HK1048991B - Method for separating lactone-containing high-molecular weight compounds - Google Patents

Description

Method for separating lactone-containing high-molecular-weight compound

Technical Field

The present invention relates to a method for separating lactone-containing high-molecular weight compounds of the same type, more particularly lactone-containing high-molecular weight compounds having different side chains, using a nonionic adsorbent resin and/or a basic activated alumina.

Background

Methods for separating cis and trans isomers of unsaturated fatty acids having the same number of carbon atoms using silver ions are known (J.chromatograpy-J.chromatograpy, 149(1978) 417-). However, it is difficult to separate the same compounds having only slightly different or partially different molecular structures in a conventional manner because they have the same or almost the same number of carbon atoms and are therefore similar to each other in physical properties such as solubility in a solvent and affinity for a solvent.

Disclosure of Invention

The present inventors have made extensive studies in search of an effective method for separating similar compounds having physical properties similar to each other without changing their chemical structures. They have surprisingly found a process for separating lactone-containing high molecular weight compounds of the same type having different side chains using a nonionic adsorption resin with a suitable eluent and/or basic activated alumina with a suitable eluent.

The present invention provides a method for separating a lactone-containing high-molecular weight compound, which comprises subjecting a mixture of at least one group having a lower alkenyl group and a lower alkoxy group as its side chain, the lactone-containing high-molecular weight compound and the like compounds thereof to either or both of the following steps in any order to separate the compounds:

adsorbing the mixture on a non-ionic adsorption resin, and eluting with an aqueous solvent containing silver ions;

step (B) the mixture was adsorbed on basic activated alumina and eluted with an organic solvent.

According to the present invention, it is preferable to separate the lactone-containing high-molecular weight compound having both a lower alkenyl group and a lower alkoxy group as its side chain and the like by both of the step (A) and the step (B). In step (A) and step (B), either step may be carried out first, but it is usually preferred to carry out step (A) first.

It is preferable to use step (A) for separating a lactone-containing high-molecular weight compound having a lower alkenyl group as its side chain and its analogous compounds.

It is preferable to use step (B) for separating a lactone-containing high-molecular weight compound having a lower alkoxy group as its side chain and its analogous compounds.

Lactone-containing high molecular weight compounds, i.e., compounds having one or more lactone rings in the molecule and a molecular weight of about 400 or above 400, to which the separation process of the present invention can be applied. They may have a sheetBasic ring structures such as cyclic, bicyclic, tricyclic, etc. More preferably, the number of constituent atoms forming the basic ring structure is 12 or more. Such monocyclic compounds include erythromycin, leucomycin, vinblastine, and the like. Such tricyclic compounds having one lactone ring include tricyclic compounds as shown in EPO 184162 and heteroatom-containing tricyclic compounds as shown in EPO 427680, EPO 532088 or WO 93/04680. A preferred tricyclic compound having one lactone ring is 1, 14-dihydroxy-12- [2- (4-hydroxy-cyclohexyl) -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 Z) (e.g., tacrolimus, substituted at position 17 with allyl, substituted at position 3 with methoxy in the cyclohexyl group of the 4-hydroxy-cyclohexyl moiety; ascomycin, substituted at position 17 with ethyl, substituted at position 3 with methoxy in the cyclohexyl group of the 4-hydroxy-cyclohexyl moiety), rapmaysins, etc. Among these compounds, tricyclic compounds are preferable, and compound Z is more preferable.

In the lactone-containing high-molecular weight compound, the lower alkenyl group as a side chain thereof may be a straight-chain or branched alkenyl group of 2 to 6 carbon atoms, such as vinyl, propenyl (allyl or 1-propenyl), butenyl, isobutenyl, pentenyl, hexenyl, or the like. Of these, vinyl and propenyl are preferred.

In the lactone-containing high-molecular weight compound, the lower alkoxy group as the side chain thereof may be a straight or branched alkoxy group of 1 to 6 carbon atoms such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, hexyloxy and the like. Among them, preferred are groups of 1 to 4 carbon atoms such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy and the like.

Particularly preferred lactone-containing high-molecular weight compounds having at least one group of lower alkenyl and lower alkoxy as their side chain, which are separated by the process of the present invention, are compounds containing compound Z as the basic chemical structure, wherein lower alkenyl is propenyl and lower alkoxy is methoxy, or compounds containing compound Z as the basic chemical structure, wherein lower alkoxy is methoxy.

The analogous compounds of the lactone-containing high-molecular weight compounds to which the separation method according to the present invention is applied, i.e., compounds having the same or substantially the same basic chemical structure as the above-mentioned lactone-containing high-molecular weight compounds, but having different substituents as their side chains. For example, the compounds of the same class as those having a lower alkenyl group as its side chain may have the same basic chemical structure but have a lower alkyl group, a lower alkoxy group, a hydroxyl group, or the like substituted for the lower alkenyl group at the same position. Such compounds of the same class include those having a basic chemical structure and somewhat different substitution positions but exhibiting similar properties as a whole.

Particularly preferred analogous compounds of the lactone-containing high-molecular weight compound having a lower alkenyl group as its side chain may be those having a lower alkyl group in place of the lower alkenyl group; the analogous compounds of the lactone-containing high-molecular weight compounds having a lower alkoxy group as its side chain may be those having a hydroxy group in place of the lower alkoxy group; the analogous compounds of the lactone-containing high-molecular weight compounds having a lower alkenyl group and a lower alkoxy group as different side chains may be those having a lower alkyl group in place of the alkenyl group and/or a hydroxyl group in place of the lower alkoxy group.

The lower alkyl side chain of the lactone-containing high-molecular weight compound may be a straight or branched alkyl group of 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. Among them, preferred are those having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, etc.

According to the present invention, a mixture of a lactone-containing high-molecular weight compound having at least one group of an alkenyl group and an alkoxy group as its side chain and a like compound thereof is adsorbed on a nonionic adsorption resin or basic activated alumina, and then the target compound is eluted from the adsorbent, may be carried out in the following manner:

for example, mixtures of lactone-containing high-molecular weight compounds having at least one group of alkenyl and alkoxy groups as their side chains and compounds of the same type obtained by fermentation processes are isolated depending on whether these compounds are produced extracellularly (i.e., extracellularly) or intracellularly (i.e., intracellularly). In the case of extracellular production, the fermented liquid mixture is separated by the process of the invention; if the compound is produced intracellularly, the bacteria are treated with a suitable solvent and the resulting mixture of extracts is then separated by the method of the invention. The stock solution or concentrate of the fermented liquid mixture or extract mixture may be injected into a separation column or similar device packed with an adsorbent. Alternatively, the fermented liquid or extract may be concentrated to dryness to obtain a residue, and then the residue is dissolved in a suitable solvent to prepare a solution, which is then injected into a separation column or the like.

If a mixture of a lactone-containing high-molecular weight compound having at least one of alkenyl groups and alkoxy groups as its side chain and a like compound is obtained by a synthetic method, a raw solution or concentrate of the reaction solution or extract may be injected into a separation column or the like packed with an adsorbent. Alternatively, the reaction solution or the extract may be concentrated to dryness to obtain a residue, and then the residue may be dissolved in a suitable solvent to prepare a solution, which may be injected into a separation column or the like.

The lactone-containing high-molecular weight compound having at least one of alkenyl groups and alkoxy groups as its side chain and the like contained in the mixture are selectively eluted depending on their affinity with the adsorbent and the eluent, and then the eluates containing the desired compound are combined and concentrated to dryness, whereby the desired lactone-containing high-molecular weight compound can be isolated.

The nonionic adsorbent resin used as the adsorbent may be a polyethylene resin having a partial structure represented by the following formula:

wherein R is hydrogen or halogen atom. In particular, Diaion is preferably used^*HP20，Diaion^*HP20SS，Sepabeads^*SP207 (manufactured by Mitsubishi chemical Co., Ltd., Japan), and the like. For example, if the basic chemical structure of the lactone-containing high-molecular weight compound is compound Z, Diaion is generally preferred^*HP20SS。

The elution of the lactone-containing high molecular weight compound adsorbed by the nonionic adsorbent resin is carried out using an aqueous solvent containing silver ions. As the silver salt contained in the aqueous solvent containing silver ions, nitrate, silver perchlorate, or the like, which exists as silver ions in water, is preferable. For example, if the basic chemical structure of the lactone-containing high-molecular weight compound is compound Z, nitrate is preferred. The concentration of silver ions varies depending on the nature of the lactone-containing high-molecular weight compound to be isolated, but may be generally 0.059 to 1.18 mol/l, preferably 0.12 to 0.59 mol/l, more preferably 0.18 to 0.47 mol/l, most preferably 0.24 to 0.35 mol/l, in terms of Ag⁺And (4) calculating.

The aqueous medium as the aqueous solvent containing silver ions may be aqueous acetone, aqueous alcohols (e.g., methanol and ethanol), and aqueous acetonitrile. For example, aqueous acetone may be preferably used in the case where the basic chemical structure of the lactone-containing high-molecular weight compound is compound Z.

The basic activated alumina which is preferable as the adsorbent may be AC12 (trade name, manufactured by Sumitomo chemical limited, japan), used in, for example, the case where the basic chemical structure of the lactone-containing high-molecular weight compound is compound Z.

The elution of the lactone-containing high molecular weight compounds adsorbed by the basic activated alumina is carried out with an organic solvent. The organic solvent used as the eluent may be a conventional solvent such as ethyl acetate, acetone, dichloromethane, a mixture of chloroform and methanol, a mixture of ethyl acetate and n-hexane, etc. For example, ethyl acetate may be preferably used in the case where the basic chemical structure of the lactone-containing high-molecular weight compound is compound Z.

The type and mixing ratio of the eluent (if the eluent is a mixed solvent) are preferably selected by preliminary analysis such as Thin Layer Chromatography (TLC) according to the properties of the lactone-containing high-molecular weight compound to be separated.

In the case where the adsorbent is a nonionic adsorbent resin, the amount of the adsorbent is preferably 50 times the weight of the lactone-containing high-molecular weight compound; in the case where the adsorbent is a basic activated alumina, the amount of adsorbent is preferably 70 times the weight of the lactone-containing high molecular weight compound.

The flow rate SV of the eluent varies depending on the particle size of the adsorbent, and may be generally about 3 to 5 ml/min (in the case of a nonionic adsorption resin), and generally about 3 to 5 ml/min (in the case of a basic activated alumina).

After several fractions were obtained, the fraction of the lactone-containing high-molecular weight compound having at least one group having an alkenyl group and an alkoxy group as its side chain can be detected with an ultraviolet-visible light detector or a differential refractive index detector or with TLC. The fractions containing the desired substance were combined and evaporated to dryness under reduced pressure, whereby the desired substance was purified.

Although the present invention is a method for separating a lactone-containing high-molecular weight compound having at least one group of an alkenyl group and an alkoxy group as its side chain, it is possible to further separate the same type of compound remaining from other same type of compound after separating the lactone-containing high-molecular weight compound. For example, a lactone-containing high-molecular weight compound having a lower alkoxy group as its side chain is separated from a mixture of the lactone-containing high-molecular weight compound and a like compound having substantially the same basic chemical structure and having a hydroxyl group as its side chain, and then the like compound is separated by elution with a solvent of different polarity by the method of the present invention.

Brief Description of Drawings

FIG. 1 shows the use of Diaion^*HP20SS and aqueous acetone as eluent.

FIG. 2 shows the use of Diaion^*HP20SS and aqueous acetone containing nitrate (0.294 mol/l) as eluent.

Best mode for carrying out the invention

The present invention will now be described in detail by way of examples, which are intended to be illustrative only and should not be construed as limiting the scope of the invention.

Preparation of example 1

Preparation of lactone-containing high-molecular-weight compound having at least one group of lower alkenyl group and lower alkoxy group as its side chain and mixture of the same by fermentation

The culture medium (100 ml) containing 1% corn starch, 1% glycerol, 0.5% glucose, 1% cottonseed meal, 0.5% dry yeast, 0.5% corn steep liquor, 0.2% calcium carbonate was adjusted to pH 6.5, poured into eight 500 ml Erlenmeyer flasks and sterilized at 120 ℃ for 30 minutes. A whole-circle culture of Streptomyces tsukubaensis No.9993 (stock No: FERM BP-927, deposited at national institute of bioscience and human technology, Japan, institute of Industrial science and technology, established according to the Budapest treaty) was inoculated into the medium in each flask, and cultured on a rotary shaker at 30 ℃ for 72 hours. This culture broth was transferred as a seed to 160 liters of the same medium contained in a 200 liter tank fermenter previously sterilized at 120 ℃ for 30 minutes and to which 0.05% Adekanol was added^*(antifoaming agent trademark, manufactured by Asahi Denka Co., Ltd., Japan) and 0.05% polysiloxane (manufactured by Shinetsu chemical Co., Ltd., Japan). Placing it inThe culture was carried out at 30 ℃ for 48 hours while stirring at a stirring speed of 200rpm under aeration at a rate of 160 liters/min. The broth (30 l) was inoculated into 3000 l of a production medium of pH 6.8 previously sterilized at 120 ℃ for 30 minutes, contained in a 4000 l tank containing 3% soluble starch, 0.8% wheat germ, 0.4% dry yeast, 0.6% corn steep, 0.1% calcium carbonate, 0.05% Adekanol^*And 0.05% polysiloxane, and then fermented at 25 ℃ for 168 hours while stirring (140rpm) under aeration (1500 liters/min).

The thus-obtained culture broth was filtered through 50 kg of celite. The mycelium cake was extracted with 1000 liters of acetone to obtain 1000 liters of extract. The cake acetone extract and filtrate (2700 l) were combined to give a crude sample.

Example 1

Using Diaion^*Separation by column chromatography using HP20SS as non-ionic adsorbent resin and eluent containing silver nitrate

Mixing 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]A mixture (200 mg) of dioctadec-18-ene-2, 3, 10, 16-tetraone (hereinafter referred to as Compound A) was dissolved in 50% aqueous acetone and diluted with Diaion^*HP20SS (20 ml) was subjected to column chromatography, and the substances contained in the mixture were adsorbed by the adsorbent. The tacrolimus, ascomycin and compound a were then eluted at room temperature using 50% (v/v) aqueous acetone containing silver nitrate (0.294 mol/l) and 60% (v/v) aqueous acetone as eluents. The loading was 9.5 g/L-R calculated as tacrolimus. The comparative experiment was carried out in the same way, but using aqueous acetone without silver nitrate as eluent. The results are shown in FIGS. 1 and 2.

Example 2

Using Diaion^*Separation by column chromatography using HP20SS as non-ionic adsorption resin and silver nitrate eluent

By Diaion^*HP20SS (20 ml) the crude sample (300 ml) obtained in preparation example 1 was subjected to column chromatography, and the substance contained therein was adsorbed by an adsorbent. The column was washed with 40% aqueous acetone (100 ml). After which tacrolimus and 17-allyl-1, 14-dihydroxy-12- [2- (3, 4-dihydroxycyclohexyl) -1-methylvinyl-1 are eluted at room temperature using 50% (v/v) aqueous acetone containing silver nitrate (0.294 mol/l) as eluent]-23, 25-dimethoxy-13, 19, 21, 27-tetramethyl-11, 28-dioxa-4-azatricyclo [22.3.1.0^4.9]A mixture of dioctadecyl-18-ene-2, 3, 10, 16-tetraone (hereinafter referred to as compound B). The combined ascomycin and compound A were then eluted separately under the same conditions with 60% (v/v) aqueous acetone.

The amounts of the respective compounds in the fractions thus obtained were measured by High Pressure Liquid Chromatography (HPLC) (mobile phase: acetonitrile/10% polyoxyethylene lauryl ether aqueous solution (Kyoto Nakalai Tesque, Japan)/water: 40/10/50; column: TOSOH TSK gel ODS-80Tm (5 μm, diameter 4.6X 150 mm); temperature: 75 ℃; detection wavelength: 210 nm; flow rate: 1.0 ml/min; injection amount: 20 μ L). The results of the separation of the mixture of Tacrolimus and compound B, the mixture of ascomycin and compound a are shown in table 1.

TABLE 1

Fraction number	Eluent	Distribution of mixture of Tacrolimus and compound B	Distribution of ascomycins	Distribution of Compound A
					0	Crude sample	100％	100％	100％
1	Passing a liquid	0％	0％	0％
					2	40% aqueous acetone (rinsing liquid)	0％	0％	0％
3	50% aqueous acetone with silver nitrate (0.294 mol/l)	99.7％	19.2％	0％
					4	60% aqueous acetone (push-out liquid)	0.3％	80.8％	90.7％
5	100% acetone (push-out liquid)	0％	0％	9.3％

Calculated from the peak area ratio to the Tacrolimus-containing mixture

As is clear from table 1, Tacrolimus, compound B, ascomycin and compound a were not washed out by passage through the liquid (fraction 1) or the flushing liquid (fraction 2), and they were completely adsorbed by the resin. Elution with silver nitrate solution (fraction 3) was achieved with Tacrolimus and compound B collected near 100% and compound a eluted below detectable limits. In the 60% aqueous acetone overhead fraction (fraction 4), the ascomycin and compound a were selectively collected close to 100%.

In summary, a mixture of lactone-containing high-molecular weight compounds having a lower alkenyl group as its side chain (Tacrolimus and compound B) and their analogous compounds (ascomycin and compound a) is adsorbed on a nonionic adsorbent resin and eluted with an aqueous solvent containing silver ions, whereby the lactone-containing high-molecular weight compounds having a lower alkenyl group as its side chain can be separated.

In addition, the above results show that lactone-containing, high-molecular weight compounds (ascomycin and compound a) having a lower alkyl group as its side chain can also be isolated.

Example 3

Column chromatography separation using basic activated alumina AC12

The fraction containing a mixture of Tacrolimus and compound B obtained in example 2 was concentrated and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and then evaporated to dryness. The resulting residue was subjected to column chromatography using basic activated alumina AC12(20 ml) (packed with ethyl acetate). Elution was performed at room temperature using ethyl acetate as eluent to give separate fractions (20 ml × 1 and 200 ml × 2).

The amounts of the respective compounds contained in the respective fractions thus obtained were measured by HPLC in the same manner as in example 2. The results of the isolation of Tacrolimus and compound B are shown in table 2.

TABLE 2

Fraction number	Amount of liquid	Tacrolimus	Compound B
				1	20 ml of	0％	0％
2	100 ml of	59.7％	0％
				3	100 ml of	10.4％	0％
All are	220 ml of	70.1％	0％

As is clear from table 2, a mixture of a lactone-containing, high-molecular weight compound (tacrolimus) having a lower alkoxy group as its side chain and a like compound (compound B) thereof was adsorbed on basic activated alumina and then eluted with an organic solvent, whereby the lactone-containing, high-molecular weight compound having a lower alkoxy group as its side chain could be isolated.

In addition, the above results show that a lactone-containing high-molecular weight compound (compound B) having a hydroxyl group as its side chain can also be isolated.

The separation of lactone-containing high-molecular-weight compounds tacrolimus having lower alkenyl groups and lower alkoxy groups as their side chains was carried out by the steps of example 2 and example 3, i.e., adsorption on a nonionic adsorbent resin followed by elution with an aqueous solvent containing silver ions, and adsorption on basic activated alumina followed by elution with an organic solvent, and the separation was carried out in the forward or reverse order on a solution of a mixture containing tacrolimus and its analogous compounds.

Industrial applications

The present invention can separate lactone-containing high-molecular weight compounds having at least one group containing a lower alkenyl group and a lower alkoxy group as a side chain from their analogous compounds by one or two steps of adsorption with a nonionic adsorption resin and elution with an aqueous solvent containing silver ions and adsorption with an alkali activated alumina and elution with an organic solvent, with an unexpectedly high efficiency.

Claims (9)

1. A method for isolating a lactone-containing compound comprising contacting a compound having a basic chemical structure 1, 14-dihydroxy-12- [2- (4-hydroxy-cyclohexyl) -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, and C_2-6Alkenyl and C_1-6At least one group of alkoxy as its side chain, lactone-containing compounds and compounds having the same basic chemical structure, each with a C_1-6Alkyl and monohydroxy in place of C_2-6Alkenyl and C_1-6Alkoxy radicalA mixture of like compounds having a group as its side chain, in any order, either or both of the following steps:

step (A): adsorbing the mixture on a non-ionic adsorption resin and eluting with an aqueous solvent containing silver ions selected from the group consisting of aqueous acetone, aqueous alcohol and aqueous acetonitrile to give C_2-6Compounds having alkenyl as their side chain and compounds having C_1-6Isolating a similar compound of alkyl; and

step (B): adsorbing the mixture on basic activated alumina and eluting with an organic solvent selected from the group consisting of ethyl acetate, dichloromethane, a mixture of chloroform and methanol, a mixture of ethyl acetate and n-hexane to have C_1-6The compound having alkoxy as its side chain is isolated from a similar compound having hydroxyl.

2. The method of claim 1, wherein the lactone-containing compound has both a C_2-6Alkenyl and a C_1-6Alkoxy as its side chain and the separation is carried out in the order of steps (A) and (B) or vice versa.

3. The method of claim 1, wherein the lactone-containing compound has one C_2-6Alkenyl as its side chain and isolated by step (A).

4. The method of claim 1, wherein the lactone-containing compound has one C_1-6Alkoxy group as its side chain and isolated with step (B).

5. The process of claim 2, wherein C is a side chain_1-6Alkenyl is propenyl, as side chain C_1-6Alkoxy is methoxy.

6. The method of claim 3, wherein C is a side chain_2-6Alkenyl is propenyl.

7. The method of claim 4, wherein C is a side chain_1-6Alkoxy is methoxy.

8. The process of claim 1 wherein the nonionic adsorbent resin has the formula

Is a partial structure represented by the formula, wherein R is a hydrogen atom or a halogen atom.

9. The method of claim 5 or 6, wherein propenyl is allyl.