JP2637997B2 - Process for producing dinucleoside or nucleoside polyphosphate or derivative thereof - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a dinucleoside or nucleoside polyphosphate or a derivative thereof which is important as a drug or a raw material thereof.

(Prior Art) Dinucleoside polyphosphate or a derivative thereof has an inhibitory action on adenylate kinase [J. Biol. Chem., 248 , 1121 (197
3)], the inhibitory effect of terminal deoxynucleotidyltransferase [Biochemist
ry) 26 , 2033 (1987)], the inhibitory effect of protein kinases [Achieves of Biochemistry and
Bio-Physics (Arch.Biochem.Biophys.) 236, 441
(1985)] and can be used as pharmaceuticals and pharmaceutical raw materials. Nucleoside polyphosphate is used as a raw material for synthesizing dinucleoside polyphosphate [European Journal of Biochemistry (Eur. J. Biochem.) 57 , 197 (1975)]. It is.

Dinucleoside polyphosphate or a derivative thereof (hereinafter referred to as Npn
N '. ) Can be obtained from the European
The Journal of Biochemistry states that adenosine 5'-monophosphate (AMP) is activated with diphenyl phosphorochloridate to produce adenosine 5'-monophosphate.
The method of condensing with tetraphosphoric acid is also known as adenosine-5 '
-A method of activating triphosphate (hereinafter referred to as ATP) with diphenyl phosphorochloridate and condensing it with ATP has been reported, and adenosine-5'-diphosphate was used in the above biochemistry. A method of activating with morpholidate and condensing it with radioactive ATP has been reported.

In addition, nucleoside polyphosphate or its derivative (hereinafter referred to as
Npn. ) As a way to get the European
The Journal of Biochemistry reports a method of synthesizing adenosine-5'-tetraphosphate by activating ATP with diphenyl phosphorochloridate and condensing it with orthophosphoric acid.

On the other hand, the magazine 126, 135 (1982), aminoacyl -tRN
It has been reported that diadenosine tetraphosphate is synthesized from ATP and amino acids by A synthase, which synthesizes dinucleoside polyphosphate and nucleoside polyphosphate having a phosphate group of tetraphosphate or more. is not.

(Problems to be Solved by the Invention) In the above-mentioned conventional method, an expensive reagent is required for activating adenosine-5'-phosphate or a derivative thereof, and NpnN 'or When synthesizing Npn, strict anhydrous conditions were required and the operation was complicated. Also, the yield of NpnN 'or Npn to be synthesized is low,
Therefore, it was difficult to isolate and purify NpnN 'or Npn.

(Means for Solving the Problems) Therefore, the present inventors have conducted intensive studies in order to improve the above-mentioned drawbacks, and as a result, ATP has a derivative (hereinafter referred to as NTP) and a phosphate chain. Anhydride (hereinafter referred to as polyphosphoric acid) or nucleoside having 4 or more phosphate groups
When 5'-polyphosphoric acid or a derivative thereof (hereinafter referred to as N'pm) and an amino acid were used as reaction substrates, and aminoacyl-tRNA synthetase was allowed to act thereon, NpnN 'was produced inexpensively and easily in high yield. Alternatively, they have found that Npn can be manufactured, and have completed the present invention.

That is, the first invention relates to adenosine-5'-triphosphate or a derivative thereof, a chain anhydride of phosphoric acid or a nucleoside-5'-polyphosphate having four or more phosphate groups or a derivative thereof, and an amino acid. A method for producing a dinucleoside polyphosphate or a derivative thereof, characterized in that dinucleoside polyphosphate or a derivative thereof is produced by using as a reaction substrate a catalytic action of aminoacyl-t-RNA synthetase.

Further, the second invention uses adenosine-5'-triphosphate or a derivative thereof, a linear anhydride of phosphoric acid, and an amino acid as reaction substrates, and uses an aminoacyl-t-RNA synthetase to act as a nucleoside polyphosphoric acid. A gist of the present invention is a method for producing a nucleoside polyphosphoric acid or a derivative thereof, which comprises producing an acid or a derivative thereof.

The derivative of ATP referred to in the present invention is an aminoacyl t-RNA.
A compound that can be converted to NpnN 'or Npn by the action of a synthase. Such ATP derivatives include N6-alkylated adenine ring, carboxyalkylated, benzoylated, carboxybenzoylated derivatives, halogenated derivatives of adenine ring, hydroxy derivatives, deamine derivatives,
Ribose includes 2'-deoxy derivatives, 3'-deoxy derivatives, 2 ', 3'-dideoxy derivatives, deoxyamino derivatives, and deoxyhalo derivatives.

The derivative of nucleoside-5'-polyphosphoric acid referred to in the present invention is a derivative in which the base portion of the nucleoside is, for example, alkylated, acylated, or halogenated, or the ribose portion is, for example, deoxylated, deoxyaminated. What is being done.

NpnN 'or Npn as used in the present invention refers to an NTP-derived AMP or a derivative thereof, in which a phosphate group and polyphosphate or a phosphate group of N'pm are bonded to each other by an anhydride. When used as a reaction substrate, diadenosine pentaphosphate, which is a diadenosine polyphosphate, is used as NpnN '.
As a result, adenosine tetraphosphate, which is adenosine polyphosphate, is produced.

If further the ATP and guanosine-5'-tetraphosphate as a reaction substrate, adenosine guanosine phosphorus pentachloride acid (Ap ₅ G) is formed as NpnN '.

The aminoacyl-tRNA synthetase used in the present invention may be any as long as it has the ability to synthesize NpnN 'or Npn, but lysyl-tRNA synthetase derived from Escherichia coli, histidyl-tRNA synthetase, phenylalanyl -TRNA synthetase, lysyl derived from yeast -tR
Specific examples include NA synthase, phenylalanyl-tRNA synthetase, fusylium-derived phenylalanyl-tRNA synthetase, leucyl-tRNA synthetase derived from thermostable bacteria such as Bacillus stearothermophilus, and phenylalanyl-tRNA synthetase derived from sheep liver cells. An example can be given. Examples of amino acids include tyrosine, alanine, leucine, isoleucine, phenylalanine, methionine, lysine, serine, valine, asparagine, aspartic acid, glycine, glutamine, glutamic acid,
Α-amino acids such as cysteine, threonine, tryptophan, histidine, proline, arginine and the like can be mentioned, and any of L-form and D-form may be used.

At this time, an aminoacyl-tRNA synthetase having amino acid specificity is used. For example, leucine-tRNA synthetase is used as leucine specificity.

To produce NpnN 'or Npn in the present invention, for example, NTP, polyphosphate or N'pm amino acid,
The reaction may be performed in the presence of an aminoacyl-tRNA synthetase. The reactor used at this time may be any reactor as long as the reaction can proceed smoothly. The amount of the enzyme, the concentration of the substrate solution, the pH, the supply speed of each substrate,
The size and shape of the reactor may be selected depending on the reaction temperature and the like. As the shape of the reactor, for example, a membrane reactor or a column reactor can be used. Among them, the membrane reactor has low molecular reaction products,
It can be used particularly effectively. At this time, since the enzyme is a high molecular substance, each enzyme can be used as it is in the reactor.

In the case of a column type reactor, the enzyme to be used may be a suitable carrier such as a derivative of a polysaccharide such as cellulose, dextran, agarose, polystyrene, an ethylene-maleic acid copolymer, or a cross-linked polyacrylamide. Derivatives of natural vinyl polymers, L-alanine, L-glutamic acid copolymers, derivatives of polyamino acids or amides such as polyaspartic acid, and derivatives of inorganic substances such as glass, alumina, hydroxyapatite, etc. It may be adsorbed and packed in a column as a so-called immobilized enzyme for use.

The above-mentioned reactor has been described on the assumption that it is operated continuously. However, other reactors can be used based on the concept as described above, and in some cases, a batch-type reactor is used. It can also be used to react by operating a batch.

Next, the reaction conditions will be described by taking as an example the case where NpnN 'or Npn is produced in a batch type reactor. Amino acids are 1 μm or more, especially 10 μm or more, and further 0.1 mM or more.
P is 10 μm or more, especially 1 mM or more, further 10 mM or more, polyphosphoric acid or N′pm is 10 μm or more, especially 1 mM or more, further 10 m
It is preferable to add NTP at a concentration of at least M, and when NpnN 'is produced using polyphosphoric acid, NTP is used in an amount of at least equivalent to polyphosphoric acid, especially at least 1.5 equivalents, more preferably at least 2 equivalents,
When n is produced, it is desirable to add polyphosphoric acid in a concentration of at least equivalent to NTP, particularly at least 1.5 equivalent, more preferably at least 2 equivalent. In the case of producing NpnN 'using N'pm, it is desirable to add N'pm at a concentration of at least equivalent, especially at least 1.5 equivalent, and more preferably at least 2 equivalent to NTP. The method of addition is not particularly limited, and may be added all at once at the start of the reaction, or may be added in portions. In addition, pyrophosphatase can be added to promote the reaction smoothly, or metal ions such as magnesium ion, manganese ion, calcium ion, cobalt ion, and cadmium ion can be added. The pH of the reaction varies depending on the enzyme, but is generally around neutral, that is, in the range of pH 5 to 11, preferably pH 6 to 8, and can be adjusted with a buffer. As the buffer, a normal buffer suitable for these pHs can be used. The reaction temperature is not particularly limited as long as the reaction does not deactivate and the reaction proceeds smoothly, but is preferably in the range of 20 ° C to 50 ° C.

From the reaction product thus obtained, NpnN 'or Npn can be easily isolated by a widely used purification method such as ion exchange chromatography.

(Examples) Next, the present invention will be described specifically with reference to examples.

Reference Example 1 6 kg of Bacillus stearothermophilus UK788 (Microtechnical Laboratories No. 5141) cells were suspended in twice the volume of 100 mM Tris / hydrochloric acid buffer (pH 7.5), and the cells were suspended using a Dynomill. After crushing, insolubles were removed by centrifugation to obtain a crude extract containing leucine-tRNA synthetase specific to leucine. 50 mM Tris buffer (pH 7.5) containing 5 mM mercaptoethanol, 2 mM sodium ethylenediaminetetraacetate and 0.1 mM phosphophenylsulfonyl fluoride in advance
The crude extract was passed through a column packed with Martrex gel blue A (manufactured by Amicon) equilibrated in step 1. The solution was obtained by adding potassium chloride to the above buffer, and the linear velocity was 60 cm · h.
Elution with ^-1 eluted leucine-tRNA synthetase. This group was collected, concentrated and desalted, resulting in about 70%
A crude enzyme solution containing leucine-tRNA synthetase specific for leucine was obtained at a yield of 1. All the above operations were performed at 4 ° C.

Reference Example 2 5 kg of Escherichia coli K12 strain (ATCC10798) was suspended in twice the volume of 100 mM Tris / hydrochloric acid buffer (pH 7.5).
After disrupting the cells using a dynomill, insolubles were removed by centrifugation to obtain a crude extract containing lysyl-tRNA synthetase specific for lysine. Contains 5 mM mercaptoethanol and 2 mM sodium ethylenediaminetetraacetate in advance
The above crude extract was passed through a column packed with Martrex gel blue A (manufactured by Amicon) equilibrated with 50 mM Tris buffer (pH 7.5), and potassium chloride was added to the above buffer. When eluted at a speed of 60 cm · h ⁻¹ , lysyl-tRNA synthetase eluted. Collect this category, concentrate,
As a result of desalting, a crude enzyme solution containing lysyl-tRNA synthetase specific to lysine was obtained at a yield of about 65%. All the above operations were performed at 4 ° C.

Example 1 20 mM ATP, 10 mM tripolyphosphate, 2.5 mM L-leucine, 10 mM magnesium chloride, leucyl-tRNA synthetase (Reference Example 1) 35 units / ml, pyrophosphatase (Boehringer Mannheim) 2 units / ml, Hepes Buffer solution (pH 7.
6) A reaction solution consisting of 100 mM was reacted at 40 ° C. for 2 hours.

The obtained reaction product was analyzed by high performance liquid chromatography using a Novapack _C18 (Waters) column, and it was found that 6 mM (60% yield) of diadenosine pentaphosphate was formed. The fact that the reaction product was diadenosine pentaphosphate was fixed by phosphorus nuclear magnetic resonance spectrum.

Example 2 Leucyl-tRNA synthetase and pyrophosphatase were converted to CN
B _r - immobilized on activated Sepharose 4B (Pharmacia, Inc.). Forty units of immobilized leucyl-tRNA synthetase and 20 units of immobilized pyrophosphatase were packed in one column.

Next, a raw material solution consisting of 8 mM ATP, 4 mM tripolyphosphate, 2.5 mM L-leucine, 10 mM magnesium chloride, and 100 mM Hepes buffer (pH 7.6) was placed at 0.6 ml / hr from the top of the immobilized enzyme-packed column.
And the reaction product was continuously withdrawn from the lower end of the column.

When the obtained reaction product was analyzed by the same method as in Example 1, 26 mM (yield 65%) of diadenosine pentaphosphate was formed.

Example 3 ATP 10 mM, tetrapolyphosphate 5 mM, L-lysine 1 mM, magnesium chloride 5 mM, lysyl-tRNA synthetase (Reference Example 2) 25 units / ml, pyrophosphatase 2 units / ml, Hepes buffer (pH 7.6) ) A reaction solution consisting of 100 mM was reacted at 40 ° C for 25 hours.

When the obtained reaction product was analyzed by the same method as in Example 1, it was found that 2.5 mM (yield 50%) of diadenosine hexaphosphate was formed. The reaction product was identified as phosphorus adenosine hexaphosphate by phosphorus nuclear magnetic resonance spectrum.

Example 4 N ⁶ - carboxyethyl ATP20mM, tripolyphosphate 10 mM, L
-Leucine 2 mM, magnesium chloride 5 mM, leucyl-tRNA synthetase 3 units / ml, pyrophosphatase 1 unit / m
l, Reaction solution consisting of 100 mM Hebes buffer (pH 7.5) at 40 ° C
The reaction was performed for 40 hours.

When the obtained reaction product was analyzed by the same method as in Example 1, it was found that 4.5 mM (yield 45%) of bis (N ⁶ -carboxyethyladenosine) pentaphosphate was formed. The reaction product is bis - were identified by phosphorus nuclear magnetic resonance spectrum to be a (N ⁶ carboxyethyl adenosine) v phosphoric acid.

Example 5 ATP 10 mM, adenosine 5'-tetraphosphate 10 mM, L-leucine 2.5 mM, magnesium chloride 10 mM, leucyl-tRNA synthetase 35 units / ml, pyrophosphatase 2 units / ml, Hepes buffer (pH 7. 5) The reaction solution consisting of 100 mM was
Allowed to react for hours.

When the obtained reaction product was analyzed by the same method as in Example 1, 5.7 mM (57% yield) of diadenosine pentaphosphate (Ap ₅ A) was produced. The reaction product was identified as phosphorus adenosine pentaphosphate by phosphorus nuclear magnetic resonance spectrum.

Example 6 Leucyl-tRNA synthetase and pyrophosphatase were converted to CN
B _r - immobilized on activated Sepharose 4B (Pharmacia, Inc.). 50 units of immobilized leucyl-tRNA synthetase and 20 units of immobilized pyrophosphatase were packed in one column.

Next, ATP 2 mM, guanosine 5'-tetraphosphate (Gp ₄ ) 10 m
A raw material solution consisting of 2 mM M, L-leucine, 5 mM magnesium chloride, and 100 mM Hepes buffer (pH 7.6) was supplied at a flow rate of 0.3 ml / hr from the upper end of the immobilized enzyme-packed column, and the reaction product was supplied from the lower end of the column. Were continuously withdrawn.

When the obtained reaction product was analyzed by the same method as in Example 1, 1.3 mM (65% yield) of adenosine anosine pentaphosphate (Ap ₅ G) was produced. The reaction product was identified as Ap ₅ G by phosphorus nuclear magnetic resonance spectrum.

Example 7 ATP10mM, guanosine-5'phosphorus pentachloride acid (Gp ₅₎ 15mM, L
A solution consisting of 3 mM lysine, 5 mM magnesium chloride, 33 units / ml lysyl-tRNA synthetase, 2 units / ml pyrophosphatase, and 100 mM Hepes buffer (pH 7.4) was reacted at 40 ° C. for 30 hours.

When the obtained reaction product was analyzed by the same method as in Example 1, it was found that 5.5 mM (yield 55%) of adenosine anosine hexaphosphate (Ap ₆ G) was produced. The reaction product, Ap ₆ G, was identified by phosphorus nuclear magnetic resonance spectrum.

Example 8 N ⁶ - carboxyethyl ATP (CE-ATP) 5mM, adenosine 5'-tetraphosphate 10 mM, L-leucine 2 mM, magnesium chloride 5 mM, leucyl -tRNA synthetase 25 Yunitsuto / ml, pyro phosphatase A synthetase 1 Unit / ml, Hepes buffer (pH7.6) 100mM
Was reacted at 40 ° C. for 15 hours.

The obtained reaction product was analyzed by the same method as in Example 1. As a result, 2.7 mM (yield 54%) of N ⁶ -carboxyethyldiadenosine pentaphosphate (CE-Ap ₅ A) was found to be produced. The reaction product was identified by phosphorus nuclear magnetic resonance spectrum to be a CE-Ap ₅ A.

Example 9 20 mM ATP, 50 mM tripolyphosphate, 2.5 mM L-leucine, 50 mM magnesium chloride, 1.5 units of leucyl-tRNA synthetase
/ ml, 0.2 units / ml pyrophosphatase, 100 mM Hepes buffer (pH 7.5) was reacted at 40 ° C for 70 hours.

When the obtained reaction product was analyzed in the same direction as in Example 1, 16.8 mM (yield 84%) of adenosine-5 'was obtained.
-Tetraphosphate was formed.

The reaction product was identified as adenosine-5'-tetraphosphate by phosphorus nuclear magnetic resonance spectrum.

Example 10 Leucyl-tRNA synthetase and pyrophosphatase were converted to CN
B _r - immobilized on activated Sepharose 4B (Pharmacia, Inc.). 15 units of immobilized leucyl-tRNA synthetase and 10 units of immobilized pyrophosphatase were packed in one column.

Next, a raw material solution containing 6 mM ATP, 20 mM tripolyphosphate, 3 mM L-leucine, 30 mM magnesium chloride, and 100 mM Hepes buffer (pH 7.6) was placed at 0.3 ml / hr from the top of the immobilized enzyme-packed column.
And the reaction product was continuously withdrawn from the lower end of the column.

When the obtained reaction product was analyzed by the same method as in Example 1, 5.3 mM (yield 88%) of adenosine-5'-tetraphosphate was found to be formed.

Example 11 ATP 5 mM, tripolyphosphate 35 mM, L-lysine 2 mM, magnesium chloride 40 mM, lysyl-tRNA synthetase 2 units / ml, pyrophosphatase 1 unit / ml, Hepes buffer (pH 7.
The reaction solution consisting of 7) was reacted at 40 ° C. for 20 hours.

When the obtained reaction product was analyzed by the same method as in Example 1, it was found that 3.6 mM (yield 72%) of adenosine-5'-tetraphosphate was formed.

Example 12 N ⁶ - carboxyethyl ATP4mM, tetrapolyphosphate 30 mM,
L-leucine 2 mM, magnesium chloride 30 mM, leucyl-tRNA
Synthetic enzyme 10 units / ml, pyrophosphatase 2 units
/ ml, 100 mM Hepes buffer (pH 7.5) at 40 ℃
For 30 hours.

When the obtained reaction product was analyzed by the same method as in Example 1, 2.7 mM (yield 68%) of N ⁶ -carboxyethyladenosine-5′-pentaphosphate was formed. Reaction product N ⁶ - were identified by phosphorus nuclear magnetic resonance spectrum to be a carboxyethyl-5'phosphorus pentachloride acid.

(Effects of the Invention) According to the present invention, NpnN 'or Npn can be easily produced at a high yield, inexpensively, and particularly in an aqueous solution.

Claims (2)

(57) [Claims] 1. A reaction substrate comprising adenosine-5'-triphosphate or a derivative thereof, a chain anhydride of phosphoric acid or a nucleoside-5'-polyphosphate having four or more phosphate groups or a derivative thereof, and an amino acid Aminoacyl t
-A method for producing a dinucleoside polyphosphate or a derivative thereof, which comprises producing a dinucleoside polyphosphate or a derivative thereof by a catalytic action of an RNA synthase. 2. A method according to claim 1, wherein adenosine-5'-triphosphate or a derivative thereof, a linear anhydride of phosphoric acid and an amino acid are used as reaction substrates, and nucleoside polyphosphate or a nucleoside polyphosphate thereof is catalyzed by aminoacyl-t-RNA synthetase. A method for producing a nucleoside polyphosphate or a derivative thereof, which comprises producing a derivative.