CS254375B1 - Biocatalyst for the preparation of purine 2'-deoxy-β-D-ribonucleosides - Google Patents

Abstract

The solution relates to the creation of a suitable immobilized biocatalyst based on alginate gel and its use for more efficient preparation of 2-deoxy-beta-D- -ribonucleosides, i.e. substances that are important biologically active substances and therapeutics. The essence lies in the fact that the immobilized component of the alginate gel-based biocatalyst consists of cells of the bacteria Escherichia coli SPT-. The said biocatalyst is preferably used for the preparation of 9-(2-deoxy-beta-D-riboturanosyl/ purines, 9-(2-deoxy-beta-D- -riboruranosyl/-6-methylpurine, 2'-deoxyadenosine or 2'-deoxyguanosine.

Description

The invention relates to a biocatalyst for the preparation of purine 2'-deoxy-beta-ribonucleic acids.

Biocatalysts based on alginate gels containing bound intact cells of microorganisms (e.g. bacteria, yeast or lower fungi) are used in modern biotechnological processes for the production of various compounds. Their essence is the fact that some enzymes retain their activity as in nutrient cells during immobilization (higher than isolated enzymes) and can therefore very effectively catalyze biotransformations of suitable substrates. The advantage of using these biocatalysts is, for example, the fact that the resulting substances are not further transformed (as can be the case in most immobilized growing cells) and also the ease of performing the reaction (and especially isolation) of the products.

Important substances prepared in this way include, for example, L-amino acids, chiral alcohols, immobilized cells used for selective transformations in a number of steroids, etc. The use of biocatalysts of this type is also important for the production of complex compounds that are difficult to produce by classical chemical processes. Such substances include, for example, 2-deoxyrbeta-D-ribonucleosides of pyrimidine and purine bases. These compounds are of exceptional importance as components of deoxyribonucleic acids, as well as precursors of their biosynthesis in nutrient cells.

Numerous substances of this type derived from chemically modified pyrimidine and purine bases are important as biologically active substances and as chemotherapeutics.

Natural deoxyribonucleosides are also of considerable importance as raw materials for the synthesis of oligodeoxyribonucleotides with applications in genetic engineering.

For the preparation of natural 2-deoxyribonucleosides, the most suitable source is deoxyribonucleic acid (DNA), obtained most preferably from fish milk. DNA cleavage must be carried out by the action of enzymes and the prices of the materials obtained in this way are high. Another possibility of preparation is the conversion from the more readily available beta-D-ribonucleosides, synthesized from ribonucleic acids; many reactions have been described for this purpose, but only the preparation of pyrimidine 2-deoxy-beta-D-ribonucleosides is well mastered.

Chemical syntheses of 2-deoxy-beta-ribonucleosides are carried out by reacting appropriate derivatives of heterocyclic bases with activated 3,5-disubstituted 2-deoxy-D-robofuranose compounds.

These reactions usually produce mixtures of anomers that need to be separated. Recently, procedures have been described that allow, in special cases, the preparation of almost pure beta-anomers of purine 2-deoxyribonucleosides with a modified base by the so-called phase transfer reaction (e.g. F. Seela and A. Kehne: Liebigs Ann. Chem. 1983, 876) or by the reaction of sodium salts of purine bases with 3,5-disubstituted 2-deoxy-D-ribofuranosyl chloride (Kazimierczuk Z., Cottam H.B., Revankar G. R. Robins R.K.: J. Amer. Chem. Soc. 106, 6 379 (1984).

A biotechnological method for the preparation of nucleosides is described, for example, in Japanese patent No. 63,393, based on reactions catalyzed by nucleoside phosphorylases contained in various microorganisms. However, this procedure is particularly suitable for the preparation of ribonucleosides.

In contrast, the synthesis of 2-deoxy-beta-D-ribonucleosides can be accomplished by the enzyme-catalyzed reaction of natural and modified heterocyclic bases (purine, pyrimidine) with 2-deoxy-beta-D-ribonucleosides in the presence of the enzyme deoxyribosyltransferase. Some microorganisms, e.g. of the genus Lactobacillus (Cardinaud R., Methods Enzymol. 51, 446 (1978) are a particularly rich source of this enzyme. In this reaction, which is carried out in an aqueous medium under almost neutral conditions, a sugar residue is transferred from a nucleoside (donor) to a heterocyclic base (acceptor). These reactions have already been used for the preparation of pyrimidine and purine deoxyribonucleosides (Holguin J., Cardinaud R.; Eur. J. Biochem. 54, 505 (1975); Holguin J., Cardinaud R., Salemink C.A.: Eur. J. Biochem. 54, 515 (1975); Baranski K.,

Bardos TJ, Bloch A, Kalman TI: Biochem. Pharmacol. 18, 347 (1969); VŘtruba I., Holý A., Wightman R.: Biochim. Biophys. Acta 324, 14 (1973); M.Ch. Huang, Hatfield K., Roetker A.W. , Ilontgomery J.A., Blakley R.L. : Biochem. Pharmacol. 30, 2663 (1981); M.Ch. Huang, Montgomery J.A., Thorpe M., Stewart E.L., Secrist III. JA, Barkley RL: Arch. Biochem. Biophys.

222, 133 (1983); Carson DA, Wasson DB, Kaye J, Ullman B, Martin Jr. DW, Robins RK,

Montgomery J.A,: Proc. Natl. Acad. Sci. U.S.77, 6 865 (1980)). The advantage of the procedure is the exclusive formation of the beta-anomer and the possibility of partial regeneration of the donor and unused base. The disadvantage of the current embodiment is the necessity to at least partially purify the necessary enzyme, which limits the practical use of the method.

Among purine deoxyribonucleosides, a special place is occupied, in addition to the cytostatic 9-(2-deoxy-beta-D-ribofuranosyl)-6-thioguanine, by 9-(2)deoxy-beta-D-ribofuranosyl) derivatives of 2,6-diaminopurine, 2-fluoroadenine and especially 9-(2-deoxy-beta-D-ribofuranosyl)-6-methylpurine. This substance exhibits high and selective toxicity towards cells infected with mycoplasma. (U.S. Patent 4,387,161). Mycoplasma infection is of great importance for maintaining the purity of tissue cultures of eukaryotic cells in the cultivation and study of viruses, in gene manipulation and in the production of antibodies and vaccines in human and veterinary medicine. 9-(2-Deoxy-beta-D-ribofuranosyl)-6-methylpurine has so far been prepared by a difficult chemical synthesis (Robins M.J., Robins R.K.: J. Amer. Chem. Soc.

87, 4934 (1965); Montgomery J.A., Hewson K.: J. Med. Chem. 11, 48 (1968) or by transdeoxyribosylation from 6-methylpurine and 2-deoxythymidine in the presence of an isolated deoxyribosyltransferase from Lactobacillus helveticus (McGarrity G.J., Carson D.A.: Exp. Cell Res. 139., 199 (1982)).

Important purine deoxyribonucleosides are also 2-deoxyadenosine and 2”-deoxyguanosine, required for the chemical synthesis of genes. Their preparation by chemical synthesis (Kazimierczuk and others, see above) or conversion from ribonucleosides (Robins M.J., Wilson J.S., Hansske F.:

J. Amer. Chem. Soc. 105, 4 059 (1983); Lessor R.A. , Leonard N.J.: J. Org. Chem. 46, 4 300 (1981)) is possible, but economically not very advantageous. For the production of 2'-deoxyadenosine by enzymatic transdeoxyribosylation, the main drawback is the poor solubility of adenine and also the necessity to rid the enzyme of contaminating adenosine aminase, which easily degrades the resulting product. The main obstacle to the use of transdeoxyribosylation for the production of 2'-deoxyguanosine is the extremely low solubility of both guanine and 2'-deoxyguanosine, which reduces yields and makes it difficult to isolate the reaction product.

The above disadvantages of the preparation of purine 2'-deoxyribonucleosides by chemical methods and enzymatic transdeoxyribosylation are eliminated by the present invention of a biocatalyst for the preparation of purine 2'-deoxyribonucleosides, which consists of an alginate gel containing mobilized cells of the bacteria Escherichia coli SPT, which can be advantageously used for the preparation of 9-(2-deoxy)-beta-D-ribofuranosyl purines of the general formula I

where R ¹ is hydrogen, methyl, amino, alkylamino, dialkylamino, where alkyl is formed by a chain with 1 to 3 carbons, hydroxy, dimethylaminomethyleneamino, methylthio or halogen, R is hydrogen, methyl, amino, dimethylaminomethyleneamino, methylthio or halogen, and R is hydrogen or amino. Its use consists in that a solution or suspension of a purine base of general formula II /11/

3 —3 “1 where R to R have the same meaning as in formula I in a final concentration of 10 mol 1 to 10 1 mol 1 1 in an aqueous buffer with a pH of 5.0 to 8.0, preferably ammonium acetate or TRIS hydrochloride, which contains a 2'-deoxy-beta-D-ribonucleoside of general formula III

where the hydrogen or methyl group _is preferably 2'-deoxyuridine, in a molar ratio of 1:1 to 1:10 based on the substance of formula II, is mixed with the biocatalyst in volumetric ratios of 100:1 to 1:1 based on the biocatalyst or is allowed to flow through a column of the biocatalyst, at a temperature of 20 °C to 45 °C, and the substance of general formula I is isolated from the mixture by filtration and chromatography, preferably on paper or on a column of hydrophobized silica gel in water.

The biocatalyst can also be advantageously used for the preparation of compounds of general formula I,

3 where R to R have the same meaning as in formula I, labeled with hydrogen, carbon and heteroatom nuclides, preferably hydrogen and carbon radionuclides, consist in reacting a substance of formula II, or a substance of formula II containing nuclides of hydrogen, carbon or heteroatoms, with a substance of formula III, or with a substance III containing hydrogen or carbon nuclides in the sugar moiety and with a biocatalyst, and the resulting product of formula I is isolated from the reaction mixture by paper chromatography or liquid chromatography.

Another example of a preferred use of the biocatalyst is its use for the preparation of 9-[2-deoxy-beta-D-ribofuranosyl]-6-methylpurine of formula IV

OH consisting in the fact that 6-methylpurine of formula V

CH ₃

is reacted with a substance of formula III in the presence of a biocatalyst and the product of formula IV is isolated from the reaction mixture by chromatography on hydrophilized silica gel, preferably octadecyl silica gel, in water.

It is also advantageous to use a biocatalyst for the preparation of 2'-deoxyadenosine of formula VI

NH,

°A>

(VI)

OH consisting in the fact that N^-dimethylaminomethyleneadenine of formula VII n=ch-n(ch ₃ ) ₂

H (VII) is reacted with the substance of formula III in the presence of a biocatalyst, the reaction mixture is diluted with an equal volume of concentrated aqueous ammonia after filtration, left for 2 to 24 hours at a temperature of 20 °C to 50 °C, evaporated under reduced pressure and the substance of formula VI is isolated from the mixture by chromatography, preferably on octadecyl silica gel in water, and for the preparation of 2'-deoxyguanosine of formula VIII

Oh ₂ n

HIM

HN

I, dt;>

(VIII)

OH consisting in the fact that N-dimethylaminomethyleneguanine of formula IX

HN |ch ₃ ) ₂ n-ch=

About to

H (IX) is reacted with the substance of formula III and with a biocatalyst, chromatography on octadecyl silica gel isolates the substance of formula X

(X) which is allowed to react with an aqueous solution containing 10 to 25 l by weight of ammonia, at temperatures of 20 °C to 50 °C, and the substance of formula VIII is isolated by crystallization after evaporation under reduced pressure.

The invention is based on the finding that a high level of deoxyribosyltransferase is present in an auxotrophic mutant of the thymine-dependent strain of Escherichia coli (SPT). This strain of bacteria has already been used for the enzymatic synthesis of radioactively labeled 5'-deoxyribonucleotides of thymine (Czech aut. osv. 172 684): a cell-free extract from the cells of this mutant catalyzes the transformation of thymine into 2'-deoxythymidine-5'-monophosphate. Using 2-deoxyribose-1-phosphate (or 2'-deoxyadenosine) as a source of deoxyribosyl function, thymine is first transdeoxyribosylated to form 2'-deoxythymidine and subsequently phosphorylated by other enzymes present in this extract. The present invention uses the same strain of SPT, but differs from the above-mentioned use in the following essential respects: (a) the biocatalyst according to the invention uses whole bacterial cells, which are, however, immobilized in a gel material, (b) Under these conditions, exclusively transdeoxyribosylation reactions are applied without subsequent phosphorylation and degradation of the resulting compounds, (o) The donor of the deoxyribosyl residue is pyrimidine 2'-deoxyribonucleoside. The novelty of the present invention lies in the fact that it uses other enzyme activities contained in these cells, which, using pyrimidine deoxyribonucleosides as donors, allows the preparation of purine 2-deoxyribonucleosides from heterocyclic bases.

Under the conditions according to the invention, the synthesis of the mentioned substances takes place specifically, so that the need to isolate or purify the respective enzyme is eliminated and the isolation of the reaction products of formula I is significantly simplified.

The preparation of the biocatalyst is very simple: A suspension of Escherichia coli SPT bacteria cells is introduced into an alginate sol in water (resulting in a biomass content of 10 to 25% by weight) and the resulting sol is converted into a gel form by dropping it into an aqueous solution of salts, e.g. calcium chloride. The biocatalyst prepared in this way is sufficiently stable and does not lose its enzyme activity after several weeks at temperatures slightly above freezing. Its regeneration after the reaction is not practical, although it can be used repeatedly for the reaction.

The enzyme reaction takes place in a heterogeneous mixture of biocatalyst and aqueous buffered solutions of both reaction substrates. The optimum pH value has been found to be pH 5.2 to 7.0 and a low ionic strength of the buffer. For preparative purposes, either a volatile buffer (e.g. triethylammonium hydrogen carbonate) or preferably 0.05 mol l ammonium acetate pH 5.8 to 5.9 can be used. Other buffer solutions can also be used, e.g. containing TRIS or phosphate or formate buffer solutions.

The reaction is carried out by gently shaking the biocatalyst with a solution of both reaction components or by pumping this solution through the biocatalyst column. Heterocyclic purine bases of formula II are in some cases poorly soluble in water, but the reaction can still be successfully carried out with the substance suspended in a buffer solution, because the resulting product of formula I is usually highly soluble in water. As a donor of the deoxyribosyl residue, e.g.

commercially available 2'-deoxythymidine of formula III (R = methyl), but the most suitable for this purpose is 2'-deoxyuridine (III, R = H). This substance is well soluble in water and is easily available either from uridine or by total synthesis from D-arabinose (Czech aut. osv. 166 523, 167 733) and is cheaper than the above-mentioned 2'-deoxythymidine.

The reaction takes place at temperatures from 20 °C to 40 °C, preferably at 37 °C. The rate and yield of the synthesis can be influenced by the ratio of the biocatalyst and reactants, the mutual ratio of the reactants, their concentration and the reaction time. To achieve optimal results and maximum efficiency of the implementation, a molar ratio of substance II (acceptor) and 2'- deoxyuridine (donor) of 1:2 to 1:3 has proven to be effective. For preparative purposes, solutions with a final concentration of up to 0.2 mol l' ¹ per donor and 0.01 mol l ^- ^ per acceptor can be used advantageously, i.e. with only five times the concentration of salts in the buffer solution. The amount of biocatalyst used depends on its granulation: in the standard implementation (diameter of geol balls 5 to 6 mm), 10 to 100 biocatalyst balls per 10 ml of reaction mixture are preferably used. This amount depends on the quality of the cell material, the relative amount of immobilized cells (relative to the volume of biocatalyst) and the chemical properties of the acceptor. The reaction rate can be increased by adding additional biocatalyst. The equilibrium state of the reaction is usually reached within 2 to 10 hours.

To stop the reaction, the incubation mixture is decanted from the biocatalyst and filtered through membrane filters to remove any released cell debris and other particles.

The mixture thus obtained usually contains four components: uracil and 2'-deoxyuridine (or thymine and 2'-deoxythymidine) in a ratio of about 1:1, the starting heterocyclic base and the reaction product. The conversion to the substance of formula I is usually 70 to 100%. The chemical properties of these components allow in most cases easy isolation of the product and regeneration of the other components of the mixture. For separation, e.g. ion exchange chromatography on cation exchange, paper partition chromatography or chromatography on hydrophobicized silica gel (e.g. octadecyl silica gel) in an aqueous medium can be used. In this procedure, the reaction mixture after ultrafiltration can be directly applied to the sorbent column without prior concentration.

Elution with water gradually washes out salts, uracil and 2'-deoxyuridine, which can be easily regenerated. Further washing with water or aqueous solutions with increasing concentrations of ethanol, methanol or dioxane gradually elutes the starting heterocyclic base of formula II and finally the pure reaction product I. After evaporation in vacuo and possible crystallization from a suitable solvent, this product is obtained in a completely pure state.

The reaction takes place with compounds substituted in positions 2,6 and 8 of the purine ring and is not accompanied by side reactions. For example, 6-methylpurine-2'-deoxyribonucleoside (IV) can be prepared in good preparative yield (see above). In the case of adenine, 2'-deoxyinoside can be formed as a deamination product of the reaction product if the reaction time is prolonged. The two substances can be easily separated; however, for the economical preparation of the important 2'-deoxyadenosine (VI), it is advantageous to use a variant in which adenine is replaced by N^-dimethylaminomethylenadenine of formula VII, which is easily available from adenine (Okumura K. et al., J. Org. Chem. 36, 1 573 (1971)). The resulting product of transdeoxyribosylation is not deaminated by adenosine deaminase. By treating this substance with a weakly alkaline medium, e.g. aqueous ammonia solutions, pure 2'-deoxyadenosine is obtained. Since the starting base VII is highly soluble in water, this variant can be used for the preparation of 2'-deoxyadenosine on a large scale.

A similar procedure can be used for the preparation of 2'-deoxyguanosine of formula VIII. In this case, the limiting factor is the very low solubility of guanine in aqueous media. By reacting guanine with dienopentylacetal of dimethylformamide at elevated temperature, N-dimethylaminomethyleneguanine (IX), which is significantly more soluble in water, is easily prepared. After carrying out the synthesis under usual conditions, the reaction product of formula X is first isolated by chromatography on octadecyl silica gel and is easily converted to 2'-deoxyguanosine by the action of aqueous ammonia solutions.

The biocatalyst according to the invention is also applicable for the preparation of purine 2'-deoxyribonucleosides labeled with nuclides (radionucleotides) of hydrogen, carbon and other atoms. Depending on the circumstances, either a labeled acceptor or donor or both labeled components (2'-deoxyuridine labeled in the sugar part) can be used and products labeled on one or both parts of the molecule can be obtained. The procedure of implementation allows for easy regeneration of labeled precursors.

The method of synthesis of purine 2'-deoxyribonucleosides, based on the use of a biocatalyst, thus enables the efficient preparation of these important substances in high yield from available raw materials, with low equipment and energy requirements, and with the possibility of easy isolation of products and regeneration of starting materials.

In the following, the invention is illustrated by examples of the preparation of the subject biocatalyst and its use for the preparation of purine 2'-deoxyrobonucleosides, without being limited thereto in any way.

Example

Preparation of a biocatalyst containing Escherichia coli SPT cells. A mixture of 0.75 g of alginate (e.g. Protanal LF, a product of Protan A/S.P.O., Drammen, Norway) and 15 ml of water is stirred until a sol is formed. 2.5 g of wet weight of biomass of Escherichia coli SPT bacterial cells (prepared according to Cs. aut. osv. 172 684) previously suspended in 10 ml of 0.9% aqueous potassium chloride solution are gradually mixed into this sol. The resulting homogeneous mixture is allowed to drip from a glass column with an internal diameter of 2.4 cm into a constantly stirred 2% solution of calcium chloride in water (100 ml) at a temperature of 20 to 25 °C. Gel balls are formed in the calcium chloride solution, which are sufficiently mechanically strong after 6 hours. The solution is decanted and the gel beads are washed by decantation with 0.05 mol 1 ammonium acetate. This material is used as a biocatalyst in the following examples.

Example 2

General procedure for the preparation of 2'-deoxyribonucleosides of formula I. To a solution of 25 µmol of heterocyclic base of formula II and 11.4 mg (100 µmol) of 2'-deoxyuridine in 5 ml of 0.05 mol 1 ¹ ammonium acetate pH 5.8, 7 beads of biocatalyst according to example 1 are added and the mixture is stirred by slow rocking at 37 °C in a bath. Aliquots of 10 µl of the incubation mixture are taken at intervals of 2 and 4 hours and analyzed by liquid chromatography on a Silasorb 300 £18 column (4 x 20 mm) in a mixture of 0.05 mol 1 ⁷ triethylammonium acetate and methanol according to the data in Table 2. Elution rate 0.6 ml/min, detection at 254 nm. The peak areas of substances I and II are integrated and the conversion to substance I is calculated. These data are given in Table 1.

Example 3

Comparison of the DNA activity of 2'-deoxyuridine and 2'-deoxythymidine. The reaction is carried out according to Example 2 using 25 µmoles of 6-methylpurine and 50 µmoles of 2'-deoxyuridine in 5 ml of buffer or using 25 µmoles of 6-methylpurine and 50 µmoles of 2'-deoxythymidine in 5 ml of buffer. 7 beads of the biocatalyst according to Example 1 are added to each mixture and stirred by rocking motion at 37 °C. After 4 hours, 10 µl aliquots of the mixtures are analyzed according to Example 2. In the reaction with 2'-deoxyuridine, the conversion of 6-methylpurine to 9-(2-deoxy-beta-ribofuranosyl)-6-methylpurine is 54.5%, in the reaction with 2'-deoxythymidine it is 52.5%.

Example 4

Preparation of 9-(2-deoxy-beta-D-ribofuranosyl)-6-methylpurine. To a solution of 1 g of 2'-deoxyuridine and 0.30 g of 6-methylpurine in 100 ml of 0.025 ml.l phosphate buffer pH 6.9 according to Sorensen, 150 beads of the biocatalyst according to example 1 are added and the mixture is stirred by rocking motion at 37 °C for 7 hours. The solution is decanted and the beads are washed with 10 ml of water. This solution is filtered through a membrane filter (Millipore 0.4 /u) and applied to a 190 ml octadecyl silica gel (30 /u) column, equilibrated with water. The column is eluted with water at a flow rate of 2 ml/min and the elution process is monitored on a Uvicord LKB (Uppsala, Sweden) at 254 nm. Uracil and 2'-deoxyuridine are eluted successively, the quality of the fractions (10 min intervals) is checked by analysis according to example 2 in 0.05 mol l ^-7 triethylammonium acetate. The 2'-deoxyuridine fractions are combined, evaporated at 40 °C/2 kPa, the residue is crystallized from ethanol-ether. 128 mg of 2'-deoxyuridine is obtained again.

Further elution with water gives 6-methylpurine (yield 90 mg after crystallization from ethanol-petroleum ether). The column is then washed under the same conditions with a mixture of methanol and water (1:9) and the main fraction absorbing in ultraviolet light is collected. After evaporation in vacuo, the residue is recrystallized from ethanol-petroleum ether. 447 mg (80%) of 9-(2-deoxy-beta-D-ribofuranosyl)-6-methylpurine is obtained, b.p. 161 °C. For (250.3) calculated: 52.79% C, 5.64% H, 22.39% N;

found 52.85% C, 5.56% H, 22.14% N. Mass spectrum: M ⁺ 250. UV-Spectrum: A _max ^{265 nm (£} max ^{7,000) at pH 7} ' Anax ^{263 nm (£} max ^{7,900) at pH 12} '

Example 5

Preparation of 2'-deoxyadenosine. To a solution of 76.4 mg (0.4 mmol) of N^-dimethylaminomethylenadenine and 182.4 mg (0.8 mmol) of 2'-deoxyuridine in 10 ml of 0.05 mol.l ammonium acetate pH 5.8, 100 beads of the biocatalyst according to example 1 are added and the mixture is incubated at 37 °C with slow shaking for 18 hours. The solution is decanted, replaced with a fresh mixture (see above) and incubated for 3 h under the same conditions. The same procedure is repeated once more. The combined decanted solutions (30 ml) are filtered according to example 3 and the filtrate is mixed with the same volume of conc. aqueous ammonia. The mixture is left to stand at room temperature for 18 h and evaporated at 40 °C/2 kPa.

The residue in 10 ml of water is applied to a 100 ml Amberlite IRC 50 column (H ⁺ form) and the column is washed with water until the UV absorption disappears. Then the UV-absorbing portion of adenine compounds is eluted with diluted (1:10) aqueous ammonia, which is evaporated under the same conditions. This residue is applied to a 90 ml octadecyl silica gel column (30 µ) in 5 ml of water and the column is eluted with water (2 ml/min). After the elution of adenine is complete, elution is continued with a mixture of methanol and water (1:9). Ultraviolet absorption indicates the elution of the product. This portion is combined, evaporated under the above conditions and crystallized from ethanol-ether. 138 mg (62%) of 2'-deoxyadenosine is obtained, identical to the authentic material (Calbiochem, USA) according to paper chromatography and liquid chromatography (see Table 1).

Example 6

Preparation of N ² -dimethylaminoethylene-2'-deoxyguanosine. To a mixture of 3 g of guanine and 90 ml of dimethylformamide, 15 ml of dimethylformamide dienopentyl acetate are added and the mixture is stirred at 110 °C under a calcium chloride stopper until a clear solution is formed. Evaporate at 60 °C/13 Pa, add 50 ml of ethanol and ca 2 g of solid carbon dioxide. After its dissolution, the mixture is evaporated again at 40 °C/2 kPa and the residue is crystallized from water. 3.5 g (85%) of N ² -dimethylaminomethyleneguanine are obtained, melting at 260 °C. For ^gH^^NgOg (206.2) calculated: 46.59%, 4.89% H,

40.76% N; found: 46.40% C, 4.82% H, 40.74% N. Mass spectrum: M ⁺ 206.

To a solution of 206 mg of this substance and 456 mg of 2-deoxyuridine in 25 ml of 0.05 mol 1 ammonium acetate pH 5.8, 120 beads of the biocatalyst according to example 1 are added and the mixture is stirred by rocking at 37 °C for 30 hours. The solution is decanted, filtered through a membrane and the filtrate is applied to a column of octadecyl silica gel (see example 3). The column is eluted first with water (2 ml/min) until uracil and 2-dexyuridine are removed (see example 3) and then with a methanol-water mixture 2 , (5:95) until the N -dimethylaminomethyleneguanine fraction is washed out. Finally, the product is obtained by eluting the column with a methanol-water mixture (9:1), which is precipitated from methanol with ether after evaporation at 40 °C/2 kPa. 161 mg (50%) of N ² -dimethylaminomethylene-2'-deoxyguanosine, mp 154 °C, is obtained.

For ^C ']_3 ^íí ]_8 ^N 6°4 ( ³²² ' ³ ) calculated: 48.44% C, 5.63% H, 26.08% N; found: 48.27% C,

5.46% H, 26.18% N. UV-Spectrum:Λ 235 nm (∆ 17,300), 290 nm (∆ 23,700) (in water).

' · ' ť _max max nax

Example 7

Preparation of 2-deoxyguanosine. A solution of 100 mg of N ^x '-dimethylaminomethylene-2'-deoxyguanosine prepared according to Example 6 in a mixture of 5 ml of water and 2 ml of conc. aqueous ammonia is allowed to stand for 18 h at 20 °C and the mixture is evaporated in vacuo. A quantitative yield of chromatographically pure (paper and liquid chromatography) 2'-deoxyguanosine identical to the authentic material (Calbiochem, USA) is obtained.

Example 8

Preparation of [' ¹ '^C-guanine]-2'-deoxyguanosine. In a 10 ml vial, 1.85 MBq of [jj-l^c^-guanine (specific activity 6.5 GBq/mmol) is evaporated under vacuum, 2 ml of 0.05 mol 1 ammonium acetate pH 5.8, 3 beads of biocatalyst according to example 1 and 1 ml of a solution of 60 µg of 2-deoxyuridine in water are added. The mixture is incubated for 60 min at 37 °C, the solution is transferred quantitatively to another 10 ml vial and concentrated in vacuum (2 kPa) over phosphorus pentoxide to a volume of about 50 µl. This concentrate is applied in three doses to a Silasorb column (see example 2) and chromatography is carried out in 0.05 mol 1 triethylammonium acetate. The obtained 2'-deoxyguanosine fractions are evaporated at 40°C/2 kPa and made up to 1 ml with water. The 2'-deoxyguanosine content, determined by measuring the radioactivity of an aliquot, is 0.74 MBq (yield 40%).

Example 9

Determination of the influence of the donor:acceptor ratio. Into four 25 ml flasks, 5 mg of 6-methylpurine and successively 8.75 mg, 17.5 mg, 26.25 mg and 35 mg of 2'-deoxyuridine are weighed. 5 ml of 0.05 .ol 1 ¹ ammonium acetate pH 5.8 and 5 beads of biocatalyst according to example 1 are added to the flasks.

After 90 minutes of incubation at 37°C, the conversion to 2'-deoxyriboside is determined as described in Example 2. The results obtained are summarized in Table 3. Increasing the donor:acceptor ratio above 2.0 does not affect the conversion under the synthesis conditions.

Example

Determination of the effect of pH on the course of the synthesis. 5 mg of 6-methylpurine and 17.5 mg of 2'-deoxyuridine are weighed into 25 ml flasks and 5 ml of 0.05 mol 1 ^L buffer (ammonium acetate pH 5.2 to 6.2 or Sorensen phosphate pH 6.9) are added. The flasks are incubated for 90 min at 37 °C after adding 5 beads of biocatalyst according to example 1 and the conversion is determined by the method according to example 2. The values obtained are given in Table 4. The optimal pH value for this synthesis is 5.8.

Table 1

Synthesis of 9-(2-deoxy-beta-D-ribofuranosyl)purines ^and Láká I

Conversion i

R

^CH 3 H H 64.5 nh ₂ H H 63.5 ¹ _CH3NH H H 64.5 (ch ₃ ) ₂ hours H H 70.0 (CH ₃ ) ₂ N-CH=N H H 75.0 _NH2 ^CH 3 H 67.0 ^NH2 nh ₂ H 46.0 ™2 H _NH2 21.0 Cl H H 63.5 Cl Cl H 33.0 II nh ₂ H 46.0 ^NH2 SCK ₃ H 72.0 OH H H 38.5 OH _NH2 H 34.0 OH (CH ₃ ) ₂ n-ch=n H 58.0 school ₃ H H 63.0 ^and See example 2; of which 11% 2'-deoxyinosine originated deamination

·.

Table 2

Liquid chromatography of compounds of formula I and II to ^and

R R' ^R.J. Sl S2 S3 S4 S5 ^NH2 H I 6.59 3.59 II 3.10 2.46 ch ₃ nh H I 6.36 II 4.55 (ch ₃ ) ₂ n H I 4.69 II 4.03 (CH ₃ ) ₂ N-CH=N H I 2.05 II 1.69 ^{NH2 CH} 3 I 4.61 II 2.79 ^{NH2 NH2} I 1.84 II 1.28 ^{NH2 SC1} 3 I 3.90 II 3.45 ^NH2 H NH _O I 3.17 ² II 1.66 H nh ₂ I 1.89 II 1.08 ^CH 3 H I 2.90 II 1.75 Cl H I 3.55 II 2.18 Cl Cl I 2.79 II 1.69 OH H I 1.88 1.09 II 0.77 0.54 OH ^NH2 I 1.02 II 0.17 OH (ch ₃ ) _O N-CH=N H I 3.93 ² II 2.26 ^SCH 3 H I 2.31 II 2.26 He deigned 0.35 0.23 2‘”-Deoxyuridine 0.98 0.53 0.41 0.12 0.08 2'-Deoxythymidine 0.85 Thymine 0.40

^and k = (t _R ~t )/t , where t _R is the retention time and t is the column dead time; see Example 2.

h-1

Mixtures methanol: 0.05 mol 1 triethylammonium acetate pH 7.0: S1 2.5:97.5, S2 5:95, S3 1:9, S4 1:4, S5 25:75.

Table 3

Effect of donor:acceptor ratio on the synthesis of 9-(2-deoxy-beta-D-ribofuranosyl)-6-methylpurine ^and

Molar ratio 2'-deoxyuridine: 6-methylpurine Conversion of 6-methylpurine, % ^l See Example 9.

Table 4

Effect of pH on the enzymatic synthesis of 9-(2-deoxy-beta-D-ribofuranosyl)-6-methylpurine ^and pH Conversion

5.2 29 5.5 31 5.8 33 6.2 28 6.9 23 7.9 18

^and See example 10.

Claims (1)

Biocatalyst for the preparation of purine 2'-deoxyribonucleosides, characterized in that it consists of an alginate gel containing immobilized cells of Escherichia coli SPT '.