JPS5963565A - Chromatograph filler and analyzing method of mixture of enantiomer using chromatograph filler - Google Patents

Abstract

PURPOSE:To obtain a method for analyzing a mixture of the enantiomer of a compd. having a specific group bound to asymmetric carbon atom by using a filler prepd. by grafting an N-carbamoyl amino acid deriv. or O-carbamoyl oxy acid deriv. to an inorg. carrier having a hydroxyl group on the surface in a liquid chromatography. CONSTITUTION:Various grafting methods are usable in preparing a chromatograph filler. For example, the filler is obtd. by grafting the organo silane, which is obtd. by bringing the amino alkyl silane expressed by the general formula (II) into reaction with the N-carbamoyl amino acid or O-carbamoyl oxy acid expressed by the general formula (III) and is expressed by the general formula ( I ) to an inorg. carrier of silica gel or the like. The filter having the residual group of an optically active N-carbamoyl alpha-amino acid or the residual group of an O- carbamoyl alpha-oxy acid is filled in a column and is used as a stationary phase of a liquid chromatography. The sepn. and analysis of a mixture of the enantiomer of the compd. having the N=-CO-group, -CONH-group, -OCO-group, -OCONH- group, -NHCONH-group or =N-CO-NH-group bound to asymmetric carbon atom are accomplished with good separability in a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is characterized by grafting a novel optically active organosilane.
The present invention relates to this chromatographic packing material and a method using the same to separate and analyze an enantiomeric mixture of a compound having one N 00- group bonded to an asymmetric carbon by liquid chromatography.

As a packing material 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, a packing material grafted with an optically active proline by Davankov et al. Ligand exchange method using (
A charge transfer complex method using a filler grafted with an optically active compound lacking E electrons by Jil-Av et al., an N-acylated amino acid ester or N-
Acylated dipeptide ester separation or Pirkl
3,5- using a filler grafted with optically active 1-(9-anthryl)trifluoroethanol by et al.
Separation of dinitrobenzoylated amino acids, amines, oxyacids, sulfoxides, etc., and separation of aromatic alcohols using fillers grafted with 8,5-dinitrobenzoylated optically active phenylglyne, etc., have been reported. However, with these methods, the compounds that can be separated are limited to a narrow range, the degree of separation is small, and it is difficult to produce fillers that have reproducible performance. However, it is difficult to obtain fillers with such properties, and it is difficult to say that any of them are practical fillers.

The present inventors have conducted intensive studies on the development of 14 grafted fillers that have a wide range of applicable compounds that can be analyzed under such circumstances, are relatively easy to manufacture, are chemically stable, and are practical. As a result of continued efforts, we have found that N is formed by bonding an optically active amino acid derivative or oxyacid derivative and aminoalkylsilane, which are carbamoylated using optically inactive isocyanate, to an inorganic carrier having a hydroxyl group on its surface.
- A chromatographic packing material to which a carbamoyl amino acid derivative or a 〇-carbamoyloxy alcohol derivative is grafted or a one N-CO- group bonded to an asymmetric carbon, -CONE
I- group, -0CO- group, 000 N 11- group, N
Not only does it show an excellent effect in separating enantiomeric mixtures of compounds having a 1-10ONH- group or -N-CONTI- group, but it can also be easily produced by ordinary chemical reactions and is also chemically stable. We have discovered that these are extremely useful fillers, and have led to the present invention.

That is, the present invention provides an N- structure in which an optically active amino acid derivative or oxyacid derivative, which is carbamoylated using an optically inactive isocyanate, and an aminoalkylsilane are bonded to an inorganic carrier having a hydroxyl group on its surface. A chromatographic packing material to which a carbamoyl amino acid derivative or an 0-carbamoyloxy acid derivative is grafted, and a -N-00- group, -0ONH- group, bonded to an asymmetric carbon by using it as a stationary phase of body chromatography, oco- group, -000N) I- group, -N 11
A method is provided for separating and analyzing enantiomeric mixtures of compounds having UON n-groups -N-0ONH-.

The N-carbamoyl amino acid derivative or O-carbamoyloxy acid derivative grafted in the present invention includes, for example, the general formula [Engine] [1], where R1, R2 and R3 are the same or different, an alkyl group, an alkoxyl group, It represents a hydroxyl group or a halogen atom, at least one of which represents an alkoxyl group, an aralkyl group, or an aryl group.

Y, 1. It represents a t-NH- group or an oxygen atom, and R5 represents an alkyl group, an aralkyl group or an aryl group.

n represents an integer from 2 to 4, and the umbrella represents an asymmetric carbon. ] Organosilanes shown in the following can be mentioned.

More specifically, in the 1-carbamoyl amino acid derivative or the 0-carbamoyloxy acid derivative represented by the general formula CI), the optically inactive isocyanate component is preferably a lower alkyl isocyanate or an aryl isocyanate, such as isopropylisocyanate, Examples include a-naphthyl isocyanate or 8°5-dinitrophenyl isocyanate.

Optically active amino acids or oxy-acid components containing asymmetric carbon atoms include a-alkyl amino acids, α-aryl amino acids, or a-aryloxy acids, such as L-valine, Either D-phenylglycine, (S)-mandelic acid or (half) can be selected depending on the purpose and use.

The aminoalkylsilane component is preferably ω-aminoalkylalkoxysilane or ω-aminoalkylhalogenosilane, such as ω-aminopropyltriethoxysilane.

Examples include ω-aminopropyltrichlorosilane.

In the present invention, 1! having a hydroxyl group on its surface!
The E 4'A 1114 body is preferably a silica-containing carrier such as silica gel, and the shape of the tu body is spherical,
In order to obtain a column for graphing with a fractured shape I (any shape C is acceptable, at most), it is preferable to use a 11" F31 column with a diameter as uniform as possible.

] ``Novel invention of the invention'' In order to produce a chromatographic filler using FJlj, the lees granod method can be used, for example, the unab J method can be used.

(/J t < times, hydroxyl shines, ij16 An inorganic carrier is reacted with an aminoalkylsilane. A method in which an optically active amino acid is carbamoylated using a secondary oxyacid and subjected to dehydration condensation or ionic bonding.

Specifically, an inorganic carrier having a hydroxyl group on its surface is coated with the general formula LID 1L2-8i-(OL+2)n-NH2[11)lOni3 [wherein 几1. R2, 几3 and n have the same meanings as above. ] An aminoalkylsilane represented by the formula is reacted by a known method to introduce an aminoalkylsilyl residue onto the surface of an inorganic carrier, and then the aminoalkylsilyl residue represented by the general formula same meaning as η
do. ] N-carbamoyl amino acids having an asymmetric carbon represented by are 〇-carbamoyloxy acids, such as N-alkylcarbamoyl-L-valine, N-arylcarbamoyl-D-phenylglycine, 0-arylcarbamoyl-
(8) - The desired filler can be obtained by reacting with mandelic acid or the like and performing dehydration condensation or ionic bonding.

In addition, N-carbamoyl amino acid or 0-carbamoyloxy acid having one asymmetric carbon as shown in the general formula [river] can be synthesized by a commonly used method. An isocyanate, such as isocyanate obtained by
- Reaction with sodium salts such as valine, D-phenylglycine, etc. in aqueous solution, or alternatively with oxyacids such as (8
)-obtained by reacting the triethylamine salt of mandelic acid in dehydrated tetrahydrofuran.

■ An organosilane obtained by reacting an aminoalkylsilane with an optically active amino acid or oxyacid that has been carbamoylated using an optically inactive isocyanate.
A method of grafting onto an inorganic carrier having hydroxyl groups on its surface.

Specifically, an organoamine represented by the general formula [■] obtained by reacting an aminoalkylsilane represented by the general formula [13] with an N-carbamoyl amino acid or 0-carbamoyloxy acid represented by the general formula [Kawa] above. The desired filler can be obtained by grafting silane onto an inorganic carrier such as silica gel.The optically active N-carbamoylated α-amino acid residues obtained by the present invention are O-carbamoylated α-oxyacids. A packing material having residues is packed into a chromatographic column according to a conventional method, and is used as a stationary phase in liquid [trilo7tography]. In the case of the grafting method mentioned above, an inorganic carrier such as silica gel is reacted with an aminoalkylsilane represented by the general formula [I[), and an aminoalkylsilyl residue 5e! is formed on the surface of the inorganic carrier. A chromatographic column is filled with the packing material prepared by the above-mentioned general formula [1] in advance in a chromatographic column according to a conventional method. A stationary phase for chromatography can also be made. In liquid chromatography using this stationary phase, appropriate elution conditions, especially the commonly used normal phase partition conditions, OCON H- group, -N
'EICON ■-g ma tko wa -N-CO-
Separation and analysis of a mixture of enantiomers of a compound having an NH- group can be performed with good resolution and in a short time.

Example 1 Silica gel (average particle size 10jtm, average pore diameter 60X, surface area 500m2/￥) was dried under reduced pressure at 180°C for 4 hours. After that, 8-aminopropyltriethoxysilane 207 was dissolved in 200mE dehydrated toluene. Add to the solution containing 8-aminopropyl silylated silica gel and stir at 60°C for 6 hours.
(abbreviated as 8). The elemental analysis value of this product is N:1.
20! , c:s. 4o%, which is the 1st floor of this thing.
On the other hand, the 3-aminopropyl group is about 0. 90mm
ol Corresponds to being grafted.

Separately, L-valine 4. 6 9 P in sodium hydroxide aqueous solution 4 0 r/! After adding tetrahydrofuran 5-, isopropyl isocyanate 841 was added while stirring, and stirring was continued overnight. After adding 10 ml of IN sodium hydroxide aqueous solution to the reaction solution, ethyl acetate 501 M/! The precipitated white crystals were collected by filtration, washed five times with 30πl of water, and dried under reduced pressure at 50°C to obtain 2,5y.

Melting point (decomposition): 154-155°C Optical rotation, [α] 0 - 7.4 (0 = 0.
8 1%, methanol) Elemental analysis value ←) Hydrogen (t) Nitrogen (t) Calculated value 5B. 45 8.97 18.85 Actual value
52.78 8.78 18.41 (09B1
8N203) Next, this compound 1. Take 1 9 9- and dissolve it in dimethylformamide 14- 17, the above AP82, 82, and l-hydroxybenzotriazole 1, 02 5'
After adding - and thoroughly degassing under reduced pressure, cool the mixture on ice while stirring. A solution of 1.5y of dicyclohexylcarbodiimide dissolved in 6 rhl of dimethylformamide is added to this, stirred at 0°C for 1 hour, and further stirred at room temperature overnight. - The graft obtained as described in
- The silica gel is centrifuged, washed with chloroform, methanol, acetone and ethyl ether, dried and N
IPC-VAL-Si
) was obtained. The elemental analysis value of this item is N: 2.78
%, C: 8.98Φ, which means that N-isopropylcarbamoyl-L-valine is about Q,
5 Indicates that 2 mmol was crafted.

The filler thus obtained has an inner diameter of 4 mm and a length of 25 mm.
The slurry was packed into a stainless steel column and heated with N under the following conditions.
-Atenthyl-DL-valine methyl ester was analyzed and the chromatogram shown in Figure 1 was obtained.

Temperature: Room temperature Mobile phase: Hexane/isopropanol (24:1) Flow rate: 1, Qm//Flash detector: Ultraviolet absorption meter (wavelength 225 nm) In Figure 1, peak number (1) is With the solvent chloroform'), (
21 is the peak of N-acetyl-D-valine isopropyl ester, and (3) is the peak of N-acetyl-L-valine isopropyl ester. The time required for peak (3) to elute was approximately 8 minutes, the separation factor was 1.46, and the area ratio of peaks (2) and (3) was 50:50.

Example 2 Pour 581 parts of trichloromethyl chloroformate into 200 parts of toluene, shake gently, and prepare granular activated carbon 1 part.
After adding and leaving overnight to slowly generate phosgene, dissolve 3,5-dinitroaniline 10FI in 50 ml of drunken ethyl f in the liquid from which the activated carbon was removed by filtration.
: Add the liquid and heat under reflux for 4 hours. , and remove excess phosgene and solvent under reduced pressure to obtain 315-dinitrophenyl isocyanate-110,5y.
I got it.

Melting point: 72°C to 74°C Elemental analysis value Carbon (5) Hydrogen ←) Nitrogen (e) Estimated value 40,21 1.45 20.09 Actual value
88,831.66 18.89 (07113N3
Separately, 2.59 F of 8((1)-mannalic acid was dissolved in 80 ml of dehydrated tetrahydrofuran, and 30 ml of triethylamine was dissolved in 80 ml of dehydrated tetrahydrofuran.
After adding 2, the above-mentioned 3,5-dinito L1 phenyl isocyanate 4.185' is added, and 1i=D I! !
j'I'll heat it up on the water bath for 5 hours. From now on, I'll reduce the heat.
Concentrate under L, add ethyl acetate-hexane (1:1) mixture 1
After melting 00r61, 5% sodium bicarbonate,
Extract 3 times with 100ml of aqueous solution, combine 1) Wash the zebra extract with 50ml of ethyl acetate-hexane (1:1) mixture,
Acidify with 6N hydrochloric acid and extract the resulting white solid substance twice 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 tetrahydrofuran-hexane (1:2) mixture to obtain 0-(
8,5-dinitrophenylcarbamoyl)-(S)-mandelic acid 2.6i was obtained as white crystals.

Melting point (min j[J'F): 195-196℃ Optical rotation: [α
] Otsu0 = 98.06 (C = 0.58%, methanol) Elemental analysis value Carbon (to hydrogen) Nitrogen ←) Calculated value
49.87 8.07 11.68 Analysis value 49.
96 8.10 11.55 (C15I(oNaO
s) Next, take this compound 1.8P and add tetrahydrofuran 5
0 ml was added and dissolved, and this was separately packed with Ar1 obtained in Example 1 as a slurry into a stainless steel column with an inner diameter of 4 WrI# and a length of 25 ffi at a flow rate of about 2 hours/win for 2 hours. Graft 0-'(8,5-dinitrophenylcarbamoyl)-(8)-mandelic acid by cycling and then condition the column with tetrahydrofuran, methanol, and chloroform. Below, this column is
) N O-M, N D -S i and f) N-acetyl-((1)-1
-(α-Naphthyl)ethylamine was analyzed and the chromatogram shown in Figure 2 was obtained.

Warm ＃：＠Purchase 1 Fuyushige ■Eye: Hekigoon/Dichloromethane/Ethanol (
, 15:4: I) Ryutan' 1. Ome/yr direction detector; Ultraviolet absorption F'i (wavelength 2541m) diagram-
2, the peak number (]) is ]N-acetyl← -1-
(/Z-naphthyl)ethylamine, (2) is each peak of N-anatyl-(+)-1-(α-naphthyl)ethylamine. The time required for peak (2) to elute is approximately 15 minutes, the separation coefficient is 1.80, and the peak area ratio of (11 and (21) is 50:50.

Example 8 A stainless steel column having an inner diameter of 4 mm and a length of 25 mm was filled with slurry of the IPO-VAT, -8i packing material obtained in Example 1, and the DNC-MND- obtained in Example 2.
8i column, and further N-isopropylcarbamoyl-D synthesized by a method similar to Example 1 or 2I.
-Phenylglycine, 0-isopropylcarbamoyl-
(n-mandelic acid, N-(8,5-dinitro7, nylcarbamoyl)-D-phenylglycine and 0-(α-
naphthylcarbamoyl)-(8)-mandelic acid, the former two according to Example 1, and the latter two according to Practical Example 2, respectively, by grafting these fillers onto the Ar8 obtained in v6 Example 1. in order IP O-J, ' ) I G 8 r
.

TPO-MND-F3i, DNO-I'RO-8i and NPC-M, abbreviated as ND8t), 3
Enantiomeric mixtures of various compounds were separated under the following conditions using each of the columns, and the separation factor ft was determined.

Temperature: Actual demand flow: l m//*t, *Detector:
The results of an ultraviolet absorption meter (wavelength: 254 nm 4 f, 1:2 2 5 nm) are shown in 117th to 14th quarters.

[Brief explanation of the drawing]

Figures 1 and 2 are chromatographs obtained in Example 1 and Example 2, respectively, where the vertical axis represents intensity and the vertical axis represents retention time. Figure-1 0510 (min)

Claims (1)

[Scope of Claims] (1) An optically active amino acid derivative or oxyacid derivative carbamoylated using an optically inactive isocyanate and an aminoalkylsilane are bonded to an inorganic carrier having a hydroxyl group on its surface. A chromatographic packing material to which an N-carbamoyl amino acid derivative or an 0-carbamoyloxy acid derivative is grafted. (2) An N-carbamoylamino derivative or a 〇-carbamoyloxy acid derivative having a hydroxyl group on its surface and grafted without a carrier has the general formula [I] [wherein R1, R2 and R3 are the same or Differently,
It represents an alkyl group, an alkoxyl group, a hydroxyl group or a halogen atom, at least one of which represents an alkyl group, an aralkyl group or an aryl group. Y represents -)1B- group or oxygen source: r, T
Lb 1. iAlA, λζ, aralkyl group represents an aryl group. 11 represents the arrangement from 2 to t, and Hachi represents an asymmetric carbon. ] 4b self with the organosilane shown! The chromatographic packing material described in item 1 for use in the traditional range. (3) For patents in which the inorganic carrier having hydroxyl crystals on its surface is silica gel, the scope of 17X, paragraph 1 or 2.
Chromatographic packing material placed in the column. (4) Claim 2 or 8, wherein in the above general formula [I], the aminoalkylsilane residue is an ω-aminopropyltriethoxysilane residue or an )-aminopropyltrichlorosilane residue. Chromatographic packing material described in . (5) In the above general formula [E], the chromatographic packing according to claim 2, 8 or 4, where 5 is an isopropyl group, a naphthyl group or an 8,5-dinitrophenyl group. agent. (6) In the above general formula [E], when R4 is an isopropyl group or a phenyl group, Y is a -Nu- group, or when IL4 is a phenyl group, Y is an oxygen atom. Chromatographic packing material according to item 2, item 8, item 4 or item 5. (7) An inorganic carrier having a hydroxyl group on its surface is bonded with an optically active amino acid derivative or an oxyacid derivative carbamoylated using an optically inactive isocyanate and an aminoalkylsilane. Using a chromatographic packing material to which one carbamoyl amino acid derivative or one 〇-carbamoyloxy acid derivative is grafted, one N-00- group, -C(
,) N pi- group, -0CO- group, -00ON) l-
A liquid chromatography analysis method characterized in that an enantiomeric mixture of a compound having a NIJOONB- group or a -N0ONIT- group is separated and analyzed. (3) An N-carbamoyl amino acid derivative or a 〇-carbamoyloxy acid derivative grafted onto an inorganic carrier having a hydroxyl group on its surface has the general formula CI] x [wherein R1, R2 and R3 are the same or different] ,
It represents an alkyl group, an alkoxyl group, a hydroxyl group or a halogen atom, at least one of which represents an alkyl group, an aralkyl group or an aryl group. N represents an -NH- group or an oxygen atom, and 5 represents an alkyl group, an aralkyl group or an aryl group. n represents an integer from 2 to 4; (9) A special method using a graph filler in which the inorganic carrier having hydroxyl groups on its surface is silica gel.
'11 Analysis method of P described in item 7 or 8 of the FJ request. Claim 8 uses a chromatographic packing material in which the aminoalkylsilane residue is an ω-a-main nobrobil l-ethoxysilane residue or an ω-aminopropyltrichlorosilane residue in the general formula CII described in Section 2. Convergence analysis method in section or section 9. aυ Claim 8 uses a chromatographic packing material in which R5 is an isoprobyl group, a naphthyl group, or an 8,5-dinitrophenyl group in the above general formula [I].
The analytical method according to Section 9, Section 9 or Section 1O. In the above general formula 〇), when Y is Iwa propyl group or phenyl group, Y is = Ni-Ni (-, or when R4 is a phenyl group, Y is an oxygen atom. ) *Claim No. 8 using agent
9, 9, 51410, or 911.