HK1050528B - Method for separating analogous organic compounds - Google Patents

Description

Process for separating analogous organic compounds

Technical Field

The present invention relates to a method for separating similar compounds, more particularly a method for separating a lactone-containing high-molecular weight compound having an alkyl group as a side chain from a lactone-containing high-molecular weight compound having an alkenyl group as a side chain by using a sulfonic acid group-containing strong cation exchange resin pretreated with silver ions.

Background

It is generally known to use silver ions to separate cis-trans isomers of unsaturated fatty acids having the same number of carbon atoms (j. chromatography, 149(1978) 417-). However, a method of efficiently separating compounds slightly different in part of the molecular structure, for example, a compound having an alkyl group as a side chain from a compound having an alkenyl group as a side chain has not been found. This is because such compounds have the same or almost the same number of carbon atoms and physical properties similar to each other, such as solubility in a solvent and affinity for a solvent.

Summary of The Invention

The present inventors have conducted extensive studies on a method for efficiently separating compounds having physical properties similar to each other without changing the compounds themselves. They have unexpectedly found a method for separating compounds having similar physical properties to each other by using a sulfonic acid group-containing strong cation exchange resin pretreated with silver ions, i.e., separation of a lactone-containing high-molecular weight compound having an alkyl group as a side chain from a lactone-containing high-molecular weight compound having an alkenyl group as a side chain, the lactone-containing high-molecular weight compounds having a common basic chemical structure.

As suitable examples of the strong cation exchange resin containing sulfonic acid groups, synthetic substrates (bases) or silica gel substrates such as gel type resins and porous resins that can be used with polar solvents but not with non-polar solvents; and highly porous resins with which both polar and non-polar solvents can be used. These resins may be selected according to the polarity of the eluent used.

The sulfonic acid group-containing strong cation exchange resin may be a benzenesulfonic acid group-containing strong cation exchange resin having a benzenesulfonic acid group at the terminal. For example, the resin may be a copolymer matrix of styrene monomer and Divinylbenzene (DVB) or a silica gel matrix.

As examples of strong cation exchange resins containing benzenesulfonic acid groups, i.e. sulfonated copolymers of styrene monomer and DVB, mention may be made of Diaion^*(SK series, RCP series, HPK series, PK series such as PK206) (trade Mark, manufactured by Mitsubishi chemical Co., Ltd., Japan), Amberlite^*(IR120B, IR200) and Duolite^*(C20, C26) (trade Mark, manufactured by Rohm)&Haas corporation), Dowex^*(50W-X8, MSC-1) (trade Mark, manufactured by the Dow chemical Co., Ltd.), Ionac^*(C-240) (trade Mark, manufactured by Sybron chemical Co., Ltd.) and Lewatit^*(S-100, SP series) (trade name, manufactured by Bayer Co.).

As examples of strong cation exchange resins containing benzenesulfonic acid groups having silica gel as the matrix, mention may be made of strong cation exchange resins coated with a benzenesulfonic acid group-containing siloxane polymer, such as Capcell Pak^*(SCX series) (trademark, manufactured by Shiseido Co., Ltd., Japan), the substrate of which is coated with a film of a silicone polymer, and then a sulfonic acid group is introduced at the terminal thereof.

Among these strong cation exchange resins containing benzenesulfonic acid groups, Diaion is particularly preferred^*(RCP series and PK series) and Capcell Pak^*(SCX series).

Silver ions useful for pretreating the strong cation exchange resin containing sulfonic acid groups may preferably be provided from various silver salts that can produce silver ions in water, such as silver nitrate, silver perchlorate, and the like.

The strong cation exchange resin containing sulfonic acid groups can be pretreated with silver ions by passing an aqueous solution of a silver salt if the resin is in the H form or by converting it to the H form if it is in the Na form, washing with water, adjusting to a pH of 3-4, and passing an aqueous solution of a silver salt. This pretreatment with silver ions is preferably carried out by charging silver salt to 1mol/L-R or more.

The purification method of the present invention can be carried out by the following steps.

(i) The mixture, i.e., the raw material containing "the lactone-containing high-molecular weight compound having an alkyl group as a side chain" and "the lactone-containing high-molecular weight compound having an alkenyl group as a side chain" to be separated may be dissolved in a suitable solvent such as acetone or the like, and fed to column chromatography packed with a strong cation exchange resin containing a sulfonic acid group pretreated with silver ions.

(ii) And, elution may be carried out with an appropriate eluent such as acetone, a mixture of ethyl acetate and methanol, and the like.

The separation process using the strong cation exchange resin containing sulfonic acid groups pretreated with silver ions can be carried out by a fixed bed system or a continuous bed system. From the viewpoint of the operation method, the fixed bed system includes a single bed system, a multi-bed system, a double bed system, a mixed bed system, an ion exchange filtration system, a circulation system and the like. As for the regeneration method, the fixed bed system includes a downflow regeneration system, an upflow regeneration system, a counter current upflow (counter current upflow) regeneration system, a counter current regeneration system, a towerless (ex-column) regeneration system, and the like. In addition, the continuous bed system includes a fluidized bed system (counter-current contact system, multistage batch system), a moving bed system (ascending type (single column type, multiple column type), descending type (single column type, multiple column type), a simulated moving bed system, an endless belt system (liquid-liquid extraction system)), and the like, among which the simulated moving bed system is effective and suitable for mass production.

In the hairThe lactone-containing high-molecular weight compound to which the separation method of the invention is applied means a compound having at least one lactone ring in the molecule and having a molecular weight of about 400 or more. They may be monocyclic, bicyclic, tricyclic, etc. More preferably they are composed of 12 or more atoms. Such monocyclic compounds include erythromycin, leucomycin, vinamycin, and the like. Such tricyclic compounds include compounds having a lactone ring, such as the tricyclic compounds described in EP 0184162; tricyclic compounds containing heteroatoms as described in EP0427680, EP0532088 or WO 93/04680. Most preferred is 1, 14-dihydroxy-12- [2- (4-hydroxy-3-methoxycyclohexyl) -1-methylethenyl]-23, 25-dimethoxy-13, 19, 21, 27-tetramethyl-11, 28-dioxa-4-azatricyclo [22.3.1.0^4.9]Dioctadecyl-18-ene-2, 3, 10, 16-tetraone. The most preferred position substituted by alkyl or alkenyl as the side chain is its 17-position, and tacrolimus when the 17-position is substituted by allyl, and ascomycin when substituted by ethyl. Further, rapamysin and the like are suitable examples.

As the alkyl side chain of the lactone-containing high-molecular weight compound, there may be mentioned a straight-chain or branched alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl and the like, among which an alkyl group having 1 to 4 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl and isobutyl is preferable.

As the alkenyl side chain of the lactone-containing high-molecular weight compound having a common basic chemical structure with the above-mentioned compounds, there may be mentioned a straight-chain or branched alkenyl group having 2 to 6 carbon atoms such as vinyl group, propenyl group (allyl group or 1-propenyl group), butenyl group, isobutenyl group, pentenyl group, hexenyl group and the like, of which vinyl group and propenyl group are preferable.

Preparation of the substances to be separated

A medium (100mL) containing 1% corn starch, 1% glycerol, 0.5% glucose, 1% cottonseed flour, 0.5% dry yeast, 0.5% corn steep liquor and 0.2% calcium carbonate was adjustedAdjusted to pH 6.5, poured into eight 500mL Erlenmeyer flasks and sterilized at 120 ℃ for 30 minutes. A platinum loop quantity of a slant culture of Streptomyces tsukubaensis No.9993 (according to the Budapest treaty, in the National Institute of Bioscience and Humantechnology, Agency of Industrial Science and Technology, Japan deposit No. FERM BP-927) was inoculated onto the medium in each flask and cultured on a rotary shaker at 30 ℃ for 72 hours. This culture was transferred as a seed stock into 160L of the same medium which was packed in a 200L jar fermenter sterilized in advance for 30 minutes at 120 ℃ and to which 0.05% Adekanol had been added^*(antifoaming agent, trademark, manufactured by Asahi Denka Co., Ltd., Japan) and 0.05% siloxane (manufactured by Shinetsu chemical Co., Ltd., Japan). It was pre-incubated at 30 ℃ for 48 hours with stirring at 200rpm under 160 l/min ventilation. 30L of this preculture were inoculated into 3000L of production medium containing 3% soluble starch, 0.8% wheat germ, 0.4% dry yeast, 0.6% corn steep liquor, 0.1% calcium carbonate, 0.05% Adekanol, at pH 6.8, pre-sterilized at 120 ℃ for 30 minutes in a 4 ton pot^*And 0.05% siloxane, and fermented at 25 ℃ for 168 hours with aeration at 1500 liters/min with stirring at 140 rpm.

The thus-obtained culture solution was filtered through 50kg of celite. The mycelium cake was extracted with 1000L of acetone to obtain 1000L of extract. The acetone extract obtained from the hypha cake and the filtrate (2700L) were combined and passed through a column (200L) of a nonionic adsorption resin "Diaion HP-20" (trademark, manufactured by Mitsubishi chemical Co., Ltd., Japan). After washing with 600L of 50% aqueous acetone, elution was carried out with 75% aqueous acetone. The solvent in the eluent was removed by evaporation under reduced pressure to give 40L of an aqueous residue. The residue was extracted twice with 40L of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to give an oily residue. The oily residue was dissolved in a mixture of n-hexane and ethyl acetate (1: 1, v/v, 3L) and subjected to column chromatography using 70L of silica gel (Merck & Co., Ltd., 70-230 mesh) packed with the same solvent.

Elution was carried out successively with a mixture of n-hexane and ethyl acetate (1: 1, v/v, 420L and 1: 2, v/v, 420L), 210L of ethyl acetate and subsequently 210L of acetone. Fractions with elution volumes from 350L to 420L (first eluate), from 490L to 840L (second eluate) and from 980L to 1190L (third eluate) were collected. The second eluent was concentrated under reduced pressure, and acetone was added to replace the solvent (50 mg/mL). This gives a starting material to be separated by column chromatography.

Brief Description of Drawings

FIG. 1 shows a process for preparing silver nanoparticles by using silver ion pretreated Diaion^*A graph obtained by performing column chromatography on RCP 160M; and

FIG. 2 shows a Capcell Pak pretreated by using silver ions^*SCX column chromatography and the resulting graph.

Best mode for carrying out the invention

The following examples are merely intended to illustrate the invention in more detail.

Example 1

By using Diaion^*RCP160M (trademark, manufactured by Mitsubishi chemical Co., Ltd., Japan)

Manufactured) column chromatography for separation

(1) Treatment of Diaion with silver salt^*RCP160M ion exchange resin

1M aqueous silver nitrate solution (3 column volumes) was passed through Diaion^*RCP160M(H⁺Form), then washed with water (4 column volumes) to remove excess silver nitrate. The ion exchange resin was equilibrated with methanol (4 column volumes) and then with a mixture of ethyl acetate and methanol (1: 1).

(2) Separation assay using a single column

Subjecting the raw material to be subjected to column chromatography using Diaion treated with silver nitrate aqueous solution^*RCP 160M. Separation was carried out using a column of 20mm * × 500mH (425 μm) (150mL) and a mixture of ethyl acetate and methanol (1: 1) as eluent, at a flow rate sv ═ 1 and at a temperature of 30 ℃ under a load of 10g/L-R converted to tacrolimus. The resulting separation flow spectrum is shown in FIG. 1.

Tacrolimus, ascomycin and 17-propyl-1, 14-dihydroxy-12- [2- (4-hydroxy-3-methoxycyclohexyl) -1-methylethenyl]-23, 25-dimethoxy-13, 19, 21, 27-tetramethyl-11, 28-dioxa-4-azatricyclo- [22.3.1.0^4.9]Dioctadecyl-18-ene-2, 3, 10, 16-tetraone (hereinafter referred to as compound A).

Example 2

By using Capcell Pak^*Separation by column chromatography of SCX UG80 (trademark, manufactured by Shiseido Co., Ltd., Japan)

(1) Separation assay using a single column

The raw material to be separated by column chromatography obtained in the same manner as in example 1 was subjected to column chromatography using a silver ion-exchanged Capcell Pak^*SCXUG 80. Separation and purification were carried out using a column of 20mm * × 250mH (20 μm) (78mL) and using acetone as an eluent under a load of 5g/L-R as tacrolimus at a flow rate sv ═ 1.5 and at a temperature of 30 ℃. The resulting separation flow spectrum is shown in FIG. 2.

(2) Continuous separation with small simulated moving bed apparatus

The raw material to be subjected to column chromatography obtained as described above was subjected to a small-sized simulated moving bed apparatus (a small-sized novel J0 system chromatography separator, TREZONE, manufactured by Organo corporation, Japan)^*) Separation was carried out using a Capcell Pak treated with an aqueous silver nitrate solution^*SCX UG 80. In the followingSeparation and purification under the conditions: eluent-acetone; the charging concentration is-25 g/L; charging method-24 g/cycle x 4 cycles; column- (20 μm)2.26L (280 mL. times.8); total load-42 g/L-R (load per batch); the amount of liquid in the active fraction was-1.5L/cycle (relative to 0.65 column volume); and a temperature of-20 ℃. The purity of each substance in the effective fraction was determined by HPLC analysis. The results are shown in Table 1.

TABLE 1

	Purity in raw Material (%)	Purity in eluted fraction (%)
			Tacrolimus	85.4	92.7
Ascomycin	5.45	0.11
			Compound A	1.30	n.d.

n.d.: not detected

INDUSTRIAL APPLICABILITY

Unexpected results were obtained in the separation by using a strong cation exchange resin containing sulfonic acid groups pretreated with silver ions for separating a lactone-containing high-molecular weight compound having an alkyl group as a side chain from a lactone-containing high-molecular weight compound having an alkenyl group as a side chain.

The separation method of the present invention is very useful because of its reproducibility, large-scale applicability and/or its economic efficiency. Thus, the present invention can be used in the chemical industry, in particular in the pharmaceutical industry, where large quantities and purity of the target compound are critical.

Claims (10)

1. Will have C by using a strong cation exchange resin containing sulfonic acid groups pretreated with silver ions_1-6Lactone-containing high-molecular weight compound having alkyl group as side chain and C_2-6A method for separating lactone-containing high-molecular weight compounds having a common basic chemical structure, wherein the common basic chemical structure of the lactone-containing high-molecular weight compounds is 1, 14-dihydroxy-12- [2- (4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23, 25-dimethoxy-13, 19, 21, 27-tetramethyl-11, 28-dioxa-4-nitrogenHetero-tricycles- [22.3.1.0^4.9]Dioctadecyl-18-ene-2, 3, 10, 16-tetraone.

2. The process of claim 1, wherein C is a side chain_1-6Alkyl and C_2-6The alkenyl groups are each substituted at the 17-position of each tricyclic compound.

3. The method of claim 1, wherein the alkyl group is ethyl or propyl and the alkenyl group is propenyl.

4. The method of claim 1, wherein the silver ions are provided by silver nitrate.

5. The process of claim 1, wherein the separation is carried out by using a simulated moving bed system.

6. The process of claim 1 wherein the strong cation exchange resin containing sulfonic acid groups is a cation exchange resin containing benzenesulfonic acid groups.

7. The method of claim 6, wherein the resin is comprised of a copolymer matrix of styrene monomer and divinylbenzene or a silica gel matrix coated with a thin film of a siloxane polymer.

8. The method of claim 1, wherein the alkenyl group is allyl.

9. The method of claim 1 wherein the silver ions are provided by silver perchlorate.

10. The process according to claim 1, wherein the lactone-containing high molecular weight compound to be isolated is selected from tacrolimus and ascomycin.