JPS6113719B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel polymerized ATP. More specifically, the present invention relates to water-soluble polymerized ATP, a method for producing the same, and a bioreactor, particularly a subreactor, that utilizes the water-soluble polymerized ATP. In the construction of a subreactor for ATP regeneration, important issues are the preparation of immobilized acetate kinase with high activity and the acquisition of so-called polymeric ATP with substrate activity immobilized on a soluble polymerized carrier. be. (For the former, the inventors have already filed a separate application.) In the subreactor, for example, a phosphate group is added by bringing ADP and water-soluble acetyl phosphate into contact with the immobilized acetate kinase complex. ATP is produced, but at this time acetic acid is produced as a byproduct. This by-product must of course be separated from the ATP produced. Furthermore, for example in the main reactor,
ATP and starting materials are supplied, and the desired product and ADP are released.
It is necessary to separate the Column chromatography, distillation, liquid-liquid extraction, and the like have already been proposed as separation methods. However, none of these methods has given satisfactory results. In particular, the distillation method is undesirable because the anidene cofactors have poor heat resistance. In any case, in the conventional methods, the substantial difference in molecular weight between the adenine cofactors and the above-mentioned by-products, etc. is small, and therefore no industrially suitable separation method has been found, which has been a major obstacle. However, the inventors of the present application have discovered that such difficulties can be overcome by polymerizing adenine cofactors while maintaining their water solubility. That is, by polymerizing the adenine cofactor, it has become possible to easily separate the adenine cofactor from the by-products in the subreactor and the products in the main reactor, for example, by simply passing through a membrane filter. be. By the way, when ATP is bonded to a water-soluble polymer carrier to polymerize, if ATP is directly bonded to the carrier, the polymerized ATP becomes larger than free ATP.
It was found that the substrate activity of However, the present inventor found that the substrate activity was maintained when ATP was bound to a polymeric carrier via a spacer having an appropriate length. The name is "ATP derivative" (see JP-A-56-154497). This has overcome many of the difficulties in polymerizing ATP. Therefore, the purpose of the present invention is to develop a new water-soluble polymer.
It is to provide ATP. Another object of the present invention is to provide a method for producing the water-soluble polymerized ATP. Yet another object of the present invention is to provide a subreactor that utilizes water-soluble polymerized ATP with substrate activity. Other objects and features of the invention will become apparent from the description below. That is, the present invention provides (1) General formula: N ⁶ -S-M {In the general formula, N ⁶ represents ATP having a substituent at the 6-position amino group of the adenine nucleus; S is a spacer; , General formula -R-X- [However, R is -(CH ₂ ) _n - (However, m = 1 ~
10), -CO- (CH ₂ ) _p - (however, p = 1 to 10), -CO-
A- (However, A is m-, p-phenylene group,

[expression] or

[Formula]) or −(CH ₂ ) _q −CONH−(CH ₂ )〓− (where q=
0 to 5, γ=1 to 10); X together with the functional group of matrix M is -CONH-, -NHCO-,

[Formula]; M is a copolymer of acrylamide or a derivative thereof and acrylic acid or a derivative thereof, or a copolymer of acrylamide or a derivative thereof and N-(6
- a water-soluble polymer matrix consisting of a copolymer with (aminohexyl) acrylamide or its derivatives}. The ATP used in the present invention is, for example, Boelinger, Mannheim,
It can be easily obtained as a commercial product from Uenouchisha. The water-soluble polymerized ATP of the present invention can be produced according to the following method. That is, (1) A water-soluble polymer represented by the general formula N ⁶ -S-M is obtained by reacting ATP with an acylating agent or an alkylating agent, optionally carrying out a rearrangement reaction, and then condensing it with a water-soluble polymer matrix. Method for producing molecularized ATP. (2) A water-soluble polymer having the general formula N ⁶ -S-M, which consists of reacting a water-soluble polymer matrix with an acylating agent or an alkylating agent, and then coupling the product of the reaction with ATP. Method for producing molecularized ATP. made by. ATP, as is well known, is calculated by the following formula: It is indicated by. By the way, when a spacer is attached to ATP, the possible positions include phosphate, sugar, and base moieties. However, it has been found that substrate activity is lost when parts other than the base, ie, the phosphate part and the sugar part, are modified. Furthermore, X-ray analysis of kinases such as myokinase and hexokinase has revealed the binding mode between ATP and these kinases [Sehurltz GE, et al. Nature, 250 , 120
(1974) and Steitz TA et al.,
J. Biol. Chem., 252 , 4494 (1977)], as a result, the binding between ATP and the kinase is almost constant regardless of the type of kinase, and when ATP binds to the enzyme, the part that is in contact with the solvent is the ATP adenine nucleus
It is known that N ⁶ , N ⁷ , etc. On the other hand, for adenine, due to its reactivity, N ¹ ,
Derivatives at the N ⁶ , C-6 and C-8 positions are contemplated. Among these, the derivative at the ^N1 position is easily transferred to the ^N6 position, and the derivative at the C-6 position is used as a starting material.
Difficult to synthesize using ATP. Therefore, considering the above results, C-8th place,
The ^N6 position is considered to be the most suitable position to attach the spacer. However, the above patent application (Japanese Patent Application Laid-open No.
154497), the substrate activity of the resulting product is best when a spacer is attached to the N ⁶ -position. Examples of the alkylating agent used in the present invention include halocarboxylic acids; cyclic compounds such as lactones and epoxides; and aldehydes. As the halocarboxylic acid, β-halopropionic acid, γ-halobutyric acid, 4-halovaleric acid, 5-halocaproic acid, 8-haloperargonic acid, etc. can be used. Lactones include β-, γ-, δ- and ε-
You can use lactones. Examples of aldehydes include dialdehydes such as malondialdehyde, succindialdehyde, and glutardialdehyde, terephthalaldehydic acid,
Aldehydic acids such as isophthalaldehydic acid, formyl acetic acid, β-formylpropionic acid, azelaic acid semialdehyde, glycolaldehyde, δ
-Oxyaldehydes such as oxyvalericaldehyde can be mentioned. Further, as the epoxide, ethylene oxide, trimethylene oxide, epichlorohydrin, etc. can be used. The acylating agent used in the present invention includes:
Diisocyanates such as acid chlorides, acid anhydrides, lactams, aliphatic diisocyanates, and aromatic diisocyanates can be mentioned. Examples of the acid chloride include aromatic dicarboxylic acid chloride such as terephthaloyl chloride and isophthaloyl chloride, glycolic acid chloride, and γ-
Oxyacid chloride such as oxybutyric acid chloride, δ-oxyvaleric acid chloride, terephthaladehydic acid chloride, isophthalaldehydic acid chloride, β
-Aldehyde acid chlorides such as formylpropionic acid chloride and the like can be mentioned. Examples of the lactam include β-propiolactam, 1-butyrolactam, δ-valerolactam,
Examples include ε-caprolactam and heptolactam. As the acid anhydride, for example, succinic anhydride, glutaric anhydride, etc. can be used. Examples of the isocyanate include tetramethylene diisocyanate, m-xylene diisocyanate, p-xylene diisocyanate, and the like. (A) Alkylation In this case, in the reaction of ATP with something other than an aldehyde, the reagent first binds to the N ¹ -position and then causes a transfer reaction to obtain an ATP derivative at the N ⁶ -position. can. In the reaction formula, (i) halocarboxylic acid (ii) Lactones (iii) Aldehyde Among these, for those having a carboxyl group at the terminal, an ATP derivative having an even longer spacer can be obtained by reacting a diamine with a carbodiimide. For example, an aminohexylcarbamoylmethyl derivative can be obtained by combining hexamethylene diamine and carbodiimide. The rearrangement reaction is known as the Deimroth rearrangement reaction, and is a rearrangement reaction of an N-alkylated or arylated iminoheterocycle compound to the corresponding alkylamino or arylaminoheterocycle compound. This rearrangement reaction easily occurs by treatment at about 70° C. for about 2 hours under alkaline conditions with a pH of about 8.5. (B) Acylation ATP can be easily acylated using a general acylating agent such as those mentioned above, and the desired coupled product of ATP and spacer can be obtained. This reaction formula is shown below. (′) Acid chloride (However, R″ is −(CH ₂ ) _b −, b=1 to 10,

【formula】

[Formula]. ) (′) Acid anhydride (′) Lactam (′) Isocyanate (However, R is −(CH ₂ ) _e −, e=2 to 10,

【formula】

【formula】

【formula】

[Formula]) The ATP derivative thus obtained can be easily purified by adding it to a column packed with Dowex 1×8 and eluting with a LiCl concentration gradient. ATP following exactly the same procedure as above
Similar ADP derivatives can be obtained using ADP instead of . ATP (or ADP) obtained as described above
The derivative is a water-soluble synthetic polymer matrix (hereinafter referred to as
This can be done by condensing the functional groups of the ATP derivative (simply referred to as a matrix) with the functional groups of each ATP derivative. The matrix used in the present invention is a homopolymer or copolymer having amino groups, carboxyl groups, etc. That is,
Copolymer of acrylamide or its derivative and acrylic acid or its derivative, acrylamide or its derivative and N-(6-aminohexyl)
Mention may be made of copolymers with acrylamide or its derivatives. These copolymers may have various proportions of each monomer, but for example, when a copolymer of acrylamide and acrylic acid is coupled with an ATP derivative having an amino terminal group, a high proportion of acrylamide may be used. was found to be preferable in order to obtain a product with high substrate activity. In other words, this means that
A certain degree of lower binding rate of the ATP derivative means that the substrate activity of the product is higher. The matrix used in the present invention can be manufactured according to any known method. The polymerized ATP of the present invention thus obtained can be purified by gel filtration using Sephadex G-50 or the like. According to another aspect of the present invention, after bonding a spacer to a predetermined matrix, the functional group of the spacer is bonded to the amino group at the 6-position of ATP, thereby achieving the desired polymerization.
You can also get ATP. The polymerized ATP of the present invention thus obtained still has substrate activity for acetate kinase, hexokinase, and polyglycerol kinase. This can be evaluated by determining the reaction parameters Km and Vmax for each of the above-mentioned kinases. Each reaction parameter is determined by coexisting reduced nicotinamide adenine dinucleotide (NADH), determining the reaction rate by tracking the absorption of NADH at 340 nm, and using the resulting reaction rate curve in accordance with a conventional method. I can do it. Hereinafter, the present invention will be explained in more detail with reference to Examples. In each embodiment, unless otherwise specified, parts represent important parts. Example [A] Synthesis of water-soluble polymerized matrix (1) 2 parts of acrylamide and 2 parts of acrylic acid are dissolved in water to a concentration of 5%, and ammonium persulfate is added to this in a ratio of 1 part to monomer.
%, and further added equimolar amount of acidic sodium sulfite, and polymerization was carried out at 40° C. under a nitrogen atmosphere. After 6 hours the polymerization product was precipitated by adding acetone, collected by filtration and dried under reduced pressure. The yield was 79%. This polymerization product is hereinafter referred to as Mx. (2) Dilute 1 part of Mx synthesized as above with water to a concentration of 5%, and add 1.6
- Add 15 parts of diaminohexane to bring the pH to 4.7
The temperature was maintained at 30°C. 0, 5, 20
and 1-ethyl- at each step of 27 hours.
2.5 parts of 3(3-dimethylaminopropyl)carbodiimide were added. After 45 hours, the reaction product was precipitated with methanol/acetone (4:1) mixed solvent, collected by filtration, and dried under reduced pressure. The yield was 150% based on Mx. Hereinafter, this product will be referred to as Mx. [η] k′ dl ^/ g Mx 0.50 ^* 0.48 Mx 0.25 1.6 * Average molecular weight 5.5×10 ⁴ (calculated from [η] = 3.73×10 ^-4 M ^0.66 ) [B] Synthesis of ATP derivative (1 ) N ⁶ -Succinyl ATP (6-S) ATP and succinic anhydride were dissolved in a 1.5% dimethyl sulfoxide solution of LiCl at a molar ratio of 1:100, and reacted with stirring at room temperature for 47 hours. . An ethanol/acetone (1:1) solution was added to the reaction solution to precipitate the product, which was collected by filtration. This residue has a pH of
2.0 aqueous solution, added to a column packed with Dowex 1-x8, washed with water, and eluted with a LiCl concentration gradient. The main fraction of the eluate was collected, poured into a concentrated ethanol/acetone (1:1) solvent under reduced pressure to precipitate it, and was filtrated to obtain the ATP derivative described above. The yield was 43%. (2) N ⁶ -(carboxymethyl)ATP(6-
CM) ATP was added to an aqueous iodoacetic acid solution so that the molar ratio of ATP to iodoacetic acid was 1:10, the pH was adjusted to 6.5 with an aqueous LiOH solution, and the mixture was kept at room temperature in the dark for 80 hours with stirring. . The product was then precipitated by the addition of cold ethanol and recovered by filtration. The recovered product was dissolved in water, the pH was adjusted to 8.5, and the mixture was treated at 70°C for 2 hours. Add this solution to PH2.0 with HCl aqueous solution.
It was added to a column packed with Dowex 1-x8 and washed with a 0.3M LiCl solution (PH2.0). The product was then eluted with a LiCl concentration gradient, and fractions having an absorption at 267 nm were collected, precipitated with a methanol/ethanol (1:1) mixed solution, and collected by filtration. The yield was 43%. (3) N ⁶ -(carboxyethyl)ATP(6-
CE) The title compound was obtained according to the method for producing 6-CM in (2) above, except that iodopropionic acid was used instead of iodoacetic acid. The yield was 46%. (4) N ⁶ -(6-aminohexylcarbamoylmethyl)ATP (6-AHCM) Dissolve 6-CM obtained in (2) above and hexamethylene diamine in water at a molar ratio of 1:10, The reaction mixture was reacted for 48 hours at room temperature in the presence of a small amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, precipitated with a methanol/acetone (1:1) mixture, and recovered by filtration. Dissolve this in water (PH
2.0) on a Dowex 1-x8 column and purified by elution with a LiCl concentration gradient as described above. The yield was 43%. (5) N ⁶ (6-aminohexylcarbamoylethyl)ATP (6-AHCE) The title compound was obtained in the same manner as in (4) above, except that 6-CE was used instead of 6-CM. The yield was 39%. (6) N ⁶ -[N-(m-aminomethylbenzyl)
[Carbamoyl]ATP (6-AMBC) ATP and m-xylene diisocyanate were dissolved in a 1.5% dimethyl sulfoxide solution of LiCl at a molar ratio of 1:50, and reacted for 8.5 hours with stirring at room temperature. Hereinafter, the product was purified and recovered according to the procedure of Example 1. Yield is 22
It was %. [C] Polymerization of ATP derivative (1) 1 part of 6-AHCM obtained in [B]-(4) above was added to 0.5
% aqueous solution and added 1.8 parts of the previously prepared Mx. Adjust the pH to 4.7, warm to 30°C, and add 3.2 parts of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) at each step of 0, 3, 7, 22, 27, and 31 hours. did. After 47 hours, the reaction solution was made neutral and subjected to gel filtration using Sephadex G-50. 0.3 as elution solvent
A molar LiCl solution was used. Fractions with absorption at 270 nm were collected, concentrated using a rotary evaporator (30°C), and the product was precipitated with a methanol/acetone (1:2) mixed solvent.
It was collected by filtration and dried under reduced pressure. This product is hereinafter abbreviated as 6-AHCM-Mx. (2) Except for using 6-CE instead of 6-CM
Water-soluble polymerization of N ⁶ -(6-aminohexylcarbamoylethyl)ATP was performed according to the same procedure as in (1). Below, this product is 6-
It is abbreviated as AHCE-Mx. (3) 6-AMBC was bonded to matrix Mx in the same manner as in (1) except that 6-AMBC obtained in [B]-(6) above was used instead of 6-AHCM. This product is the following 6-
It is abbreviated as AMBC-Mx. (4) Dissolve 1 part of N ⁶ -(carboxymethyl)-ATP (6-CM) obtained in [B]-(2) above in water to make a solution with a concentration of 0.5%, and add 1.8 parts of Mx to this. Added. The pH was adjusted to 4.7, heated at 30°C, and 0.8 part of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide was added at each stage of 0, 6, 23, and 30 hours. After carrying out this reaction for 47 hours, the solution was made neutral,
Gel filtration was performed using Cuffadex G-50. A 1 molar LiCl solution was used as the elution solvent. Collect the flow-through fraction with absorption at 270 nm and concentrate it using a rotary evaporator (30 nm).
°C), precipitated with methanol/acetone (1:2) mixed solvent, collected by filtration, and dried under reduced pressure. Hereinafter, this product is 6-CM-
Abbreviated as Mx. (5) Except for using 6-CE instead of 6-CM
Following exactly the same operation as (4), the matrix
6-CE was coupled to Mx. This product is hereinafter abbreviated as 6-CE=Mx. (6) Following similar operations, 6-CM, 5-
CE, 6-S ADP derivative to Mx, and 6
-SATP derivatives were also bound to Mx. (7) Similarly, 6-AHCM and 6-AHCE
ADP derivatives were polymerized using Mx. The polymerized ATP thus obtained and
The properties of ADP are given in Table 1. In Table 1,
The binding rate per ATP or ADP derivative is
It was calculated from the absorbance of λ _nax of each product.

[Table] [D] Substrate activity of polymerized ATP The substrate activity of polymerized ATP produced as described above for acetate kinase (AK), hexokinase (HK), and polyglycerate kinase (PGK) was constantly determined. Measured according to the law. For comparison, free ATP was also measured in the same way. The acetate kinase used in this example is Bacillus stearothermophilus (Bacillus stearothermophilus).
stearothermophilus), and hexokinase and polyglycerate kinase were obtained as commercial products from Boelinger Mannheim. The results are shown in Table 2.

【table】

Claims (1)

[Claims] 1 General formula: N ⁶ -S-M {In the general formula, N ⁶ represents ATP having a substituent at the 6-position amino group of the adenine nucleus; S is a spacer, General formula -R-X- [However, R is -(CH ₂ ) _n - (however, m = 1 to 10),
-CO- (CH ₂ ) _p - (however, p = 1 to 10), -CO-
A- (However, A is m-, p-phenylene group, [Formula] or [Formula]) or - (H ₂ ) _q -CONH-(CH ₂ ) _r - (However, q = 0 ~
5, r=1 to 10); X together with the functional group of matrix M is -CONH-, -NHCO-,
[Formula]] represents; M is a highly aqueous polymer consisting of a copolymer of acrylamide or its derivative and acrylic acid or its derivative, or a copolymer of acrylamide or its derivative and N-(6-aminohexyl)acrylamide or its derivative; Water-soluble polymerized ATP represented by } showing the molecular matrix. 2 R is −(CH ₂ ) ₂ −, −CO−(CH ₂ ) ₂ −, −CO−
NH−( _CH2 ) ₆₋ , The water-soluble polymerized ATP according to the first line of the claims. 3. ATP is reacted with an acylating agent selected from acid halides, acid anhydrides, lactams, or isocyanates, or an alkylating agent selected from halocarboxylic acids, lactones, or aldehydes, optionally carrying out a rearrangement reaction, and then forming a water-soluble polymer matrix. The general formula N ⁶ − consists of condensation with
A method for producing water-soluble polymerized ATP represented by SM. In the general formula N ⁶ -S-M, N ⁶ represents ATP having a substituent at the 6-position amino group of the anidene nucleus. ; S is a spacer and has the general formula -R-X- [wherein R is -(CH ₂ ) _n - (m=1 to 10),
-CO-( _CH2 ) _p- (however, p=1 to 10), -CO-A
- (However, A is m-, p-phenylene group, [Formula] or [Formula]) or - (CH ₂ ) _q -CONH- (CH ₂ ) _r - (However, q = 0 to
5, r=1 to 10); X together with the functional group of matrix M is -CONH-, -NHCO-,
M represents a highly water-soluble copolymer of acrylamide or its derivative and acrylic acid or its derivative, or a copolymer of acrylamide or its derivative and N-(6-aminohexyl)acrylamide or its derivative; A molecular matrix is shown. 4. React the water-soluble polymer matrix with an acylating agent selected from acid halides, acid anhydrides, lactams, or isocyanates or with an alkylating agent selected from halocarboxylic acids, lactones, or aldehydes, and then react the product of the reaction with ATP. The general formula N ⁶ −S− consists of coupling
A method for producing water-soluble polymerized ATP represented by M, and in the general formula: N ⁶ -S-M, N ⁶ represents ATP having a substituent at the 6-position amino group of the adenine nucleus; S is a spacer and has the general formula -R-X- [wherein R is -(CH ₂ ) _n - (m=1 to 10),
-CO-( _CH2 ) _p- (however, p=1 to 10), -CO-A
- (However, A is -, p-phenylene group, [Formula] or [Formula]) or - (CH ₂ ) _q -CONH- (CH ₂ ) _r - (However, q = 0 ~
5, r=1 to 10); X together with the functional group of matrix M is -CONH-, -NHCO-,
M represents a highly water-soluble copolymer of acrylamide or its derivative and acrylic acid or its derivative, or a copolymer of acrylamide or its derivative and N-(6-aminohexyl)acrylamide or its derivative; A molecular matrix is shown.