JPH04202194A - Production of oxazopyrroloquinoline - Google Patents

Abstract

PURPOSE:To obtain at low cost the title compound of low nephrotoxicity having anti-cataractal activity, anti-allergic activity, etc., by adding ammonia, etc., to a pyrroloquinolinequinone-contg. solution. CONSTITUTION:(A) A pyrroloquinolinequinone(PQQ)-contg. culture solution prepared by e.g. incubating in a medium with methanol as carbon source methanol-assimilable bacteria capable of extracellularly producing PQQ is incorporated with (B) pref. 5-100 molar times (based on PQQ) of ammonia (e.g. in the form of ammonium sulfate) and (C) pref. 30-1000 molar times of formaldehyde (e.g. in the form of formalin) followed by reaction at a pH of 4-8 at 25-50 deg.C, thus obtaining the objective compound of the formula.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing oxazopyrroquinoline,
More specifically, the present invention relates to a method for producing oxazopyrroquinoline from pyrroloquinoline quinone.

Oxazopyrroloquinoline (hereinafter abbreviated as OPQ) is also known as 2,8,104-licarboxy IH-oxazo[5,4
-h]-pyrrolo[2,3-f]quinoline, and its chemical structural formula is as follows.

○PQ is a derivative of pyrroloquinoline quinone (hereinafter referred to as PQQ), and is an important substance that can be developed as a pharmaceutical in the future. PQQ was discovered in 1979 as a coenzyme of methanol dehydrogenase of methanol-assimilating bacteria.

[Prior art and problems to be solved by the invention] PQQ is present not only in bacteria but also in eukaryotic molds, yeast, and even sister mammals, and plays an important role as a coenzyme. There is. In addition, in recent years, cell proliferation-promoting effects (
JP-A-61-58584, JP-A No. 63-233783
JP-A No. 63-41421), anti-cataract effect (JP-A-63-41421).

Publication No. 63-48215, No. 64. -29313 Publication), liver disease preventive and therapeutic action (Japanese Unexamined Patent Publication No. 1987-19271)
7), wound healing effect (JP-A-63-152309)
(Japanese Patent Application Laid-Open No. 17493/1983), antiallergic effect
(Japanese Patent Application Laid-open No. 1567-1987), reverse transcriptase inhibitory effect
No. 24, JP-A No. 1-29313) and glyoxalase inhibitory effect-anticancer effect (JP-A No. 63-21)
5628, JP-A-1-29313), and many other physiological activities have been revealed.

However, in recent years it has been revealed that PQQ has nephrotoxicity (Watanabe et al., Hiroshima J, Me.
d.

Sei:, Vol. 38, No. 1, 2, pp. 49-51 (1989) The development of safe PQQ derivatives with low toxicity and nephrotoxicity is desired.

The present inventors conducted nephrotoxicity and acute toxicity tests on various PQQ derivatives and found that oxazopyrroquinoline (OPQ) significantly reduced these toxicities.

OPQ can be obtained by reacting high-purity PQQ with glycine (1989 Chemical Society of Japan Abstracts 2m B54), or by reacting glycine with a culture solution containing PQQ (patent application 2001). -292459) is known.

However, in either method, in order to improve the reaction yield, it is necessary to add an excess amount of glycine to PQQ, and a production method using cheaper raw materials has been expected.

[Means and effects for solving the problem] As a result of various studies on cheaper manufacturing methods for OPQ, the present inventors added ammonia and formaldehyde to a solution containing PQQ and reacted in the presence of oxygen. The present invention was completed based on the discovery that it can be efficiently converted to OPQ.

That is, the present invention provides a method for producing oxazopyrroquinoline, which involves adding ammonia and formaldehyde to a solution containing pyrroloquinoline quinone, and producing oxazopyrroquinoline from the pyrroloquinoline quinone in the solution.
It is.

The PQQ-containing solution used in the present invention is as follows: ] Bacteria capable of assimilating methanol and capable of producing PQQ extracellularly are cultured in a medium using methanol as a carbon source. Culture solution 2 containing PQQ obtained by culturing in a medium using methanol as a carbon source Solution 3 obtained by dissolving PQQ-containing powder obtained by recovering PQQ-containing culture solution by acid treatment, salting-out treatment, etc. in water or buffer solution, high purity PQQ. , water or a solution obtained by dissolving it in a buffer solution.

Ammonia and formaldehyde are added to the PQQ-containing solution obtained in this manner and reacted with the PQQ contained in the solution under aerobic conditions. However, if the solution is a culture solution containing PQQ and already contains a sufficient amount of ammonia, only formaldehyde may be added.

Examples of the ammonia added at this time include ammonium salts such as ammonium sulfate and ammonium chloride, aqueous ammonia, and ammonia gas, but for practical purposes, aqueous ammonia and ammonium salts are used.

As formaldehyde, formaldehyde aqueous solution,
Examples include formaldehyde gas, but in practical terms,
An aqueous formaldehyde solution (formalin) is used.

The amount of these compounds added is not particularly limited as long as it is a stoichiometric amount or more, but in practice, the amount of ammonia added is 1 to 1000 with respect to the PQQ contained in the solution.
It is preferably 5 to 100 times the amount by mole, particularly preferably 5 to 100 times by mole.

Formaldehyde is preferably 1 to 3000 times the mole of PQQ contained in the solution, particularly 3
The amount is preferably 0 to 1000 times by mole.

The pH of the reaction solution is preferably in the range of 3 to 10, and preferably in the range of 4 to 10.
8 is particularly preferred. Also, as the reaction progresses, T)H
In order to maintain the pH of the reaction solution, ammonia, caustic potassium, caustic soda, etc. are added to lower the pH of the reaction solution.
It is necessary to adjust H.

The temperature is preferably from 15°C to 80°C, and for practical purposes, particularly 25°C.
~50'C is preferred.

In addition, there is no particular limit to the oxygen concentration in the reaction solution, but in order to complete the reaction in a short time, it is necessary to
m or more is preferable. For this purpose, measures such as aerating air, oxygen, or a mixed gas thereof, stirring the mixture, and increasing the pressure of the reaction tank are used. By reacting under such conditions, PQQ changes to OPQ.

When a PQQ-containing culture solution is used as the PQQ-containing solution, the reaction product solution obtained in this way contains solid substances such as bacterial cells and proteins denatured by formaldehyde. Solids are removed by conventional solid-liquid separation means such as filtration or centrifugation to obtain a supernatant.

When OPQ is generated at low pH such as p, H3-5,
Since the produced OPQ may exist as a precipitate in the reaction solution, it is necessary to make the pH of the reaction solution above neutral and once dissolve the produced OPQ, and then obtain a supernatant.

OPQ is separated and recovered from the obtained supernatant.

OPQ can be separated and collected from the supernatant by methods known per se. For example, ion exchange chromatography, gel filtration of concentrates, dissolution extraction of freeze-dried products, or affinity chromatography can be used.

Identification of OPQ requires elemental analysis, nuclear magnetic resonance spectroscopy,
Means such as infrared absorption spectroscopy and mass spectrometry are used.

Furthermore, OPQ can be quantitatively determined by high performance liquid chromatography.

[Examples] The present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 100 mg of PQQ and 80 mQ of distilled water were placed in a 20M beaker, and the pH of the solution was adjusted to pH 7.0 by adding sodium hydroxide to dissolve PQQ. In this beaker, 10 n of 37% formaldehyde aqueous solution and ammonium chloride 500 mg was added and dissolved, and the pH of the solution was adjusted to 7.0 by adding sodium hydroxide, and then distilled water was added to bring the total amount to 100 mg.

When this solution was reacted at room temperature for 24 hours while being stirred using a stirrer, 30.9 mg of OPQ was produced in the solution.

Example 2 Per pure water IQ, (NH4) 2S○43. Og, K
H2PO41.4g, Na2HPO,,2.1g, Mg
SO4・7H,00,2g, CaC1,・2H203
0mg, FeC6H507・H2O30mg, M
nCl24H205mg, ZnSO4・7H205mg
, CuSO4.5H, 00.5 mg and 8 ml of methanol were dissolved and 200 mQ of the solution was adjusted to pH and H of 7.1, and then put into a 19-volume Erlenmeyer flask, sterilized at 120'C for 20 minutes, and used as a culture medium. .

Hyphomicrobium methyloborum DSM
1869 was inoculated, and cultured with a rotary shaker at 220 revolutions/min at 30°C. This culture solution was used as a seed mother solution.

Per pure water IQ, (NH4) 2S 04 1,
Og, MgSO4・7H901, Og, KH2PO
Medium 15Q to which 41.4 g was added was placed in a 30Q culture tank and sterilized.

FeSO4・7H7H2O75 per 10 relatives of pure water
, ZnSO4・7H20150mg, Cacl,・
2H20150mg-NaC1150mg, MnSO4
・4~5H2045mg, H2BO33mg, Cu
SO4・5H201,5mg, COCl2・2H201
,5mg, KI 1.5mg, (NH4)6Mo
702.・A mineral solution containing 1.5 mg of 4H20 was sterilized.

After the temperature of the 30Q capacity culture tank decreased to 30'C, 10 mΩ of this mineral solution was added aseptically to the culture medium in the tank, and aqueous ammonia was added aseptically to adjust the pH of the culture solution to 6.8. Adjusted to.

To this culture tank, 150 ml of methanol and 200 ml of the above seed mother liquor were added aseptically, and the aeration rate was 10 Q/m i.
n, stirring number 30 rpm, temperature 30°C, culture pH 6
．． Culture was carried out while adding ammonia water so that the concentration of the cells was 8. As the bacteria proliferated, the methanol concentration in the culture solution decreased, but this was detected by analyzing methanol in the exhaust gas by gas chromatography, and it was found that the methanol concentration in the culture solution was 0.1 to 0.5. Methanol was supplied in an amount equal to % by weight.

By such a method, 3. The cells were cultured in an OQ culture tank for 10 days to obtain a culture solution 15Q containing 361 mg/2 of PQQ. To this PQQ-containing culture solution, 150 mQ of 37% formaldehyde aqueous solution was added, 45 marrow of 25% ammonia water was added, the pH was adjusted to 7°0, and the number of stirring was 25 Orp.
When the reaction was carried out at an aeration rate of 5 Q/min, the pH of the reaction solution decreased, so 25% ammonia water was added and the reaction was continued for 2 hours while controlling the pH in the reaction solution to 7.0. As a result, the oPQi degree in the reaction solution was 185 mg/Ω. The amount of 25% ammonia water added was 71 in total.

Example 3 In the same manner as in Example 2, Hyphomicrobium methyloborum DSM 1869 was cultured for 10 times in a 302 volume culture tank.
I did it for days. Thereafter, the culture solution was centrifuged, 760 g of sodium chloride was added to the obtained cultured synovial fluid, the pH was adjusted to 3.0 with hydrochloric acid, and the mixture was left standing at 5°C overnight, and the precipitate was removed by centrifugation. By collecting and drying it,
16.2 g of powder was obtained. 1 g of this powder contained 0.27 g of PQQ.

Dissolve 12 g of this powder in 300 mQ of distilled water, and
The pH was adjusted to 7 using H, and three 200 m
Q: Dispense into Erlenmeyer flask. This solution is 1070
It contained mg/Q of PQQ. .

In each Erlenmeyer flask, (1) 0.37%, (2) 1
%, (3) 3 to give a formaldehyde concentration of 2%.
Add 7% formaldehyde aqueous solution, add 0.5 g of ammonium chloride to each Erlenmeyer flask, and add Na
The p value was adjusted to H7 using OH, and the reaction was carried out at 25°C for 24 hours.

The content of ○PQ in the reaction product liquid is shown in Table 1.

Table 1 [Effects of the Invention] According to the present invention, oxazopyrroquinoline can be produced from PQQ using a cheaper method.

Claims (1)

[Claims] A method for producing oxazopyrroquinoline, which comprises adding ammonia and formaldehyde to a solution containing pyrroloquinoline quinone, and producing oxazopyrroquinoline from the pyrroloquinoline quinone in the solution.