JPS6210690B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula (wherein, R is a divalent hydrocarbon group which may contain a substituent, and Y is a phosphoric acid residue selected from monophosphoric acid, diphosphoric acid, and triphosphoric acid) An adenine nucleotide derivative or a salt thereof in which the amino group at the 6-position of the nucleus is carbamoylated is chemically bonded via its terminal amino group to a polysaccharide or a polymer substance having a repeating structural unit with a carboxyl group bonded as a side chain. The invention relates to a polymeric material bound to adenine nucleotides. The substance according to the present invention is novel and can be used as an adsorbent in affinity chromatography, a reaction medium in enzyme reactions, etc. due to the action of the adenine nucleotide derivative represented by the formula (I) bound as a ligand. In the enzyme industry, there is a strong demand for the development of polymeric substances bound to adenine nucleotides as adsorbents for so-called affinity chromatography and reaction media in enzyme reactions, which are used for the separation and purification of necessary enzymes. However, all of the methods proposed so far have been accompanied by significant difficulties in preparation, have poor yields of the target product, and
Furthermore, in actual use, there are drawbacks such as extremely low affinity for enzymes and the bonded adenine nucleotide being easily detached during use, so that a satisfactory product has not yet been obtained. For example, among polymeric substances bound to adenine nucleotide derivatives as ligands, the one represented by the following formula is known to be the most suitable for the above-mentioned purpose [M.
Lindberg et al., Eur.J.Biochem., 53, 481
(1975)]. (In the formula, Y is monophosphoric acid, diphosphoric acid, or triphosphoric acid, and [SP] represents sepharose.) This polymeric substance shows good affinity for various kinases and is stable under actual usage conditions. Although it has ^the characteristic of However, in one reaction step, it takes a long time of 5 days or more to complete the reaction, so it cannot be said to be of practical use. The inventors of the present invention have conducted intensive research to develop a product that has excellent usability properties and is easy to prepare.
The inventors have discovered that a polymeric substance containing the adenine nucleotide derivative represented by the general formula (I) as a ligand is suitable for this purpose, and have completed the present invention. In the present invention, the adenine nucleotide derivative represented by the general formula (I) that is bound to a polymeric substance as a ligand is a novel compound that has not been described in any literature, and is produced as shown below. In addition,
In the above general formula, specific examples of the divalent hydrocarbon group R include ethylene, propylene, butylene, amylene, hexylene, heptylene, octylene,
_C2 - _C20 , preferably _C4 - _C8 alkylene such as dodecylene; _C4 - _C20 , preferably _C4 - _C12 alkenylene such as butenylene, hexenylene, dodecenylene; arylene groups such as phenylene, xylylene, etc. These include halogen atoms such as chlorine, bromine, and iodine, alkoxy groups such as methoxy, acyl groups such as acetyl and acryloyl,
It may be substituted with substituents such as alkoxycarbonyl such as acetoxy and nitro groups.
Examples of these salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts, alkaline earth metal salts such as barium salts, other metal salts such as aluminum salts, and ammonium salts. . To produce the adenine nucleotide derivative represented by the general formula (I), first the corresponding
Using AMP, ADP or ATP as a raw material, diisocyanate (NCO-R-
NCO) (wherein R is the same as above).
In this case, the reaction temperature is 30-100℃, preferably 50℃
The temperature is ~80°C, and the solvent used is an inert solvent that dissolves the starting material, such as hexamethylphosphoramide, dimethylsulfoxide, or dimethylformamide, and that does not react with the diisocyanate. The amount of diisocyanate to be used per 1 mol of raw material adenosine phosphate may be 1 mol stoichiometrically, but generally the ratio is 30 to 200 mol. This ratio depends on the reaction temperature and the degree of reactivity of the diisocyanate, and it is preferable to carry out the reaction with as little diisocyanate as possible, but generally a large excess is required. Next, after the completion of this reaction, the adenine nucleotides with a pH of 3 or less, preferably 1 to 2, are not decomposed and the remaining isocyanate is gelled by the reaction while cooling to room temperature or lower temperature, usually 10 to 0°C. It is poured into a mixed system of water and an organic solvent under acidic conditions, adjusted to a pH range that does not cause this reaction. In this case, the solvent is one that is immiscible with water but inert and miscible with isocyanate;
Examples include hydrocarbons such as benzene and hexane, halogenated hydrocarbons such as chloroform, ethyl acetate, and ethers and ketones. The amount of this organic solvent is 50 to 200 parts by weight per 100 parts by weight of water. The amount of this mixed solution is 5 to 100 parts by weight, preferably 20 to 40 parts by weight, per 1 part by weight of the reaction solution. In this way, the raw material compound AMP,
Compounds in which the amino group at position 6 of the adenine nucleus in ADP and ATP are carbamoylated with a carbamoyl group having an amino group at the terminal are obtained. The reaction in this case is expressed by the following formula. (In the formula, [AP] represents an adenosylic acid residue) When carrying out the reaction of the present invention, it is necessary to carry out the hydrolysis reaction shown in reaction (2) promptly in order to obtain the target product in good yield. It is. If water is simply added to the reaction system, the entire system will become gel-like.
To avoid such inconveniences, a mixture of acidic water and a water-insoluble organic solvent is stirred vigorously at as low a temperature as possible, and the mixture is reacted (1).
Add the reaction solution obtained in step. In the reaction of the present invention, advantageously, when ADP is used as a raw material, in addition to the desired ADP derivative, approximately equivalent amounts of AMP and ATP derivatives are obtained as products. It is. Considering that it is generally difficult to produce ATP derivatives, it is of great industrial significance that ATP derivatives can be easily produced using ADP as a raw material according to the present invention. In the present invention, the adenine nucleotide derivative thus obtained is chemically converted into a polysaccharide or a polymer substance having a repeating structural unit with a carboxyl group bonded as a side chain by utilizing the reactivity of its terminal amino group. to combine. The polysaccharide used may be starch, cellulose, dextran, agarose, or any other water-soluble or insoluble polysaccharide. When soluble, the molecular weight can be any size, from a few thousand to a few hundred thousand. These polysaccharides are formed by polymerizing monosaccharides such as glucose and galactose via glycosyl bonds. When adenine nucleotide derivatives are attached to these polysaccharides, the attachment is carried out as follows. The hydroxyl group of the polysaccharide is activated with bromic cyanide to form an imidocarbonate derivative. This imidocarbonate group reacts with the amino group of the general formula (I) to form an isourea bond, an imidocarbonate bond, or a urethane bond. The reaction is shown below. ([A]—R ₁ —NH ₂ represents the adenine nucleotide derivative used.) This binding reaction is known and is described, for example, in a book (Immobilized Enzyme, Chibata et al., Kodansha, 1974). There is. Therefore, the polymer of the present invention is a monosaccharide such as glucose or galactose by using the adenoid nucleotide derivative represented by the general formula (I) or a salt thereof using the terminal amino group of the substituent at the ^N6 position. It is a polymeric substance that is bonded to the hydroxyl group of a polysaccharide with a molecular weight in the range of several thousand to several hundred thousand by glycosyl bonds through isourea bonds, imidocarbonate bonds, or urethane bonds. In this case, 10 to 100 μmol of adenine nucleotide derivative is bound per 1 g of polysaccharide;
This corresponds to one adenine nucleotide derivative molecule being bound to every 600 to 60 monosaccharide residues constituting the polysaccharide. Note that the bonding order is completely random. Further, in the case of a polymer having a repeating structural unit to which carboxyl groups are bonded, the polymer substance to be used is one having a known structure such as polyacrylic acid or carboxymethyl cellulose. The molecular weight of these polymers may be any size from several thousand to hundreds of thousands. When bonding an adenine nucleotide derivative to a polymer having these carboxyl groups, the bonding is carried out as follows. When the carboxyl group is activated with water-soluble carbodiimide and reacted with the adenine nucleotide derivative of general formula (I), it reacts with the amino group to form an amide bond. This coupling reaction is known and described, for example, in the aforementioned texts. Therefore, the polymer of the present invention has the general formula (I)
A polymer substance in which an adenine nucleotide derivative represented by the formula or a salt thereof is bonded to a carboxyl group-containing polymer with a known structure using the terminal amino group of the N ⁶ -position substituent as the carboxyl group hair amide group. . For example, in the case of polyacrylic acid, a polymer substance containing 50 to 100 μmol of adenine nucleotide derivative per gram can be obtained.
This corresponds to one adenine nucleotide derivative molecule bound per 280 to 140 carboxyl group residues. Note that the bonding order is completely random. The adenine nucleotide derivative-bonded polymer substance according to the present invention is extremely easy to synthesize, and moreover, under normal conditions of use, its N ⁶ -
The carbamoyl bond is extremely stable, and the bound adenine nucleotide derivative will not be released. Furthermore, it has a strong affinity for related enzymes, and when used as an adsorbent in affinity chromatography, for example, it can be used as an adsorbent for various kinases. It shows a remarkable effect on the separation of dehydrogenase and dehydrogenase. Furthermore, when applied as an immobilized coenzyme to an enzyme reactor (bioreactor), it exhibits high coenzyme activity against various kinases. Next, the present invention will be explained in more detail with reference to Examples. Example A: Synthesis of adenine nucleotide derivative A-(1) N ⁶ -[N-(6-aminohexyl)
Synthesis of carbamoyl]-AMP/Li salt Dissolve 2 g of adenosine 5'-monophosphate (AMP) in 50 ml of hexamethylphosphoramide (HMPA), add 17 ml of hexamethylene diisocyanate (HDC), and dissolve at 70°C. The reaction was allowed to proceed for 2 hours. Next, this reaction mixture was poured into a mixture of 750 ml of water acidified with hydrochloric acid (PH1) and 750 ml of chloroform under ice cooling and stirring vigorously. The reaction solution was transferred to a separatory funnel and allowed to stand at room temperature overnight, then the aqueous layer was removed, and the chloroform layer was extracted again with water acidified with hydrochloric acid at pH. Combine both water layers and add 1N
PH7 with LiOH and 40℃ with evaporator
It was concentrated with When about 1 part of a 1:1 mixture of ethanol and acetone was added to the concentrated solution to about 100 ml under ice cooling, a white precipitate was formed. This precipitate was collected by centrifugation and dried under reduced pressure. The obtained solid was dissolved in water again,
It was purified by ion exchange chromatography using Dowex 1×2 (C ⁻ ) as an adsorbent. In this case, the elution is 0.05MLiC solution (PH5) 1
and 0.2M LiC (PH2) 1 using a linear gradient elution method. Combine the eluate from 500ml to 800ml, adjust the pH to 7 with 1NLiOH,
After concentrating this, 1: ethanol and acetone
1 mixture was added to this concentrated solution to form a white precipitate, which was centrifuged and dried under reduced pressure.
0.39 g of N ⁶ -[N-(6-aminohexyl)carbamoyl]-AMP.Li salt monohydrate (A) was obtained as a white powder. Next, a part of this Li salt was dissolved in water and acidified with formic acid to form a white precipitate, which was centrifuged and dried under reduced pressure to obtain the free acid monohydrate (B) as a white powder. Compounds (A) and (B) obtained as above are
It showed a single spot in thin layer chromatography (TLC) and a single peak in high performance liquid chromatography (HSLC), and was recognized as a single substance. Table 1 summarizes the Rf values and retention times. Compound (B) was analyzed after being neutralized with LiOH, so it showed the same value as compound (A). Table 1 also lists the starting materials for reference.
Values for AMP are also shown. The developing solvents shown in the table are as follows. a; Isobutyric acid: 1M aqueous ammonia (5:3) (saturated with EDTA) b: 0.1M potassium phosphate (PH6.8): ammonium sulfate: 1-propanol [100:
60:2 (V/W/V)] c; 2M formic acid: 0.5MLiC (1:1) d; 1MLiC e; 2% boric acid: 2MLiC (2:1) f; 3% boric acid: 3MLiC (2:1)

[Table] Compounds (A) and (B) are both amorphous solids and do not show clear melting points, but the decomposition points are 215-225℃ for compound (A) and 215-225℃ for compound (B).
The optical rotation of compound (A) was [α] ¹³ _D = -29.7° (C = 0.74, H ₂ O). It was confirmed that the elemental analysis values of compounds (A) and (B) each agreed with the theoretical values. moreover,
Functional group analysis confirmed that compounds (A) and (B) have one amino group in their molecules. Furthermore, in the C ¹³ nuclear magnetic resonance spectrum, it was confirmed that compounds (A) and (B) had one amino group in the molecule. Further, in the C ¹³ nuclear magnetic resonance spectrum, compound (A) shows six signals between 26.5 ppm and 41.3 ppm. This confirmed the presence of a hexamethylene group in the molecule. Furthermore, the UV spectra of compounds (A) and (B) confirmed the presence of a carbamoyl group bonded to the amino group at the 6-position of the adenine nucleus within the molecule. A-(2) N ⁶ - [N-(6-aminohexyl)
Carbamoyl] - ADP Li salt and N ⁶ - [N
-(6-aminohexyl)carbamoyl]-
Synthesis of ATP/Li salt 1g of adenosine 5′-diphosphate (ADP) in 50ml
of HMPA, 20 ml of HDC was added thereto, and the mixture was reacted at 75°C for 1.5 hours. The reaction solution was treated in the same manner as in Example (A)-1,
N ⁶ -[N-(6-aminohexyl)carbamoyl]-ADP Li salt [1000ml to 1200ml elution in ion exchange chromatography, compound (C)] 0.19g and N ⁶ -[N-(6-
Aminohexyl)carbamoyl]ATP・Li
Salt [in ion exchange chromatography]
From 1600 ml to 1900 ml of elution, 0.26 g of Compound (D) was obtained as a white powder. Regarding these compounds (C) and (D), the above A
The results of TLC and HSLC analysis are shown in Table 2 in the same manner as in the case of -1.

[Table] Compounds (C) and (D) are both amorphous solids and do not show clear melting points, but compound (C)
At 220°C or higher, compound (D) decomposes at 225°C or higher, and the optical rotation of compound (C) is [α] ¹³ _D = -21.9° (C = 0.82, H ₂ O), compound
(D) showed [α] ¹³ _D = -27.2° (C = 1.69, H ₂ O). It is known that the phosphoric anhydride bond at the end of ADP and ATP is cleaved by heat treatment in dilute acid, and both convert to AMP. ) and (D), each was dissolved in water to a pH of 3 and treated at 100°C for 5 hours. The resulting product was purified and isolated using ion exchange chromatography, and precipitated with formic acid. This was collected and dried under reduced pressure to obtain a white solid. It was confirmed by TLC and IR spectra that the products obtained from compound (C) and compound (D) were both identical to compound (B). Furthermore, the results of phosphorus analysis showed that compound (C) had two phosphorus atoms in its molecule, and compound (D) had three phosphorus atoms in its molecule. Further, in compounds (C) and (D), the presence of a carbamoyl group was confirmed by UV spectrum analysis, and the presence of a hexamethylene group was confirmed by nuclear magnetic resonance analysis. Furthermore, compound (C) is effective against pyruvate kinase and acetate kinase.
Compound (D) exhibits coenzyme activity as ADP.
showed coenzyme activity as ATP for hexokinase and glycerokinase. Its degree of activity is 20 and 82, 63 respectively at maximum rate when compared with original ADP or ATP.
and 87%. B: Binding of adenine nucleotide derivatives to polymer substances B-(1) Binding of N ⁶ -[N-(6-aminohexyl)carbamoyl]-AMP to Sepharose, a commercially available agarose gel. 4 g of Cepharose 4B to 6 ml of water. suspended,
The pH was adjusted to 11 with 0.5N NaOH, and 120 mg of BrCN dissolved in 6 ml of water was added thereto. After adding 0.5N NaOH to compensate for the drop in pH and keeping the pH at 10 for 15 minutes, suction filtrate the resulting solid.
It was washed with 50 ml of 0.1M NaHCO ₃ and the washing liquid was removed by suction filtration. Note that all of the above operations were performed under ice cooling. Next, the Sepharose 4B gel activated with BrCN in this way was treated with N ⁶ -[N-(6-aminohexyl) obtained in A-(1) above.
Carbamoyl] - 4ml of 20mg of AMP Li salt
Add to solution in 0.1M NaHCO ₃ at 4°C.
The mixture was stirred and reacted for 15 hours. Next, after collecting this by suction filtration, water,
It was thoroughly washed with 1M NaC and water in that order, and filtered with suction to obtain about 4 g of wet gel. As a result of phosphorus analysis, this gel contains the active ingredient
N ⁶ -[N-(6-aminohexyl)carbamoyl]-AMP is 1.81 per gram of wet gel.
It was confirmed that they were bound at a ratio of μmol. (Hereinafter, this product will be referred to as Cepharose 4B-immobilized AMP.) B-(2) N ⁶ for Cepharose 4B - [6-
Binding of N ⁶ -[N-(6-aminohexyl)carbamoyl]-ADP and N 6 -[N-(6-aminohexyl)carbamoyl]-ATP In B-(1) above, N ⁶ - as a raw material
Each Li of [N-(6-aminohexyl)carbamoyl]-ADP and N ⁶ -[N-(6-aminohexyl)carbamoyl]-ATP
The reaction was carried out in the same manner except that salt was used.
Sepharose 4B immobilized ADP (active ingredient content
1.01 ₁ μmol/1g wet gel) and Cephalose
4B immobilized ATP (active ingredient content 0.67μmol/1g
wet gels) were obtained. B-(3) N ⁶ for dextran - [N-
(6-aminohexyl)carbamoyl]-
ATP binding Soluble dextran (Dextran T40)
Dissolve 100mg of in 3ml of water and add with 0.5N NaOH.
The pH was adjusted to 11, and 0.4 ml of a solution of 100 mg of BrCN dissolved in 2 ml of water was added, and 0.5 N NaOH was added to maintain the pH at 11. After about 15 minutes, the drop in pH stopped and at this point the pH was 9.5 due to 0.1N HC.
After adjusting ^to
1 ml of an aqueous solution containing mg was added, and the mixture was left at room temperature for 16 hours with stirring. Then 0.2M
After adding 1 ml of ethanolamine hydrochloride (PH8.5) and leaving it for 1 hour, gel chromatography was performed using a BiOGel P-6 column.
Collect the high molecular fraction and add it to 0.1M
Dialysis was performed overnight against triethanolamine buffer (PH7.6). As a result of phosphorus analysis of dialysis fluid, 10.2 μmol ATP per 1 g of dextran
Soluble dextran-immobilized ATP bound to
It was confirmed that . B-(4) N ⁶ -N- for polyacrylic acid
(6-aminohexyl)carbamoyl]-
Binding of ATP Dissolve 100 mg of polyacrylic acid in 3 ml of water,
Adjust the pH to 4 with 0.5N NaOH, and add 200% of ethyldimethylaminopropyl cabodiimide hydrochloride to this.
Immediately add N ⁶ -[N-(6-aminohexyl)carbamoyl]-ATP Li salt 20
mg was dissolved in 0.5 ml of water, added, and left at 40°C overnight while stirring. This reaction solution was
-Purified by gel filtration and dialysis treatment in the same manner as in (3). As a result of phosphorus analysis of the dialysis fluid, it was confirmed that polyacrylic acid-immobilized ATP binding 50 μmol of ATP per 1 g of polyacrylic acid was obtained. C: Application example C-(1) Application as an adsorbent 5g of wet gel of Sepharose 4B-immobilized AMP prepared in B-(1) above was packed in a column (column bed volume 6.5ml) with 25mM triethanolamine buffer (PH7). .6) and after equilibration, alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), pyruvate kinase (PK), hexokinase (HK), and 3-phosphoglycerate kinase (PGK) were 4.2 and 2.9, respectively. , 3.6, 2.7, 4.9 (units) and a solution containing 3 mg of serum albumin.
Add 0.2 ml from the top of the column, first add 50 ml of the above buffer solution with mercaptoethanol added to make it 1mM, then add KC to the above buffer solution with mercaptoethanol added to make it 1M. Linear gradient elution was performed with the solution and the solution not added (60 ml of each solution). The eluate was fractionated into 4.8 ml portions using a collector, and the serum albumin concentration in each fraction was measured by absorbance at 280 mμ, and the activity of each enzyme was measured by a conventional method. As a result, amibumin and HK were eluted in fraction No. 2, LDH was eluted before and after fraction No. 6, and PGK was eluted in fraction No. 6.
is fraction No. 18 and 19, PK is fraction No. 18 and 19, ADH is fraction No. 23 and 24.
was eluted. Sepharose 4B similarly prepared in B-(2)
We conducted experiments using immobilized ADP and Sepharose 4B-immobilized ATP as adsorbents, and found that HK, LDH, PK, PGK,
It was confirmed that ADH was well separated from each other. C-(2) Application as an immobilized coenzyme The soluble dextran-immobilized ATP prepared in previous B-(3) has 49% of unmodified ATP for hexokinase and glycerokinase, respectively.
and showed 70% activity. Furthermore, it acted as a medium for the next reaction in the coupled enzyme reaction system of acetate kinase (AK) and hexonase (HK). This immobilization in this coupled enzyme reaction system
The rotational speed of ATP (recycling of ATP type and ADP type) is at a concentration of 0.1mM.
7.5cycles/hr, while unmodified ATP
showed a rotation speed of 38.2 cycles/hr at the same concentration. Next, 3 ml of a solution containing AK (1.8 units), HK (8 units), and this immobilized ATP (0.1 mM) was transferred to a continuous ultrafiltration device equipped with an ultrafiltration membrane that cuts off molecules with a molecular weight of 10,000 or more. When a substrate solution containing 100mM glucose and 7.5mM acetyl phosphate was poured into the cell at a flow rate of 11ml/hr, the effluent passing through the membrane contained 0.27mM glucose-6-phosphate. The concentration remained almost constant during continuous ultrafiltration for 6 hours. The total amount of effluent is
It was revealed that glucose-6-phosphate was constantly produced even when the volume was 60 ml and 20 times the amount of the cell liquid was flowed.

Claims (1)

[Claims] 1. General formula (wherein, R is a divalent hydrocarbon group that may contain a substituent, and Y is a phosphoric acid residue selected from monophosphoric acid, diphosphoric acid, and triphosphoric acid) A polymer substance having an adenine nucleotide bonded thereto, which is obtained by chemically bonding a nucleotide derivative or a salt thereof to a polysaccharide or a polymer substance having a repeating structural unit to which a carboxyl group is bonded as a side chain via its terminal amino group.