JPS63251091A - Production of epipodophyllotoxin - Google Patents

Abstract

PURPOSE:To efficiently produce epipodophyllotoxin useful as an intermediate of an anticancer agent, by reacting epipodophyllotoxin with an enzymic agent consisting of 1-beta hydroxylase which is a cultivation product of a microorganism, viable bacterium cell or treated product of a bacterium cell. CONSTITUTION:An enzymic agent of 1beta hydroxylase is prepared from a microorganism belonging to the genus Penicillium or the genus Syncephalastrum. Deoxypodophyllotoxin is reacted with the above-mentioned enzymic agent of 1beta hydroxylase to provide epipodophyllotoxin expressed by the formula by an enzymic reaction. After the enzymic reaction is over, all kinds of chromatography methods such as adsorption chromatography and distribution chromatography or means such as countercurrent distribution are applied to the reaction liquid to separate and recover the epipodophyllotoxin. Liquid cultivation method is normally and preferably used as the cultivation of microorganism for preparing the enzymic agent of 1beta hydroxylase.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to epipodophyllotoxin (epi-podophyllotoxin).
phyllotoxin, hereinafter abbreviated as rEPTJ.

, ).

[Conventional technology]

EPT (the following structural formula [I]) is an epimer of bodophyllo-l-xin (the following structural formula [■], hereinafter abbreviated as rPTJ), and is a compound of etoposide and teniposide (tenj), which has strong antitumor activity. , p
It is an important compound as a synthetic intermediate such as oqj, de).

Sushi 1″i3 0H As shown in the above structural formula [■], although PT is a compound with a complex structure, it can be chemically synthesized (for example, J, OrH.

Chem,, 31.4004 (1966), J, Org.
, Chern.

46, 2862 (1981), J. Am, Che.
m,Soc,.

103.620.8 (1981), etc.).

On the other hand, EPT can also be synthesized chemically; for example, a method is known in which EPTti is produced through 6 reaction steps using aryltetralone as a starting material (
(See Japanese Patent Application Laid-open No. 61-238758). Also, deoxypodophyllotoxin (deoxypodophyllotoxin)
Otoxin, the following structural formula [■], hereinafter abbreviated as rDPTJ. ) in the presence of benzoyl peroxide in carbon tetracarbon,
A method for producing EPT by reacting it with N-bromosuccinimide under reflux and then hydrolyzing it is also known (Ch
em, Pharm.

Bull, 34.2056 (1986)).

[Problem that the invention seeks to solve]

However, the above chemical manufacturing method of EPT has
There are many problems such as long reaction process, complicated reaction and difficult to control, (3) low overall yield, and (2) production of by-products.

[Means for solving problems]

As a result of repeated research aimed at establishing a method for enzymatically producing EPT from DPT, the present inventors discovered that 1β hydroxylase derived from filamentous fungi specifically converts DPT to EPT. The present invention was completed based on this discovery.

That is, the present invention is a method for producing EPT from DPT by an enzymatic conversion reaction, which is characterized in that DPT is reacted with an enzyme agent of 1β hydroxylase to produce EPT. This relates to a manufacturing method.

The present invention will be explained in detail below.

In the present invention, the following terms are used with the following meanings.

``1β hydroxyl e element J refers to DPT by introducing a hydroxyl group into the 1β position of DPT (see structural formula (III) above).
Refers to an enzyme that catalyzes the conversion reaction from EPT to EPT.

The term "enzyme preparation for 1β hydroxylase" means an enzyme preparation derived from a microorganism (eg, a culture, a live cell, a treated cell, etc.) containing at least 1β hydroxylase activity.

The term "culture" refers to a culture of a microorganism having 1β-hydroxylase activity, in which the culture medium and cultured bacterial cells are unseparated.

The term "live bacterial cells" refers to cultured bacterial cells separated from a culture or washed bacterial cells thereof.

``Bacterial cell processed material J refers to dried bacterial cells derived from live bacterial cells,
It refers to cell membrane and/or cell wall-denatured bacterial cells, crushed bacterial cells, bacterial cell extracts, protein fractions containing 1β-hydroxylase, or purified products thereof.

DPT The DPT used as a raw material compound in the method of the present invention is not limited in any way to its preparation method, and may be produced by chemical synthesis or extracted and isolated from natural products. It is fine, and it doesn't matter how refined it is.

As a chemical synthesis method for DPT, for example, after lithiation of halogen-substituted carbon in a silylmethyl derivative,
A method is known in which DPT is obtained by reacting an aromatic aldehyde to obtain a trialkylsilylmethyl derivative, and then reacting the derivative with maleic anhydride (for details, see JP-A-61-93185; Unexamined Japanese Patent Publication 1986-1993
(See Publication No. 187).

As a natural substance containing DPT, for example, A. ovigera, which belongs to the family of.
Seeds of I-, ) are widely known. Hasunohagiri is
A coastal evergreen tree that grows in the subtropics such as the Noshin Islands and the Ogasawara Islands.
It grows widely in the tropics, and its seeds are easily available. DPT from seeds of Hasunohagiri
As a single extraction method, a solvent extraction method using an organic solvent such as methanol is adopted (for details, see Pharmacology: 1t Magazine,
- Noriichiten, 674 to 677 (1979)).

-]-] Enzyme preparation of 1β hydroxylase used in the method of the present invention can be prepared from microorganisms belonging to the genus Cervicalium lIl' or Syncephalusdora 11.

Examples of microorganisms of the genus Penicillium having 1β-hydroxylase activity include Penicillium citreoviride (P,
citreo-viride), Penicillium ethinulonalgiobens (P. eehinulonalgi)
ovense), Penicillium aculiatum (P,
acul, eatum), Penicillium lividum (1
', 1ividum), Penicillium melini (P,
melini), Penicillium paraherquei (P
, paraherquei), Penicillium purpurescens (P, purpurescCns), etc., and examples of Syncephalastrum microorganisms include Syncephalastrum fridinosum (S, ful
iginosum), Synthephalastrum racemosa ls (S, raeemosum), etc. can be used.

More specifically, Penicillium lividum IAM7200 and Synthephalus 1 and Rum frisinosum AHU9452 can be exemplified as representative strains of the microorganisms mentioned above.

However, these strains are merely illustrative strains, and include strains of microorganisms belonging to the above-mentioned genera that are newly isolated from the natural world, known cultured strains other than the exemplified species,
and 4T strains of mutant strains obtained by conventional microbial mutagenesis methods, such as physical treatments such as ultraviolet rays, X-rays, and γ-ray irradiation, and chemical treatments with cross-agents such as nitrosoguanidine However, as long as it contains 1β-hydroxylase activity, it can be used in the method of the present invention.

As described above, the method of the present invention is characterized by utilizing 1β-hydroxylase, which is widely distributed in various microorganisms belonging to the genus Penicillium and Syncephalus 1 to Ramum. This includes the use of enzymes derived from all microorganisms belonging to the genus Penicillium or Syncephalastrum.

For the cultivation of the above-mentioned microorganisms for preparing an enzyme preparation for 1β-hydroxylase, liquid culture is usually advantageous, and stationary, shaking, and aerated agitation culture methods can be employed as the culture method.

The medium used for liquid culture may be any medium as long as it contains components that allow the above-mentioned microorganisms to grow and grow. For example, carbon sources include glucose,
Sucrose, maltose, starch powder, starch liquefaction liquid, molasses, soluble starch, glycerin, ethanol,
Monohydric or polyhydric alcohols such as sorbitol, fatty acids such as acetic acid, propionic acid, stearic acid, and citric acid.

Organic acids such as succinic acid may be used alone or in combination of two or more. As a nitrogen source.

In addition to organic nitrogen sources such as pegone, cornstarch liquor, soybean flour, yeast extract, meat extract, and urea, sulfuric acid,
Inorganic nitrogen sources such as ammonium salts such as nitric acid, hydrochloric acid, and carbonic acid, ammonia gas, and aqueous ammonia are used alone or in combination.

In addition, micronutrients necessary for the survival of the bacteria, such as sulfur, are added.
- Various salts such as Wau 11 salt, potassium salt, calcium salt, magnesium salt, manganese salt, zinc salt, iron salt, phosphate, sulfate, biotin, thiamine, riboflavin,
Various substances such as vitamins such as nicotinic acid, pantothenic acid, and vitamin B12, and amino acids may be added as medium components, individually or in combination, in two appropriately selected substances.

In addition, in the present invention, wheat bran, defatted soybean, rice bran,
Corn, rapeseed meal5 Even natural solid raw materials such as wheat, rice, and rice husks can be used alone or in combination after being finely ground7Culture conditions are usually such that the pH of the medium at the initial stage of culture is 5 to 8. , the culture temperature can be selected from the range of 15 to 40°C, preferably 20 to 35°C. The culture time is usually 1 to 5 days, preferably 2 to 5 days.

The method of the present invention uses the above-mentioned microbial culture, live microbial cells, or treated microbial cells as an enzyme agent for 1β-hydroxylase.

Viable bacterial bodies are obtained from the above-mentioned microbial culture by centrifugation, sedimentation,
It can be prepared by collecting bacteria by a conventional solid-liquid separation method such as aggregation separation, and further washing the cells with water or an appropriate buffer as necessary.

The bacterial cell-treated product is prepared by subjecting the living bacterial cells to (a) physical treatment (for example, freeze-thaw treatment, freeze-drying treatment, heat treatment under strongly alkaline conditions, grinding treatment,
(Ultrasonic treatment, osmotic pressure difference treatment, etc.), (b) Chemical or biochemical treatment (e.g., enzyme treatment with lysozyme, cell wall lytic enzyme, etc., contact treatment with a solvent or surfactant such as toluene, xylene, etc.), etc. It can be obtained by performing various treatments. In addition, enzyme preparations are prepared by subjecting the bacterial cell extract to normal enzyme separation and purification treatments (for example, salting out treatment, isoelectric focusing precipitation treatment, organic solvent precipitation treatment, various chromatography treatments, dialysis treatment), etc. be able to.

In addition, for enzyme preparations for 1β-hydroxylase, live bacterial cells or treated bacterial cells are bound to a solid phase carrier using conventional enzyme and microorganism immobilization methods (e.g., entrapment method, crosslinking method, adsorption method, etc.). It also includes immobilized products.

As a method for causing a 1β-hydroxylase enzyme agent to act on DPT, if a culture is used as the enzyme agent, it can be used at any time during the culture, preferably at the time when the microbial cells have grown sufficiently. A method can be adopted in which DPT is added to the culture in a continuous or batch manner, and when live bacterial cells or treated bacterial cells are used as the enzyme agent, the living bacterial cells or treated bacterial cells are added to water or a phosphate buffer. A method in which DPT is continuously or batchwise added to a solution suspended or dissolved in a common buffer solution such as a solution, or DP is added to an immobilized product of live bacterial cells or treated bacterial cells.
A method of contacting with a T substrate solution can be adopted.

Enzyme reaction conditions can be appropriately set depending on the enzyme agent used. Usually, the concentration of DPT in the reaction solution is 10
It may be within the range of 0.01 to 1 mM, preferably 0.01 to 1 mM. The reaction temperature is 10-50°C, preferably 20-50°C.
40'C1 reaction time is 1-72 hours.

When dissolving DPT in the reaction solution, DPT may be used in a solid state, but in consideration of uniform dissolution and diffusion in the reaction solution, methanol, ethanol, ethyl acetate, chloroform, acetone, hexane, dichloromethane, It is preferable to use it in a dissolved or suspended state in an organic solvent such as dichloroethane or a mixture of the organic solvent and water.

After the enzymatic reaction, separation and purification of EPT from the reaction solution may be carried out by appropriately combining known methods or methods adapted therefrom. Specifically, various chromatographic methods such as adsorption chromatography and partition chromatography, methods that utilize distribution between two liquid phases such as countercurrent distribution and countercurrent extraction, concentration, cooling, addition of organic solvents, etc. This can be carried out by appropriately combining methods that utilize differences in solubility.

〔Effect of the invention〕

The method of the present invention has the excellent effect of overcoming the drawbacks observed in conventional chemical production methods and stereospecifically converting DPT to only EPT.

〔Example〕

EXAMPLES The present invention will be explained in more detail by way of Examples below, but these are merely illustrative of specific embodiments of the present invention and do not limit the scope of the present invention.

In addition, as a test method for the presence or absence of EPT generation, 1
The reaction solution treated with the β-hydroxylase enzyme was subjected to thin layer chromatography (developing solution: n-hexane: ethyl acetate = 1
:1) or a method of measuring ultraviolet absorption at 280 nm was used.

Example 1 Penicillium lividum I in the medium (A) IQ shown below.
AM7200 was inoculated and cultured at 28°C for 48 hours.

Medium (A); NaNO33-00gK, HPO
41,00g FeC1,・6H200, 61g KCI 0.50g MgSO4・7H200-50g Soluble starch 30.00g to 100100O Next, 1% (W/V) DPT ethanol solution 10+++
Q was added to the culture and allowed to react at 28°C for 72 hours. After the reaction, the bacterial cells were removed by suction filtration, the resulting culture solution was extracted with ethyl acetate, and the solvent was distilled off. The ethyl acetate extract was subjected to silica gel column chromatography, eluted with an elution solvent (n-hexane:ethyl acetate-1=1), and only the two fractions with absorption at UV 280 nm were collected.
Concentrate.

A pale yellow solid was obtained. This was recrystallized from ethanol-water to give 25.2 ml of colorless needle-like crystals of EPT (yield 24.2%). Physical and chemical properties of this colorless needle crystal (1H-N
MR spectrum, IR spectrum, [:α]D, melting point) are Chem, Pharm, Bull, 34-,
2056 (1986).

Example 2 Using Syncephalastrum frisinosum AHU9452, 81.3 square crystals of EPT (yield 78.2%) were obtained in the same manner as in Example -.

The physical and chemical properties of this colorless needle crystal are also described in the above literature.
It matched that of T.

Example 3 Penicillium lividum IA, M7200 was inoculated into the described medium (A) IQ and cultured at 36° C. for 48 hours for 6 incubations, after which bacterial cells were collected from the culture solution by centrifugation.

Next, the collected live bacteria were added to 0.1M phosphate buffer (Pu6
．． 3) into suspension IQ prepared by suspending it.

10 mfl of 1% (W/V) DPT ethanol solution was added and reacted at 36°C for 72 hours.

After the reaction is complete, the bacterial cells in the reaction solution are removed by centrifugation.
The reaction solution was then extracted with ethyl acetate.

After evaporating the solvent, the same procedure as in Example 1 was carried out to obtain 50.0 cm of colorless needle crystals of EPT (yield: 48.1%). The physical and chemical properties of this colorless acicular crystal are CheIIl, Pharn+.

Bull, 34.2056 (1986).

Claims (1)

[Claims] 1) A method for producing epipodophyllotoxin from deoxypodophyllotoxin by an enzymatic conversion reaction, comprising:
1. A method for producing epipodophyllotoxin, which comprises making epipodophyllotoxin act on deoxypodophyllotoxin with an enzyme agent of 1β hydroxylase to produce epipodophyllotoxin. 2) The method for producing epipodophyllotoxin according to claim 1, wherein the enzyme agent for 1β-hydroxylase comprises a microbial culture, live bacterial cells, or treated bacterial cells. 3) The microorganism is Penicillium
Genus or Syncephala
3. The method for producing epipodophyllotoxin according to claim 1 or 2, wherein the epipodophyllotoxin is a microorganism belonging to the genus Strum.