JPS6047280B2 - Method for producing purine nucleotide derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a novel aminophenyl group-containing purineuc-10-reotide derivative, and more specifically, to a method for producing a ligand for a carrier for affinity chromatography. Separation and purification of various enzymes using

In addition, the nucleic acid derivative produced by the present invention is also expected to be used as a nucleic acid 15-based pharmaceutical intermediate. Conventionally, for the separation and purification of enzymes, ion exchange chromatography such as DEAE-cellulose columns, adsorption chromatography such as hydroxyapatite columns, and gel filtration chromatography such as Sephadex columns have been used.

However, in recent years, new principles have been developed that utilize the specific affinity between biological substances such as enzymes and enzyme inhibitors, hormones and carriers, and antigens and antibodies to selectively separate one biological substance from the other. As a result, affinity chromatography, or affinity chromatography, has come into widespread use. The greatest advantage of affinity chromatography is its highly specific adsorption, which cannot be seen with other chromatography methods, and its high separation ability. However, this advantage also brings about the disadvantage that the prepared carrier can only be used for the purification of a single substance and is not versatile. For example, affinity chromatography carriers immobilized with specific substrates for one enzyme reaction are extremely effective for purifying that enzyme, but cannot be used for other enzymes; It was necessary to synthesize a specific carrier for each case. Therefore, it has been desired to develop a carrier that can be used for general purposes while maintaining high selectivity in affinity chromatography.
Nicotinamide adenine dinucleotide (NAD+) is a common coagulant of dehydrogenases such as alcohol dehydrogenase and lactate dehydrogenase.

So N. Attempts have been made to create affinity chromatography using AD+ as two ligands, and thereby to separate and purify dehydrogenase enzymes from each other. However, when an enzyme solution is run through these columns, if the solution contains an enzyme substrate or phosphodiesterase2, the immobilized NAD+ may be converted to NADH (reduced form of NAD+) or pyridine or nicotine. Disadvantages include loss of binding capacity and irregularity of the column due to cleavage of the amide mononucleotide.
3. On the other hand, adenosine triphosphate (AT
P) is also a common substrate for kinases such as pyruvate kinase and acetate kinase. Therefore, an attempt was made to mutually separate and purify the kinases using an affinity chromatography column using ATP as a ligand, similar to NAD+. It is. However, since ATP is a substrate, sometimes immobilized ATP during chromatography is adenosine diphosphate (
ADP). For example, glycogen phosphorylase, threonine decarboxylase, siokinase, etc. are known as enzymes that use adenosine-5-phosphate (AMP) and 4C itself as inhibitors, cofactors, and substrates, but they are not very common.

However, AMP is a basic component of NAD+ or ATP molecules, generally has a strong specific adsorption ability with dehydrogenases and kinases, and is not a true cofactor or substrate, so it is difficult to perform chromatography as described above. The loss of the ligand is less than that of NAD+ and ATP, and it is considered to be an excellent ligand for affinity chromatography. The present inventor synthesized various cleotide derivatives and investigated their properties as ligands for affinity chromatography. The carrier for affinity chromatography that serves as a ligand for group-containing purine nucleotide derivatives has extremely excellent properties for the separation and purification of enzymes such as dehydrogenases and kinases, and that the derivatives reduce nitrophenyl-containing purine nucleotide derivatives. The nitrophenyl group-containing purine nucleotide derivative used as the starting material of the present invention is a compound represented by the general formula and (n=0, 1, 2 or 3). or a salt thereof, such as a derivative obtained by dehydration condensation of P-0H of purine nucleotide monophosphate ester with a nitrophenyl group-containing alcohol such as P-nitrophenol or P-nitrophenylcarbinol, or a derivative of purine nucleotide monophosphate ester. A derivative having a nitrophenyl-containing substituent such as a P-nitrophenyl group or a P-nitrobenzyl group in the purine base moiety is employed.

The reduction method in the present invention may be any conventional method, for example,
Pd-C. Alternatively, hydrogenation may be carried out using a reducing catalyst such as Raney nickel, or sodium thiosulfate may be used.

In some cases, the nitrophenyl group-containing purine nucleotide derivative used as the starting material of the present invention is a new compound. It can be easily synthesized using the θ method.

X: Halogen atom Y: Halogen atom, -HSO4 n: 0,1,2,3 To synthesize (■), for example, a tertiary amine such as triethylamine or sodium hydroxide is used as a base, and ethanol or methanol is used. Alternatively, I and (2) may be reacted in a water-soluble organic solvent such as dimethylformamide, or in some cases, I and (1) may be reacted in a water-soluble medium using a basic buffer.

To protect the 2″ and 3″ positions of ■, for example, acetone,
This can be carried out by a conventional 2''●3''-alkylidene reaction using 2,2-dimethoxypropane and P-toluenesulfonic acid. Phosphorylation of 5'' in (2) can be carried out by a conventional method using, for example, phosphorus oxychloride in triethyl phosphoric acid, or using cyanoethyl phosphoric acid or cyclohexylcarbodiimide. Deprotection can be carried out by a conventional method. However, in the case of deisopropylidenation, for example, PHL.
This can be done completely by heating in an aqueous solution of No. 5 at 70°C for 4 minutes. In addition, various derivatives can be similarly synthesized by replacing the sugar moiety of the ribonucleoside with, for example, xylose, arabinose, or glucose during the above synthesis reaction.

7 The derivative produced according to the present invention can be used as a carrier for affinity chromatography in the separation and purification of enzymes, for example, by the following method.

A carrier for affinity chromatography generally consists of an insoluble support, a spacer, and a ligand.

As the insoluble support, for example, agarose, Sepharose (manufactured by Pharmacia), cellulose, Sephadex (manufactured by Pharmacia), porous silica beads, and polyacrylic imide gel are used. Examples of spacers include Aba(CFI2)n-(n=1,2,...6,...
), or a polymethylene chain such as
2CH2CO primary peptide bond, 1- -゛-
Various spacers containing an ether bond such as 0H are used.

The binding of the insoluble support and the spacer can be carried out, for example, by the commonly used Bromsian method (Nature, Vol. 214, 13).
02, 1967) and the dicyclohexylcarbodiide method. The spacer and the ligand can be bonded by a conventional method, for example, between the P-amino group of the benzene ring of the ligand (the derivative produced in the present invention) and a functional group such as a carboxyl group, an oxirane group, a bromoacetyl group, or an active ester group of the spacer. (Journal of Biochemistry, Vol. 245, 3059)
Page, 1970, Biochemistry, Volume 11, 229
1 page, 197 details, method dispute in temple enzymology,
3 volumes). In addition, recently, many supports for affinity chromatography, in which an insoluble support and a spacer are integrated, have been commercially available. For example, agarose having an active ester group (Activated CH manufactured by Pharmacia)
-SephaOse4B) (introduced into the water-acid group of agarose) or EpOxy- manufactured by Pharmacia Co., Ltd.
Activated SephaOse 4B (in which hydroxyl groups of agarose are introduced) is available, and these can also be used.

As an example, ActivatedCh-SephaO
A reaction example using se4B (manufactured by Pharmacia) is shown below.

Alcohol dehydrogenase (ECI, l, l, l), lactet dehydrogenase (ECI, l, l, l), lactate dehydrogenase (
ECI, l, l, 27), malate dehydrogenase (
Dehydrogenases such as ECI, l, l, 37), glyceraldehyde 3-phosphate dehydrogenase (ECI, 2, l, l2), pyruvate kinase (EC2, 7, l
, 4O), acetate kinase (EC2, 7, 2, l), various enzymes such as phosphorylase, threonine decarboxylase, and siokinase can be separated and purified. Actual chromatography operations can be performed according to general methods, but in particular, NAD+, NADH (NAD+
(reduced form), elution using AMP, etc. is effective. Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 16 - Chlorpurine ribonucleoside 15.7y
．． ．． 19.5 V of P-nitrobenzylamine hydrochloride and 20.8 Y of triethylamine were added to 300 ml of ethanol and heated at 60° C. for 1 hour with stirring. The reaction solution became a homogeneous and transparent solution. After cooling the reaction solution, it was left in the refrigerator overnight. Separate the generated crystals and make approximately 100 mt of sake liquid.
After concentrating to
-Puribonucleoside 12.1y (yield: 55%)
I got it. The melting point of the crystal was 129-130°C, and its structure was confirmed by nuclear magnetic resonance absorption spectrum (NMR) in deuterated dimethyl sulfoxide. In other words, the absorption by the two hydrogen atoms of the purine ring is δ = 8.52, 8
．． 27 as a singlet, the absorption by the four hydrogen atoms of the benzene ring is δ = 7.64, and 8.23 is the doublet, the absorption by one hydrogen atom at the 1″ position of the ribose is δ = 5.9.
Observe each on 7th. The results of elemental analysis of the crystal are shown below. Analysis value C5O. 62%, H4.62%, N2O. 7
5% calculated value (as Cl7Hl6O6N6) C5
O. 75%, H4.5l%, N2O. 89%N6-(p
-nitrobenzyl)-purine ribonucleoside 5.0
7y12,2-dimethoxypropane 3.5ml and p-
21.7y of toluenesulfonic acid and 100m of dry acetone
1, stirred overnight at room temperature, and reacted! The liquid was weighed down and poured into saturated ice water, and the solution was stirred for about 3 minutes at room temperature.

The resulting precipitate? The mixture was separated, thoroughly washed with water, and dried to obtain 4.30y of white N6-(p-nitrobenzyl)-2'',3''-isopropylidene purine ribonucleoside.
No starting material was found in the crystals by 3 silica gel thin layer chromatography.

The melting point of the crystal was 180-181°C, and its structure was confirmed by NMR in deuterated dimethyl sulfoxide. That is, two 40 hydrogen atoms on the purine ring at δ = 8.44s8.27, four hydrogen atoms on the benzene ring at δ = 8.21 and 7.60, and ribose at δ = 6.19. One hydrogen atom was observed in ``, and six hydrogen atoms of the isopropylidene group were observed at δ = 1.34 and 1.56. The results of elemental analysis of the crystal are shown below. Analysis value C53.95%,
H5.28%, Nl8.85% calculated value (C2OH2.
(as O6N6) C54.29%, H5. Ol%
,Nl9. OO%″N6-(p-nitrobenzyl)-2
',3''-isopropylidenepurin ribonucleoside 3
．． Add 6y to 15ml of triethyl phosphoric acid and lower the temperature to 0°C.
While maintaining the temperature, 3.87 fI of phosphorus oxychloride was added dropwise, and stirring was continued for 3.5 hours.

The reaction solution became homogeneous, and analysis by silica gel thin θ layer chromatography showed that the raw material creoside had completely disappeared. After adding the reaction solution to 200 ml of ice water,
Adjust the pH to 8.0 with a 4N caustic soda aqueous solution,
Stirring was continued for an hour. The reaction solution was then diluted with hydrochloric acid to PHL. 5 and heated at 70° C. with thorough stirring in the evening.
This operation completely removed isopropylidene. After adjusting the pH of the reaction solution to 4.5 with a 4N aqueous solution of caustic soda, activated carbon was added to the reaction solution until no ultraviolet absorbing substance was present. Approximately 100 ml of activated carbon was packed in a column, washed thoroughly with water to desalt it, and then filled with 100ml of activated carbon containing 10% ethanol. After eluting the nucleotides adsorbed on activated carbon with approximately 1.5 e of precipitated ammonia water, the nucleotides were eluted under reduced pressure at 40°C or below for approximately 100 ml of aqueous ammonia.
After concentrating to TrLt and decolorizing with a small amount of activated carbon, the Oki liquid was further concentrated to about 20 ml, a small amount of ethanol was added, and the mixture was left in the refrigerator. The white crystals produced are separated from each other, washed with a small amount of cold ethanol-water mixed solvent, and dried to form N6-(
p-Nitrobenzyl)-5-adenylic acid sodium salt 9
83 mg was obtained. The crystals were analyzed by cellulose thin layer chromatography (Avicel SF, manufactured by Funakoshi Pharmaceutical Co., Ltd.), and were found to have RfO. RfO. with 6λ developing solvent B (n-butyl alcohol diacetic acid: water = 5:2:3). 68 single spots were given. Note that under the same conditions, AMP was developed with developing solvent A at RfO. RfO with 2N developing solvent B
．． It showed 29. Ultraviolet absorption is PH7. λ in an aqueous solution of O
MaX272rlml (δMax239OO). Its structure in heavy water was found to be δ=8.3λ8.
Absorption by purine ring hydrogen atom at 05, benzene ring hydrogen atom at δ = 7.94, 7.35, hydrogen atom at 1″ position of ribose at δ = 6.0, hydrogen atom at benzyl position at δ = 4.70 It was confirmed that
Pd-ClOOmg was added and hydrogenation was carried out at normal pressure.

After gas absorption has stopped, separate the reaction solution. The liquid was concentrated under reduced pressure. Cellulose thin layer chromatography analysis revealed that the raw material had completely disappeared. The concentrated residue was dissolved in a small amount of water and left in the refrigerator. The resulting crystals were separated and dried to obtain N6-(
387m9 of sodium salt of p-aminobenzyl)-5''-adenylic acid was obtained.The crystals showed a single spot at FRfO.44 in developing solvent A and RfO.37 in developing solvent B in cellulose thin layer chromatography analysis. The ultraviolet absorption was λMaX27Orlml at pH7.O.
(δMax2O8OO), λMax238nml (δM
axll6OO). Its structure was determined by NMR analysis in heavy water with δ = 8.32, purine ring at 8.06, and δ = 7.0.
This was confirmed by the observation of absorption by hydrogen atoms at the benzene ring at 4 and 6.66, the ribose position at δ=5.99, and the benzyl position at δ=4.46. Activate
dCH-SephalOse4B (manufactured by Pharmacia)
5y was swollen and washed with 1mM hydrochloric acid 1e.

163 m9 of N6-(p-aminobenzyl)-5''-adenylic acid sodium salt was dissolved in 12 mt of 0.1 M sodium bicarbonate buffer and the pH was adjusted to 80. The solution was cooled to 4°C and Swelled and washed Ac
Add tivatedCH-SephaOse4B,
Slow stirring was continued for 3 hours. Separate the resin and remove the resin.
1M sodium bicarbonate buffer (PH=8.0) 200
After washing with 0.1M Tris hydroxymethyliminomethane (Tris) buffer (PH=8.0) 200TT
It was soaked in tt and left at room temperature for one hour, and then removed. 0.5r
V1 salt-containing 50■ false acid soda buffer 200ml1105
After washing with 200ml of 50rnM Tris buffer containing M NaCl and repeating this washing three times, the prepared resin was
rr1Mα-sodium glycerophosphate buffer (PH=
6.8) and stored at below 8°C.

Covalently bonded to the resin. N6-(p-aminobenzyl)-
The amount of 5″-adenylic acid is about 2 to 1 TrLL of resin.
It was 4 micromol. 7CW1 x 7.5TIT of 3ml of carrier for Affinity chromatography containing N6-(p-aminobenzyl)-5''-adenylic acid as a ligand.
100111M sodium phosphate buffer (PH=7.0) was applied to the column to equilibrate the column.

Glucose-6-phosphate dehydrogenase (obtained from yeast, EC.l,l,l,49), alcohol dehydrogenase (obtained from yeast, EC.l,l)
, l, l), chlorate dehydrogenase (obtained from puta muscle, EC. l, l, l, 27), phosphorylase b (obtained from rabbit muscle, EC. 24, l, l)
) was dissolved in 100rT1M sodium phosphate buffer (PH7.O) 200Pe, and this enzyme mixture was charged to the column at a flow rate of about 2mt/8.
First, 100rT 1M sodium phosphate buffer (P
H=7.0) was flowed, the eluate was collected in 1.6 ml portions, and then 25 ml of a solution of 1 mM NAD+ added to the above buffer was flowed. Furthermore, 1mM NADH was added to the above buffer solution and 25mL was allowed to flow. Finally, add 25% ethylene glycol and 1M potassium chloride to the above buffer solution for 25 minutes.
ml was run at room temperature. The enzyme activity of each fraction was measured, and the results are shown in FIG. It can be seen from FIG. 1 that the enzyme mixture was separated with extremely good resolution. The activity of phosphorylase b was slightly decreased due to the high concentration of ethylene glycol. Each enzyme activity was determined by commonly used methods. That is, glucose-6-phosphate dehydrogenase (C6PDH), phosphorylase b, is a
From the increase in absorbance at 0r1m, alcohol dehydrogenase (ADH), lactate dehydrogenase (
LDH) was measured from the increase and decrease in absorbance of NADH at 340r1m (Methods of Enzymatic Analysis Volumes 1 and 2).

Example 2 500 ml of adenosine-5″-phosphoric acid-p-nitrophenyl ester sodium salt was mixed with 1 part of water and methanol.
1 dissolved in 50 mt of mixed solvent, 5% Pa-ClOOm9
was added to perform hydrogenation at normal pressure.

After the gas absorption was completed, analysis using cellulose thin layer chromatography 7 revealed that the p-nitro compound had completely disappeared. After filtering the reaction solution, the solution was concentrated under reduced pressure, and the residue was mixed with 2 ml of water.
0 ml and lyophilized to obtain 450 ml of sodium salt of adenosine-5''-phosphate-p-aminophenyl ester as a white powder.The crystals were analyzed by cellulose thin layer chromatography using RfO in developing solvent A.
．． 55, RfO. in developing solvent B. It showed 32. Ultraviolet absorption is entered at PH = 7.0 MaX26Orlml (δ
Maxl52OO), λMaX236llml(δMa
xl26OO). Its structure was determined by NMR in heavy water, with purine rings at δ = 8.12 and 8.14, and δ = 6.74.
, benzene ring at 6.44, ribose 1 at δ=6.00
This was confirmed by the observation of absorption by the hydrogen atom at the `` position.ActivatedCH-SephaOse4B (
(manufactured by Pharmacia) 4y was swollen and washed with 800 ml of 1 mM hydrochloric acid.

Adenosine-5″-phosphate-P-aminophenyl ester sodium salt (130ml) was dissolved in 8ml of 0.1M sodium bicarbonate buffer, and the pH was adjusted to 8.0.The solution was cooled to 4°C, and the above cellulose was dissolved. After stirring slowly for 3 hours, the mixture was treated in the same manner as in Example 1 to obtain a carrier for affinity chromatography using adenosine-5''-phosphoric acid-P-aminophenyl ester as a ligand. The amount of ligand covalently bonded to the resin is
It was about 2 to 4 micromoles per 1 part. The carrier thus obtained gave good results in enzyme separation experiments similar to those in Example 1 (experiments conducted at lower temperatures than in Example 1).

[Brief explanation of the drawing]

FIG. 1 shows the results of enzyme separation by affinity chromatography in Example 1.

Claims (1)

[Claims] 1 A nitrophenyl group-containing purine nucleotide represented by the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (n = 0, 1, 2, or 3) 1. A method for producing an aminophenyl group-containing purine nucleotide derivative for a ligand of a carrier for affinity chromatography, which comprises subjecting the derivative to a reduction reaction to convert its nitrophenyl group into an amino group.