JP2000515627A - Chiral non-particulate solvent - Google Patents

Abstract

  (57) [Summary] The present invention relates to non-particulate solvents modified with chiral groups, for example on the basis of monolithic (monolithic) base supports or derived polyamide membranes. These solvents can be used at high flow rates while separating the enantiomers.

Description

The present invention relates to chiral non-particulate solvents and their use for resolving enantiomers. Many chiral separating materials for resolving enantiomers are known from the prior art. These are all particulate separation materials. Known chiral separation materials consist of the chiral compound itself (eg, cellulose triacetate), or the chiral separation effector is applied to the support or chemically attached to the support. It is also possible to add to the eluate a chiral separation effector which interacts with the stationary phase (dynamic application). Many chiral separating effectors are known; the most important group of known chiral separating effectors are: a) amino acids and derivatives thereof, such as L-phenylalanine or D-phenylalanine, esters or amides or oligos of amino acids or acylated amino acids. peptide; b) a natural or synthetic polymers having an asymmetric or asymmetry in the main chain; among the protein (e.g., an acidic alpha _{1-glycoprotein,} bovine serum albumin, cellulase;. J .Chrom 264, 63~68 page ( 1983), J. Chrom. 269 , pp. 71-80 (1983), WO 91/12221), cellulose and cellulose derivatives and other polysaccharides and derivatives thereof (eg, cellulose tribenzoate, cellulose tribenzyl ether, trisphenyl) Carbamate cellulose, tris-3-chlorobenzo EP 0 147 804, EP 0 155 Cellulose perfume, tris (3,5-dimethylphenylcarbamic acid) amylose, tris (3,5-dimethylbenzoic acid) cellulose, tris (3,5-dimethylphenylcarbamic acid) cellulose; 637, EP 0 718 625); c) cyclodextrins and cyclodextrin derivatives (eg J. High Resources. Chrom. & Chromat. Comm. 3 , pp. 147-148 (1984); EP 0407412; EP 0 445 604). D) a polymer having an asymmetric center in the side chain (eg EP 0 249 078; EP 0 282 770; EP 0 448 823) e) a polymer which polymerizes around a chiral structure (eg an “imprint” polymer (eg J . .Chromt 707, 199~203 page (1995);.. J.Chromat 694 , 3~ 13 pages (1995)) chiral separation material of the prior art good be used in column chromatography particulate solvent The column packing material used for this requires a considerable operating pressure to achieve an acceptable flow rate, and the physical stability of the particulate solvent bed is not very good. The objective is to provide a method for resolving enantiomers with more stable solvent packings that allows higher flow rates, and also to provide more stable chiral modified solvents. Therefore, it was found that a non-particulate solvent can be used and a high elution rate can be provided. The above objective is accomplished by providing a non-particulate solvent comprising a chiral separation effector. Non-particulate solvents are, in particular, solvents based on monolithic (integral structure) carriers and solvents based on porous membranes. The present invention uses a porous molded body as a substrate, in particular, a porous molded ceramic body having macropores and mesopores communicating with each other in a macropore wall, and the diameter of the macropores is A chiral non-particulate solvent having a median larger than 0.1 μm and having a mesopore diameter having a median of 2 to 100 nm as a base or a porous shaped polyamide as a base. I will provide a. The present invention provides for the use of the chiral non-particulate solvents of the present invention for chromatographic resolution of enantiomers. The present invention also provides a method for chromatographic resolution of enantiomers using the chiral non-particulate solvents of the present invention. Figures 1 to 3 show elution diagrams for various applications; experimental details are given in Examples AC. Surprisingly, the H / U curve was found to be flat when using non-particulate, especially monolithic, solvents. In addition, it has been found that the length of the separation step is short for non-particulate solvents with a low differential pressure. As a result, preparative chromatography separation methods using non-particulate solvents can be significantly optimized compared to using particulate solvents; the economics of these methods are significantly improved. The term "non-particulate solvent" as used for the present invention indicates a difference from the above-mentioned known particulate solvents in which the solvent bed is made up of individual, discrete particles. Both monolithic solvents and membranes modified using separation effectors are included in the term non-particulate solvent. Monolithic solvents are known in principle from the literature; among others are the porous shaped ceramic bodies disclosed in WO 94/19 687 and WO 95/03 256. F. Svec and JMFrechet (1992) Anal. Chem. 64 , pp. 820-822 and S. Hjerten et al. (1989) JChromatogr. 473 , pages 273-275, the term monolith solvent also broadly includes shaped bodies made of polymers. Particularly preferred as base support is a porous shaped ceramic body disclosed in WO 95/03 256, having macropores and mesopores interconnected in the macropore wall, wherein the macropores Has a median of greater than 0.1 μm, and the diameter of the mesopores has a median of 2 to 100 nm. These base supports can be modified by methods known per se and provide suitable solvents for the resolution of the enantiomers according to the invention. Suitable denaturing methods are known to those skilled in the art and are described in handbooks such as Unger KK. (Editor) Porus Silica, Elsevier Scientific Publishing Company (1979) or Unger KK Packings and Stationry Phases in Chromatogarapic Techniques, Marcel Dekker (1990). Adsorption membranes modified using separation effectors are disclosed in WO 91/03 506, DE 196 27 302.1 and WO 96/22 316, and also in PCT / EP97 / 02768. It is particularly preferred to use the modified polyamide membranes disclosed in WO 96/22 316, DE 196 27 302.1 and PCT / EP97 / 02768. Methods for making these films are given in these publications. These membranes can also be denatured using chiral separation effectors. Polyamides particularly suitable as base polymers for membranes are known to those skilled in the art, Such a film retains its shape under the reaction conditions used at DE 195 01 726.9 and DE 19 624 813.2 (reaction temperatures below 80 ° C.), whereas the other method of modifying polyamide is melt Alternatively, the reaction of such a membrane is preferred because it is performed in a solution. Apart from the azalactone groups and the epoxy groups, the modified polyamides disclosed in the above-mentioned application can contain in particular carboxyl groups, amino groups or hydroxy groups. These groups can attach the chiral separation effector to the polyamide in a manner known per se. For example, the optically active monomers disclosed in EP 0 249 078, EP 0 282 770 and EP 0 448 823 can be polymerized in a manner known per se to the polymerizable modified polyamide disclosed in the above-mentioned application. Adsorption or chemical bonding of the chiral separation effector can be achieved, for example, by first introducing a functional group such as an amino group, a carboxyl group, a carbonyl group, a hydroxy group or an epoxy group or an azalactone group into the base support. . Next, for example, a chiral separation effector containing an amino group (eg, optically active amines, amino acids, amino acid esters and amides, oligopeptides, proteins, amino sugars disclosed in EP 0 249 078) is converted to a water such as carbodiimide. It can be attached to a base support modified with a carboxyl group using a removing agent. These chiral separation effectors can also be attached using a base support modified with azalactone or epoxy groups. Chiral separation effectors containing carboxyl groups (eg, amino acids or N-acylated amino acids, oligopeptides, proteins, optically active carboxylic acids) have been modified with amino or hydroxy groups using a water scavenger such as carbodiimide to retain the base. Can be bound to the body. Chiral separation effectors containing hydroxyl groups (eg, polysaccharides and their derivatives, cyclodextrins and their derivatives) can be attached to carboxyl-modified base supports using water-removing agents such as carbodiimides. In addition, the chiral separation effector can be attached to a suitably modified base support using a bifunctional reagent (eg, diisocyanate). Chiral solvents containing cyclodextrin chemically attached as chiral separation effectors can be obtained from the preferred base supports by the method disclosed in EP 0 445 604. These methods and their customary variants are known to those skilled in the art and are described in handbooks and review articles. For the purposes of the present invention, a "chiral modified non-particulate solvent" or "modified non-particulate shaped body" is a non-particulate base support that includes a chiral separation effector. Here, the chiral separation effector can be chemically bound or adsorbed or present as a dynamic application. It has been found that use of these preferred solvents allows the flow rate to be varied over a wide range without adversely affecting the separation properties. Utilizing this property, the flow rate can be tailored to the elution profile without compromising the separation performance. This significantly reduces the time required for the separation, and provides significant advantages, especially for preliminary separations or routine analysis. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the above to its fullest extent. Accordingly, the preferred embodiments should be considered as illustrative only and not in any way limiting of the disclosure. The entire disclosures of all the above and below applications, patents and publications and corresponding applications, as well as DE 196 29 206.9 filed on July 19, 1996 and DE 197 26 152.3 filed on June 20, 1997, are by way of reference. Incorporated in the present application. Examples The following examples illustrate the invention but do not limit it in any way. In the following, room temperature means a temperature of 15 to 30 ° C. Example 1 Preparation of a Chiral Solvent Chemically Linked with β-Cyclodextrin Prepared as described in EP 0 710 219 using a method similar to that described in Example 2 of EP 0 445 604 Containing the chemically bonded β-cyclodextrin by reacting the porous shaped body thus obtained with a reaction solution comprising β-cyclodextrin, p-nitrophenyl chloroformate and 3- (2-aminoethyl) aminopropyltrimethoxysilane. To obtain a chiral solvent. For this purpose, the reaction solution is pumped through the shaped body. As a result, a modified monolith molded article in which β-cyclodextrin is chemically bonded is obtained. Example 2 Preparation of a C ₁₈ -RP Solvent A porous shaped body prepared as described in EP 0 710 219 is chemically modified with methyloctadecyldichlorosilane; Is pumped away. The result is a modified monolith sculpture, modified with a C ₁₈ -alkyl group and suitable as stationary phase for enantiomer resolution using dynamically applied solvents (see Examples B and C). Example 3 Reaction of Polyamide Hollow Fiber Membrane with Vinyl Dimethylazalactone 2.56 g of 1,8-diazabicyclo [5.4.0] unde-7-cene (DBU) and 32 ml of vinyldimethylazalactone were converted to 160 ml of dimethyl Dissolve in formamide. Polyamide hollow fiber bundle (polyamide 6, number of yarns 64: diameter of each fiber: inner diameter 0.2 mm, outer diameter 0.5 mm; average pore opening: 0.5 μm) is 300 to 10 mm The solution is then pumped through the hollow fiber bundle at room temperature and at a flow rate of 2 ml / min for 24 hours. Next, the denatured hollow fiber bundle is washed with dimethylformamide, acetone, ethyl acetate and acetone, and dried at 50 ° C. in a vacuum dryer. Example 4 Production of γ-globulin on Polyvinyl Hollow Fiber Membrane Activated with Vinyldimethylazalactone 1 g of γ-globulin was dissolved in 100 ml of Tris buffer (50 mM, pH 7.4) and described in Example 1. Is circulated in the hollow fiber bundle modified as described in Example 3 using a pump at room temperature using a pump (flow rate: 5 ml / min). During this procedure, the protein concentration in the solution and the degree of its decrease were continuously measured by UV spectrophotometry; after 2 hours, the protein concentration in the solution circulated by the pump was constant. After washing the hollow fiber bundle with Tris buffer (50 mM, pH 7.4) and 0.1 M acetic acid, the protein covalently bound in the hollow fiber was measured: 66.5 mg γ-globulin added chirality to the main chain. It is a polymer that has A hollow fiber module with a chirally modified polyamide membrane is obtained. Similarly, for example, bovine serum albumin can bind to a polyamide membrane activated by azalactone or epoxy groups. As a result, a chiral solvent suitable for resolving the enantiomer similar to the solvent described in J. Chrom. 264 , pp. 63-68 (1983) is obtained. Use Example A: Resolution of racemic macalin over a chiral solvent containing chemically bound β-cyclodextrin The modified monolithic shaped body (83 × 7.2 mm) prepared as described in Example 1 was used as the solvent. And the racemic macalin is resolved under the following conditions. Sample: cromakalin (0.2 mg / ml in ethanol) Injection volume: 5 μl Eluent: methanol / water (20/80; volume ratio) Temperature: room temperature Flow rate: 1.0 ml / min Detection: 254 nm The elution diagram is shown in FIG. Use Example B : Resolution of racemic ltalidone using dynamic application of β-cyclodextrin The modified monolith shaped body (RP-18; 83 × 72 mm) prepared as described in Example 2 was used as a solvent, Chlorthalidone is split under the following conditions. Sample: chlorthalidone (0.44 mg / ml) Injection volume: 5 μl Eluent: 25 mM sodium phosphate aqueous solution (pH 2) containing methanol / 10 mM β-cyclodextrin (20/80, volume ratio) Temperature: room temperature Flow rate 1.0 ml / min Detection: 254 nm The elution diagram is shown in FIG. Use Example C : Resolution of racemic prominal using dynamic application of β-cyclodextrin The modified monolithic shaped body (RP-18; 83 × 7.2 mm) prepared as described in Example 2 was used as a solvent, The semiprominal is split under the following conditions. Sample: Prominal (0.55 mg / ml) Injection volume: 5 μl Eluent: 25 mM sodium phosphate aqueous solution (pH 2) containing methanol / 10 mM β-cyclodextrin (20/80, volume ratio) Temperature: room temperature Flow rate 1.0 ml / min Detection: 254 nm The elution diagram is shown in FIG.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) G01N 30/48 G01N 30/48 D (81) Designated country EP (AT, BE, CH, DE, DK, ES) , FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), JP, US (72) Inventor Wierland, Gerhard DE-64625 Bensheim im Bangert 19 (72) Inventor Cabrera, Karin Germany Day 63303 Drei Eich Mühlweg 14 (72) Inventor Zagnii, Christina Germany Day 64331 Wei Tästadt Kastaneenweg 33 (72) Inventor Dix, Edith Germany Day 64297 Darmstadt Eberstadt Kirch Strasse 16

Claims (1)

[Claims]   1. A chiral non-particulate solvent based on a porous shaped body.   2. Pores with interconnecting macropores and mesopores in the walls of the macropores A shaped ceramic body having a macropore diameter of more than 0.1 μm. A porous molding cell with a median and a median pore diameter of 2 to 100 nm 2. The chiral solvent according to claim 1, wherein the chiral solvent comprises a lamic body.   3. The chiral solvent according to claim 1, wherein the base is a porous shaped polyamide.   4. The method according to claim 1, wherein the enantiomer is chromatographed. Use of chiral non-particulate solvents.   5. The use of the chiral non-particulate solvent according to any one of claims 1 to 3 is characterized. A method for chromatographic division of a mirror image.