CS202047B2 - Process for preparing macromolekularized derivatives of adenine - Google Patents

Description

The invention relates to the binding of functionalized adenine derivatives by covalent bonding to macromolecular compounds. Functionalized adenine derivatives disclosed in U.S. Pat. No. 202,046 have an ω-carboxyl side chain at position 8 and are derived from adenine core-containing compounds such as nicotinoylamide, adenine dinucleotide (NAD ⁺ ), nicotinoylamidadenine dinucleotide phosphate (NADP ⁺ ), adenosine triphosphate (ATP), adenosine diphosphate , adenosine monophosphate. (AMP), adenosine or adenine. Most of these compounds are of great importance in biochemistry.

The present invention provides a process for the preparation of macromolecularized adenine derivatives containing at least one unit of formula (I)

is derived from a compound selected from nicotinoylamidadenine dinucleotide, nicotinoylamidadenine dinucleotide phosphate, adenosine monophosphate, cyclic adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, adenosine and adenine, which comprises functionalized adenine derivatives of formula II

Saw ^{1LS '(CH.!.' CC '0H 1} TIL) wherein n is an integer from 2 to 4, are reacted at a temperature of 5-50 ° C in which n is an integer of 2-4, and the radical environment of water, optionally in mixtures with a water-miscible organic solvent in the presence of a water-soluble or insoluble carbodiimide condensation agent with a polymer containing at least one primary or secondary amino group selected from polylysine, ω-aminoalkylpolyanilamides, polysaccharide esters of ω-aminoalkylcarbamic acids, polyvinylamine and ω-amino esters ω-aminoalkylamides of polyglutamic acid, aminoalkylsilanized glass microspheres and polyethyleneimine.

An example of a water-soluble carbodiimide is N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride, insoluble carbodiimide may be N, N'-dicyclohexylcarbodiimide. As water-miscible solvents, for example, pyridine, tetrahydrofuran, dioxane and others can be used. It is preferred to work at room temperature.

Functionalized derivatives of, for example, NAD, but also the other compounds mentioned above, can be used as non-diffusible coenzymes or in affinity chromatography after binding to water-soluble or water-soluble macromolecular substances.

When bound to water-soluble macromolecular substances, the resulting products can be used as non-diffusible water-soluble macromolecularized coenzymes. These substances extend the possibilities of using known enzyme systems in which the enzyme is physically incorporated in insoluble porous structures such as fibers; polyacrylamide gel, microcapsules and the like which are impermeable to macromolecules. As a result of the physical incorporation of the water-soluble macromolecularized coenzyme into the enzyme or polyenzymatic system, in fact, both the enzyme and the coenzyme remain in intimate contact and the dispersion of the coenzyme out of the closed structure is prevented. This phenomenon is not possible with natural coenzyme due to its low molecular weight. When bound to water-insoluble macromolecular compounds, the products can be used in affinity chromatography or for enzymatic reactions in heterogeneous phase, whereby coenzyme can be regenerated.

Example 1

Preparation of Nicotinoylamide-8- (polyethyleneimine-3-carbonylethylthio) adenine dinucleotide.

/

I, n = 2, —N = polyethyleneimine residue \

- the rest of NAD ⁺

To 0.76 ml of an aqueous solution of polyethyleneimine (33% w / v) adjusted to pH 5 with conc. hydrochloric acid add 39 mg of nicotionylamide-8- (carboxyethylthio) adenine dinucleotide

= residue of NAD + dissolved in 0.5 ml of water, and 40 milligrams of N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride dissolved in 0.5 ml of water.

The reaction mixture adjusted to pH 4.8 by addition of 2 M HCl was stirred at room temperature for 48 hours while maintaining pH 4.8 during the first three hours by addition of 0.1 M HCl. The reaction mixture diluted to 20 ml with water is then transferred to a centrifuge tube and precipitated with 20 ml of phosphate buffer at pH 6. Centrifuge for 10 minutes at 39,000 g and the solution is freed from the polymer precipitate by decanting.

To further purify the polymer, it is dissolved in 4 ml of 2 M sodium chloride solution and 0.05 M acetate buffer at pH 5.5, and the solution thus obtained is supplemented with 16 ml of water, reprecipitating 20 ml of 1 M phosphate buffer at pH 6, centrifuged for 10 minutes at 39,000 g and the polymer was separated by decantation.

This purification procedure is repeated at least four times.

Redissolved product in 2 M sodium chloride solution and 0.5 M acetate buffer at pH 5.5 was dialyzed against 1 L portions of 1.ICH HCl for four days, changing the solution every day.

Freeze-drying of the dialysis residue yielded 223 mg of polymeric NAD derivative having an A _{max of} 276.5 nm.

The total amount of NAD bound to the polymer is determined by measuring the absorbance at 276.5 nm, assuming that the extinction coefficient is the same as that of the NAD derivative II (ε 18,800 Mcm ^-1 ).

The determination of coenzymatically active NAD bound to the polymer is performed by quantitative enzymatic reduction of yeast alcohol dehydrogenase in 0.1 M tris buffer at pH 9 in the presence of 0.2 methanol and 0.5 M semicarbazide hydrochloride.

The spectrophotometric measurement at 340 nm of the NADH derivative produced shows that 115 μΐηοΐ of enzymatically reducible NAD is bound to each gram of polymer, which corresponds to 90% of the total NAD bound to the polymer.

The macromolecular NAD thus obtained is coenzymatically active with many dehydrogenases. For example, with yeast alcohol dehydrogenase, the rate of enzymatic re202047 of the macromolecular NAD is 50% compared to natural coenzyme. HCl 42 μΐηοΐ, coenzyme 0,1 μπιοί (based on bound and enzymatically reducible NAD), enzyme 0,2 μ%, pH 9,0, incubation temperature 25 ° C. The reduction rate is determined by increasing the absorbance at 340 nm.

Example 2

Preparation of Nicotinoylamide-8- (polylysine-2-carbonylethylthio) adenine dinucleotide> /> 1, n = 2 - N = residue of lysine poles

To 50 milligrams of polylysine hydrobromide, approximately 50,000 molecular weight dissolved; 40 ml of nicotinoylamide-8- (carboxyethylthio) adenine dinucleotide is added in 0.5 ml of water

= residue of NAD ⁺ dissolved in 0.5 ml of water, and 40 milligrams of N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride dissolved in 0.5 ml of water.

The reaction mixture was adjusted to pH 4.7 with 0.1 M NaOH, stirred for 24 hours at room temperature while maintaining the pH at 4.7 during the first three hours by the addition of 0.1 M HCl.

Under the same conditions, an additional 40 mg of carbodiimide in 0.5 ml of water was added and stirring was continued for 24 hours.

The reaction mixture diluted to 15 ml with water was transferred to a centrifuge tube and precipitated with 10 ml of 0.15 M phosphate buffer at pH 6.

It was centrifuged for 10 minutes at 19,000 g and the solution was decanted from the polymer precipitate.

For further purification, the polymer is dissolved in 1 ml of 2 M NaCl solution and 0.05 M acetate buffer at pH 5.5, the solution thus obtained is supplemented with 15 ml of water and reprecipitated with 10 ml of 0.15 M pyrophosphate buffer pH 6, then is centrifuged at 39,000 g for 10 minutes and the polymer is isolated by decantation.

This purification procedure is repeated at least four more times. The product dissolved in 2 M sodium chloride solution and 0.05 M acetate buffer pH 5.5 was dialyzed over 48 hours against 500 ml of 3 M sodium chloride solution. '

Dialysis is then carried out against 2 mole fractions

1. ΙΟ ^-4 M HCl for four days, changing solution daily.

Freeze-drying the dialysis residue yielded 34 mg of NAD polymer derivative having an A _{max of} 276.5.

The total amount of NAD bound to the polymer is determined by measuring absorbance at

Assuming that the extinction coefficient is the same as for derivative II NAD (ε 18 800 M ⁴ cm ⁴ ), the coenzymatically active NAD bound to the polymer is determined by quantitative enzymatic reduction of yeast alcohol dehydrogenase in 0,1 M tris- buffer at pH 9 in the presence of 0.2 M ethanol and 0.05 M semicarbazide hydrochloride.

The spectrophotometric determination at 340 nm shows that the NADH derivative thus formed contains 157 µmol of enzymatically reducible NAD bound per gram of polymer, corresponding to 90% of the NAD bound to the polymer.

The NAD thus converted to a macromolecule substance is coenzymatically active with a number of dehydrogenases.

Example 1 a d 3

Preparation of Nicotinoylamide-8- (aminohexylsepharoso-2-carbonylethylthio) adenine dinucleotide

I, n = 2 — N aminohexyl-

500 mg of aminohexylsepharose 4B is swelled in 0.5 M NaCl, then washed with 200 ml 0.5 M NaCl and then with water. The gel obtained is stirred in 2 ml of water, then 40 mg of nicotinoylamide-8- dinucleotide (cxyethylthio) adenine card is added.

dissolved in 0.5 mL of water and adjusted to pH 4.7 with 1 M NaOH. 40 mg of N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride dissolved in 0.2 ml of water are added to the suspension stirred with a mechanical stirrer at room temperature and stirring is continued for 24 hours after stirring at 2047, the pH being within the first three hours. hours at 4.7 by adding 0.1 M HCl.

Under the same conditions, three additional portions of a solution of carbodiimide (40 mg in 0.2 mL of water) were added at 24 hour intervals for a total reaction time of 96 hours. The gel is then filtered and washed with 1 M sodium chloride solution and 1. ⁴ M HG1 until complete disappearance of UF absorption (NAD, which is not bound). 1.8 ml of polymeric NAD derivative is obtained in the form of a wet gel, λ _max 276.5 nm.

The UF spectrum was obtained by suspending the gels in an aqueous 50% sucrose solution (wt., Which prevents gel deposition). The total amount of NAD bound to the polymer is determined by measuring absorbance at 276.5 nm assuming the extinction coefficient is the same as the NAD derivative II. (ε 18 800 M ¹ cm ¹ ) Coenzymatically active polymer-bound NAD is determined by quantitative enzymatic reduction of yeast alcohol dehydrogenase by suspending the gel in a 50% aqueous sucrose solution (w / w) containing 0.1 M tris.HCl buffer, 0, 2 M ethanol and 0.5 M semicarhazide hydrochloride adjusted to pH 9.

From a photometric measurement at 340 nm it is apparent that 21 μπιοί of the enzymatically reducible NAD is bound to each gram of dry NADH derivative, which corresponds to 80% of the total bound NAD per polymer.

Example 4

Preparation of Nicotinoylamide-8- (polyethyleneimine-2-carbonylethylthiojadenine dinucleotide phosphate)

II, n = 2

= rest of NADP /

I, n = 2 —N = polyethyleneimine residue

= rest of NADP

To 0.38 ml of a 38% w / v aqueous solution of polyethyleneimine adjusted to pH with concentrated hydrochloric acid is added 20 mg of nicotinoylamide-8- (carboxyethylthio) adenine dinucleotide phosphate dissolved in 0.5 ml of water, and 20 mg of N-ethyl -N'- (3-dimethylaminopropyl) carbodiimide hydrochloride dissolved in 0.2 ml of water The reaction mixture adjusted to pH 4.8 by addition of 2 M HCl is stirred at room temperature for 24 hours and the pH is maintained at 0 for the first three hours. 1 M HCl Under the same conditions, an additional 20 mg of carbodiimide in 0.2 ml of water is added and stirring is continued for 24 hours, then the reaction mixture diluted to 10 ml with water is transferred to a centrifuge tube and precipitated with 10 ml of 1M phosphate buffer. pH

6. Centrifuge at 39,000 g for 10 minutes and separate the solution from the polymer coagulate by decanting. For further purification, the polymer is dissolved in 2 ml of 2 M NaCl solution and 0.05 acetate buffer at pH 5.5, the solution thus obtained is supplemented with 8 ml of water and reprecipitated with 10 ml of 1 M phosphate buffer at pH 6, centrifuged during 10 minutes. minutes at 39,000 g and the polymer is separated by decantation. This purification procedure is repeated at least four times. The product was redissolved in 2M NaCl solution and 0.05M acetate buffer at pH 5.5, dialyzed against 1 liter aliquots of 1 * 10 < ⁴ > M HCl for four days, renewing the solution daily. Freeze drying of the dyalysis residue yielded 83 mg of the polymer derivative of NADP, _λmax 276.5 nm. The total amount of NADP bound to the polymer is determined by absorbance at 276.5 nm, provided that the extinction coefficient is the same as for the NAD II derivative (ε 18,800 M-icm-i).

Coenzymatically active polymer-bound NADP is determined by quantitative enzymatic reduction of yeast glucose-6-phosphate dehydrogenase in 86.3 mM triethanolamine buffer pH 7.6 containing 6.7 mM MgCl 2 and 1.2 mM glucose-6-phosphate. From the spectrophotometric determination of NADP formed at 340 nm it is apparent that 23 µmol of enzymatically reducible NADP derivative did not bind to each gram of polymer.

The NADP thus obtained converted to the macromolecules of the substance is coenzymatically active with numerous dehydrogenases, for example 6-phosphogluconate dehydrogenase and L-glutamate dehydrogenase.

Claims (1)

OBJECT OF INVENTION A process for the preparation of macromolecularized adenine derivatives comprising at least one unit of formula I tu, adenosine and adenine, characterized in that the functionalized adenine derivatives of formula II wherein n is an integer from 2 to 4 and the remainder of the formula is derived from a compound selected from dinucleotide nikotinoylamidadeninu, dinucleotide phosphate nikotinoylamidadeninu, adenosine monophosphate, cyclic adenosine monophosphate, adenosine diphosphate, adenosintrifosfáS _J / K _N ^s - ^{s' lc} K'i ^c00H III) n is an integer from 2 to 4, are reacted at a temperature of 5-50 ° C in water, optionally in admixture with a water miscible organic solvent, in the presence of a water-soluble or insoluble carbodiimide condensation agent, with a polymer containing at least one primary or secondary amino group selected from polylysine, ω-aminoalkylpolyanilamides, polysaccharide esters of ω-aminoalkylcarbamic acids, pol yvinylamine, ω-aminoalkyl esters and ω-aminoalkylamides of polyglutamic acid, aminoalkylsilanized glass microspheres and polyethyleneimine.