JPH06205685A - Method for producing vitamin D3 derivative - Google Patents

Abstract

(57) [Summary] (Corrected) [Constitution] The enzyme P450 _C24 derived from the rat kidney P450 _C24 producing E. coli strain is expressed by the following formula. (In the formula, R ₄ represents H or OH, and R ₅ is R ₄ is H.
Represents OH, and when R ₄ is OH, R ₅ is H or O
Represents H. In the presence of ferredoxin and NADPH-ferredoxin reductase, the compound represented by the formula (Wherein, R ₁ is OH, R ₂ is .R ₃ representing a = O H or R ₁ and R ₂ total, represents H when R ₁ is OH, R ₂ represents H, R ₁ and When R ₂ represents ═O, R ₃ represents H or OH, R ₄ represents OH when R ₁ represents H, and OH represents when R ₂ represents H, and ═O represents R ₁ and R _{2 as a} whole. vitamin D ₃ derivative production method of represented by represents.) H or OH when. [Effect] The obtained compound is effective for treating bone lesions such as osteoporosis.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY According to the present invention, a vitamin D ₃ derivative effective for the treatment of bone lesions such as osteoporosis is added to rat kidney P4.
It has become possible to provide a simple production method using the enzyme P450 _C24 derived from a 50 _C24- producing Escherichia coli strain and ferredoxin and NADPH-ferredoxin reductase.

[0002]

2. Description of the Related Art Vitamin D ₃ derivatives are effective in treating bone lesions such as osteoporosis, which has been a problem as geriatric diseases. Currently, these compounds are manufactured by a complicated chemical synthesis method, and thus are efficient. Development of a manufacturing method is desired.
On the other hand, it is known that vitamin D ₃ derivatives are physiologically biosynthesized by mitochondrial P450 molecular species.

Active vitamin D ₃ is known as a hormone having a calcium metabolism regulating action. When the serum calcium level becomes normal, the side chain of active vitamin D ₃ is oxidized and metabolized to maintain the in vivo concentration, which contributes to the regulation of serum calcium level. Many side-chain oxidative metabolites have been isolated and identified so far, but 1α, 25 (OH) ₂ D ₃ is known to have a calcium metabolism regulating action.
Compared with the above, the calcium metabolism regulating effect was considerably weaker in all cases, and all metabolites other than 1α, 25 (OH) ₂ D ₃ were considered to be inactive. However, 24,25 (OH) ₂ D
₃ became 1α, 25 (OH) bone mineralization through joint action with ₂ D _3, secretion regulation of parathyroid hormone (PTH), and shown to exhibit effects such as cartilage growth. Moreover, there are no side effects such as hypercalcemia, and the blood concentration is 25 (O
H) It has the second highest level after D ₃ and has a long half-life, which makes it clear that it is an important metabolite. Similarly, 24oxo25 (O which is a vitamin D ₃ derivative in which the 24-position is oxolated
H) D ₃ also showed physiological activity such as intestinal absorption of calcium and bone formation similar to 24,25 (OH) ₂ D ₃ described above in rats fed a vitamin D ₃ deficient diet, and osteoporosis. It has been reported to be effective in treating bone lesions such as.

P450 is a heme protein that exists widely in the living world from microorganisms to mammals, and catalyzes a one-atom oxygen addition reaction using various fat-soluble compounds as substrates as a terminal enzyme of the electron transfer system. Terminal enzyme P45
Since 0 has various molecular species and each shows different substrate specificity, a very wide range of fat-soluble compounds can be hydroxylated. Mammalian P450-dependent electron transfer system is divided into microsomal type and mitochondrial type based on its constituent enzymes. In the former, NADPH-cytochrome P450 reductase (reductase) containing flavin adenine dinucleotide and flavin mononucleotide as coenzymes in the molecule supplies electrons from NADPH to P450, and in the latter, flavin adenine dinucleotide is supplemented. NADPH-ferredoxin reductase contained in the molecule as an enzyme and ferredoxin contained in the molecule as a non-heme iron coenzyme contain P450 electrons from NADPH.
To catalyze the hydroxylation reaction on various lipophilic substrates.

1α, 25-dihydroxyvitamin D ₃ (1
Rat kidney P450 _{C24 that} is induced and synthesized by α, 25 (OH) ₂ D ₃ ) (active vitamin D ₃ ) is known as an enzyme that catalyzes the hydroxylation of the 24th side chain of the vitamin D ₃ derivative. P450 _C24 is one of the mitochondrial P450 molecular species, and is synthesized in the cytoplasm as a precursor consisting of 514 amino acids. Then, the translocation signal existing in the amino terminal part is recognized and incorporated into the inner mitochondrial membrane,
Mature P4 consisting of 479 amino acids and having a molecular weight of about 53 kD
It becomes 50 _C24 . The rat kidney P450 _C24 gene is a plasmid pCC24-8 (Ohyama et al., (1991) FEBS 278, 1
95-198).

[0006]

An object of the present invention is to provide a simple method for producing a vitamin D ₃ derivative which is effective for treating bone lesions such as osteoporosis by using an Escherichia coli strain producing a mitochondrial P450 molecular species. It is to be. The present inventors reconstructed an electron transfer system using the enzyme produced by a mitochondrial P450 _C24- producing Escherichia coli strain and two enzymes of bovine adrenal ADX and bovine adrenal ADR necessary for expression of the activity of the enzyme. Vitamin D rebuilding system
_The present invention was completed as a result of conducting sincere research for the purpose of utilizing it for the production of the _three derivatives.

[0007]

The present invention provides rat kidney P45
The enzyme P450 _C24 derived from the 0 _C24- producing Escherichia coli strain has the following general formula 3

[Chemical 3] (In the formula, R ₄ represents H or OH, and R ₅ is R ₄ is H.
Represents OH, and when R ₄ is OH, R ₅ is H or O
Represents H. ) The compound represented by the formula (4) is allowed to act in the presence of ferredoxin and NADPH-ferredoxin reductase.

[Chemical 4] (Wherein, R ₁ is OH, R ₂ is .R ₃ representing a = O H or R ₁ and R ₂ total, represents H when R ₁ is OH, R ₂ represents H, R ₁ and When R ₂ represents ═O, R ₃ represents H or OH, R ₄ represents OH when R ₁ represents H, and OH represents when R ₂ represents H, and ═O represents R ₁ and R _{2 as a} whole. Where R ₄ represents H or OH, and R ₄ represents the same atom or atomic group in the compound represented by the general formula 3 and the compound represented by the general formula 4. The present invention provides a simple method for producing a vitamin D ₃ derivative represented by the formula (1) and the compound represented by the general formula (4).

The present invention will be described in more detail below. CDN encoding rat kidney P450 _C24 used in the present invention
A is already known and can be isolated by a usual operation method. As a method for cloning the cDNA, for example, the method described in JP-A-4-207196 can be used. Alternatively, all genes can be chemically synthesized. The expression plasmid expressing P450 _C24 of the present invention can be constructed by inserting the rat kidney P450 _C24 gene into an appropriate expression vector by a conventional method. Expression vectors include those that contain genetic information that can be replicated in host cells, that can autonomously grow, that are easy to isolate and purify from the host, and that have a detectable marker such as drug resistance. It is suitable. Various vectors are commercially available, and for expression in E. coli, for example, expression vectors containing promoters such as lac, tac, trp (these are commercially available as expression vectors or promoter cartridges from Pharmacia, LKB, etc.). ) Can be used. Restriction enzymes and the like used for vector cleavage are also commercially available, and methods for using these are known. Further, the structure of the expression plasmid is not limited, and any structure may be used as long as it can be stably retained in the host cell, but pKC24R described in Japanese Patent Application No. 3-320351 is particularly preferable. As a host, E. coli, such as Escherichia coli JM109, HB101, DH1,
Examples include the K12 strain, and the plasmid pKC24R
The most preferred combination with is a lac I ^q strain (a lactose repressor high-producing strain) represented by the JM109 strain.

Transformation of a host with an expression plasmid containing the rat kidney P450 _C24 gene can be carried out by a known method by mixing E. coli competent cells obtained by calcium treatment with DNA. Escherichia coli competent cells having transformability are also commercially available. When a commercially available Escherichia coli JM109 strain competent cell is used, the expression plasmid solution is added to the JM109 strain competent cell and cultivated, and this culture solution is spread on a plate containing an antibiotic such as ampicillin to obtain vector-derived cells. A transformant carrying the expression plasmid having acquired the antibiotic resistance can be selected.

The resulting transformed Escherichia coli is cultured, and IPTG (isopropylthio-β-D-galactoside) is added in the late logarithmic growth phase to induce expression of _P450C24 . Since the expressed P450 _C24 is present in the membrane fraction of E. coli,
The transformant is disrupted by sonication and the like, and an E. coli membrane fraction containing _P450C24 can be prepared by centrifugation. The Escherichia coli membrane fraction thus obtained and a reconstitution system in which ferredoxin and NADPH-ferredoxin reductase required for expression of P450 _C24 activity were added to P450 _C24
The activity can be measured. As the ferredoxin and NADPH-ferredoxin reductase used in the reconstitution system, for example, bovine adrenal ADX and ADR can be used, and these can be used according to the method of Orme-Johnson et al. ((1969)).
J. Biol. Chem. 244, 6143-6148) and Nonaka et al. ((1985) J. Biochem. 98, 257-260) can be used to purify bovine adrenal glands. Further, as ferredoxin and NADPH-ferredoxin reductase, in addition to bovine adrenal gland ADX and ADR described in the examples of the present invention, spinach-derived ones capable of transferring electrons to _P450C24 may be used.

The reaction conditions in the production of the vitamin D ₃ derivative using the P450 _C24 reconstitution system are as follows. When the enzyme P450 _C24 is added to the reaction system, it is preferable to use a membrane fraction containing the enzyme P450 _C24 prepared from transformed Escherichia coli so that the final concentration in the reaction system will be 1-10 mg / ml. The enzymes ADX and ADR prevent the reaction by these enzymes from becoming the rate-limiting enzyme P450.
Add in a large excess relative to _C24 . ADX is 0.15-8 μM with respect to the amount of P450 _C24 added, ADR is
It is preferably added in the range of 0.02 to 0.8 μM. The coenzyme NADPH is preferably used in a range that does not interfere with the expression of enzyme activity. 25 (OH) D ₃ or, l [alpha], the case of using the 25 (OH) ₂ D ₃ as a substrate, preferably employed in the range of 5 -100 [mu] M. 25 (OH) D ₃ and 1α, 25 (OH) ₂ D ₃
Are commercially available from Funakoshi and Wako Pure Chemical, respectively. A metal chelating agent EDTA is preferably added to the reaction system for the purpose of preventing inhibition of the enzyme activity by the metal salt. The reaction pH is preferably 6-8. The buffer used in the reaction system is not particularly limited as long as it can maintain the above pH,
Use normal buffer such as Tris-HCl buffer, phosphate buffer, etc.
It can be used at a concentration of 100 mM. Reaction temperature is 10 ℃
A range of 40 ° C is preferred. The reaction is stopped by adding a solvent capable of extracting the product. Hydrophobic organic solvents such as dichloromethane, benzene, toluene, ethyl acetate, chloroform and the like can be used as the addition solvent, but dichloromethane, which is particularly excellent in extraction efficiency, is preferable. The produced vitamin D ₃ derivative can be easily taken out from the reaction system by distilling off the solvent.

[0012]

EFFECT OF THE INVENTION Rat kidney P450_C24From the producing E. coli strain
The crudely purified enzyme was bovine adrenal ADX and bovine adrenal ADR.
25 (OH) D in the reconstitution system of₃And 1α, 25 (O
H)₂D ₃To 24 Oxo 25 (OH) D₃And 24 oxo 23,25
(OH)₂D₃, 24oxo 1α, 25 (OH)₂D₃And 24
Kiso 1α, 23,25 (OH)₃D₃The first time we can manufacture
I found it. According to the present invention, P45 produced in E. coli
0_C24Ferredoxin, NADPH-ferredoxin
By combining with reductase, bones such as osteoporosis
Vitamin D is effective in treating lesions₃Derivatives can be manufactured
Noh Comparing with the complicated chemical synthesis methods currently used
More easily, 24 oxo 25 (OH) D₃And 24 oxo 2
3,25 (OH)₂D₃, 24oxo 1α, 25 (OH)₂D₃And 2
4oxo 1α, 23,25 (OH)₃D₃Vitamin D such as₃Invitation
It became possible to manufacture conductors.

[0013]

EXAMPLES The present invention will be described in detail below based on examples. The present invention is not limited to the examples, and it is possible to make ordinary changes in the technical field of the present invention. Note that quantitation of vitamin D ₃ derivative is the product in the examples HPLC - it was by analysis.
The analysis conditions are as follows. 1. Column: μBondapak C18 (φ4X300mm) 2. Flow rate: 1.0 ml / min 3. Column temperature: 50 ° C 4. Detection wavelength: 265 nm 5. Mobile phase conditions: 0 to 20 minutes: 50% to 100%
Agricultural gradient of acetonitrile 20 minutes to 25 minutes: 100% acetonitrile 25 minutes to 26 minutes: Linear gradient of 100% to 50% acetonitrile 26 minutes to 34 minutes: 50% acetonitrile

Example 1 Method for Producing 1α, 24,25 (OH) ₃ D _{3 In} a reconstitution system with bovine adrenal ADX and bovine adrenal ADR, 1α, 24 was prepared using a membrane fraction prepared from a P450 _C24- expressing strain. , 25 (OH) ₃ D ₃ was produced by the method as shown below. A strain not producing P450 _C24 was used as a negative control. The cell fraction from the transformed E. coli strain was according to the following method. The microbial cells were recovered by centrifuging the culture solution with an OD660 of around 1, and using the MOPS buffer (50 mM M
OPS, pH 7.5, 100 mM potassium chloride, 1 mM DTT, 1 mM
After washing with EDTA), 1/20 volume of MO of the culture solution
The cells were suspended in PS buffer (containing 0.2 mg / ml lysozyme). This was ice-cooled for 30 minutes to obtain spheroplasts, and PMSF (phenylmethylsulfonylfluoride), leupeptin and pepstatin were added to the final concentrations of 1 mM, 0.1 μg / ml and 0.1 μg / ml, respectively, to The cell membrane was disrupted by sonication. 1,200 xg, 10
Unbroken cells were removed by centrifugation for 5 minutes, and the obtained supernatant was centrifuged.
After ultracentrifugation at 225,000 xg for 30 minutes at ℃, it was separated into a soluble fraction (supernatant) and a membrane fraction (precipitate). Membrane fraction has a final concentration of 6
After washing with MOPS buffer containing mM MgCl ₂ , it was finally suspended in 50 mM potassium phosphate (pH 7.5) /0.25 M sucrose. The production of 1α, 24,25 (OH) ₃ D ₃ was carried out under the following conditions. 100 mM Tris-HCl (pH 7.
5), 1 mM EDTA, 20 μM 1α, 25 (OH) ₂ D ₃ , 2 μ
A membrane fraction (containing 1 mg protein) prepared from transformed Escherichia coli was added to a reaction mixture consisting of MADX, 0.2 μM ADR and 1 mM NADPH to make the total volume 0.5 ml, and the reaction was carried out at 37 ° C. for 60 minutes. Stop the reaction by adding 1.5 ml dichloromethane,
The mixture was stirred for 1 minute with a vortex mixer and then centrifuged to dry 1.3 ml of a dichloromethane layer. This was dissolved in a small amount of acetonitrile and analyzed by HPLC. As a result, the conversion rate to 1α, 24,25 (OH) ₃ D ₃ was 15% after the reaction time of 60 minutes. In addition, in the membrane fraction of the control strain, 1α, 24,25 (
No conversion to OH) ₃ D ₃ was observed.

Example 2 24 Oxo 25 (OH) D ₃ and
Method for producing 24oxo23,25 (OH) ₂ D _{3 Using} 24,25 (OH) D ₃ as a substrate, two kinds of vitamin D ₃ derivatives shown in FIG. 1 were produced according to the following method. The membrane fraction was prepared from the transformed Escherichia coli strain by the method described in Example 1. 20 μM 24, 25 (OH) ₂ D ₃ , 100 m
M Tris-HCl (pH7.5), 1mM EDTA, 4μM AD
A membrane fraction (containing 2.5 mg protein) prepared from transformed Escherichia coli was added to a reaction mixture consisting of X, 0.4 μM ADR and 1 mM NADPH to bring the total volume to 0.5 ml, followed by reaction at 37 ° C. for 60 minutes. As a result, it was observed the generation of 24-oxo-25 (OH) D ₃ and 24-oxo-23,25 (OH) ₂ D _3. The structures of these compounds were confirmed by the fragment cleavage pattern by EI-MS. The conversion rates of 24oxo 25 (OH) D ₃ and 24oxo 23,25 (OH) ₂ D ₃ are 20% and 3 respectively.
It was 1%. In addition, even if a strain that did not produce P450 _C24 was reacted as a negative control, the formation of oxo form was not observed.

Example 3 1α, 24,25 (OH)₃D₃, 24
Kiso 1α, 25 (OH)₂D₃And 24 oxo 1α, 23,25 (O
H)₃D₃Manufacturing method of 1α, 25 (OH)₂D₃As a substrate, the three types of vinyl shown in Fig. 2 are used.
Tamin D₃Preparation of derivatives according to the following method
did. 100 mM Tris-hydrochloric acid (pH 7.5), 1 mM EDTA,
20 μM 1α, 25 (OH)₂D₃, 2 μM ADX, 0.2 μM A
Add P450 to the reaction mixture consisting of DR and 1 mM NADPH._C24
Membrane fraction prepared from producing E. coli (containing 22 mg protein)
) Was added to bring the total volume to 11 ml, and the mixture was reacted at 37 ° C. for 60 minutes. 35 ml
Stop the reaction by adding dichloromethane and vortex the mixture.
After stirring for 1 minute or more with a kiser, centrifuge and separate the dichloromethane layer.
It was separated and dried. Dissolve this in a small amount of acetonitrile
And analyzed by high performance liquid chromatography. That conclusion
Fruit, 92μg of 1α, 25 (OH)₂D ₃From 14 μg of 1α, 24,
25 (OH)₃D₃, 6 μg of 24oxo 1α, 25 (OH)₂D₃,
 6 μg of 24oxo 1α, 23,25 (OH)₃D₃Generated by
It was

[Brief description of drawings]

FIG. 1 shows the metabolic pathway of 24,25 (OH) ₂ D ₃ by P450 _C24 .

FIG. 2 shows the metabolic pathway of 1α, 25 (OH) ₂ D ₃ by P450 _C24 .

Claims (2)

[Claims] 1. An enzyme P450 _C24 derived from a rat kidney P450 _C24- producing Escherichia coli strain is represented by the following general formula 1 (In the formula, R ₄ represents H or OH, and R ₅ is R ₄ is H.
Represents OH, and when R ₄ is OH, R ₅ is H or O
Represents H. ) A compound represented by the following general formula (2) is characterized in that it is allowed to act in the presence of ferredoxin and NADPH-ferredoxin reductase. (Wherein, R ₁ is OH, R ₂ is .R ₃ representing a = O H or R ₁ and R ₂ total, represents H when R ₁ is OH, R ₂ represents H, R ₁ and When R ₂ represents ═O, R ₃ represents H or OH, R ₄ represents OH when R ₁ represents H, and OH represents when R ₂ represents H, and ═O represents R ₁ and R _{2 as a} whole. And R ₄ represents the same atom or atomic group in the compound represented by the general formula 1 and the compound represented by the general formula 2. However, in the general formula 1, compound with general formalized represents a compound different from the compounds represented by 2.) vitamin D ₃ derivative production method of which is represented by represented. 2. The method according to claim 1, wherein a membrane fraction of a rat kidney P450 _C24- producing Escherichia coli strain is used.