner to O-{(R)-1-(α-naphthyl) obtained in Example 3. ethylcarbamoyl}-(S)-mandelic acid in the same manner as in Example 2.
mm, packed in a stainless steel column with a length of 25 cm.
Using each column (hereinafter abbreviated as PEC-PHG-Si and NEC-MNI-Si) prepared by grafting APS within the column, enantiomeric mixtures of the following compounds were separated under the following conditions. Then, the separation coefficient was determined. Temperature: Room temperature Flow rate: 1 ml/min Detector: Ultraviolet absorption meter (wavelength 254 nm) The results are shown in Tables 1 to 4.

[Brief explanation of drawings]

Figures 1, 2, and 3 are chromatograms obtained in Example 1, Example 2, and Example 3, respectively, where the vertical axis represents intensity and the horizontal axis represents retention time.

【table】

【table】

【table】

【table】

【table】

【table】

【table】

Claims (1)

[Scope of Claims] 1. A chromatographic packing material in which a compound formed by bonding an optically active isocyanate derivative and an aminoalkylsilane is grafted onto an inorganic carrier having hydroxyl groups on its surface. 2 A compound grafted onto an inorganic carrier having a dihydroxyl group on its surface has the general formula [] [In the formula, R ₁ , R ₂ and R ₃ are the same or different and represent an alkyl group, an alkoxyl group, a hydroxyl group or a halogen atom, and R ₁ , R ₂ and
At least one of R ₃ is an alkoxyl group or a halogen atom. X represents [Formula] [Formula] [Formula] or [Formula]. Here, R ₅ represents an aryl group, an alkyl group or an aralkyl group. R ₄ represents an alkyl group or an aralkyl group, and Ar represents a phenyl group or a naphthyl group which may have a substituent. However, when Ar represents a naphthyl group, R ₄ represents a methyl group. n represents an integer from 2 to 4, and * represents an asymmetric carbon. ] The chromatographic packing material according to claim 1, which is an organosilane represented by the following. 3. The chromatographic packing material according to claim 1 or 2, wherein the inorganic carrier having hydroxyl groups on its surface is silica gel. 4 In the above general formula [], Ar is a phenyl group, R ₄ is a methyl group or 4-methylbenzyl group, or Ar is a naphthyl group and R ₄ is a methyl group, Claim 2 Chromatographic packing material according to item 1 or 3. 5. Claims 2, 3, or 4, wherein the aminoalkylsilane residue in the general formula [] is an ω-aminopropyltriethoxysilane residue or an ω-aminopropyltrichlorosilane residue. chromatographic packing material. 6 Claim 2 in _which X in _the above general formula [] is -NHCO-CH( _C6H5 )-NH- or _-NH3.OCO -CH( _C6H5 )-NH- Chromatographic packing material according to item 1, 3, 4 or 5. 7 Claim 2 in _which X in _the above general formula [] is -NHCO-CH( _C6H5 )-O- or _-NH3.OCO -CH( _C6H5 )-O- Chromatographic packing material according to item 1, 3, 4 or 5. 8 -CONH- group bonded to an asymmetric carbon using a chromatographic packing material in which a compound formed by bonding an optically active isocyanate derivative and an aminoalkylsilane to an inorganic carrier having a hydroxyl group on its surface is grafted. , -OH group, -OCO- group, -
A liquid chromatography analysis method characterized by separating and analyzing a mixture of enantiomers of a compound having an OCONH- group or a [Formula] group. 9 A compound grafted onto an inorganic carrier having a hydroxyl group on its surface has the general formula [] [In the formula, R ₁ , R ₂ and R ₃ are the same or different and represent an alkyl group, an alkoxyl group, a hydroxyl group or a halogen atom, and R ₁ , R ₂ and
At least one of R ₃ is an alkoxyl group or a halogen atom. X represents [Formula] [Formula] [Formula] or [Formula]. Here, R ₅ represents an aryl group, an alkyl group or an aralkyl group. R ₄ represents an alkyl group or an aralkyl group, and Ar represents a phenyl group or a naphthyl group which may have a substituent. However, when Ar represents a naphthyl group, R ₄ represents a methyl group. n represents an integer from 2 to 4, and * represents an asymmetric carbon. ] The analysis method according to claim 8, which uses a chromatographic packing material which is an organosilane represented by the following. 10. The analytical method according to claim 8 or 9, wherein the inorganic carrier having hydroxyl groups on its surface is a chromatographic packing material which is silica gel. 11 Using a chromatographic packing material in which Ar is a phenyl group and R ₄ is a methyl group or 4-methylbenzyl group, or Ar is a naphthyl group and R ₄ is a methyl group in the above general formula [] An analytical method according to claim 9 or 10. 12 Claims 9 and 10 using a chromatographic packing material whose aminoalkylsilane residue is an ω-aminopropyltriethoxysilane residue or an ω-aminopropyltrichlorosilane residue in the above general formula [] or the analytical method described in Section 11. 13 A chromatographic _packing material in which X in the above general formula [] is -NHCO-CH( _{C6H5 )} -NH- or _-NH3.OCO -O-CH( _C6H5 )-NH- The analysis method according to claim 9, 10, 11 or 12, using the method. 14 Chromatographic packing material in _which X in the above general formula _[ ] is -NHCO-CH( _C6H5 )-O- or _-NH3.OCO -O-CH( _C6H5 )-O- The analysis method according to claim 9, 10, 11 or 12, using the method.