CS254375B1 - Biocatalyst for preparation of purine 2-deoxy-beta-d-ribonucleosides - Google Patents

Abstract

The solution is to create a suitable one immobilized biocatalyst on alginate gel and its use to make 2-deoxy-beta-D- -ribonucleosides, ie substances that are important biologically active substances and cherapeutics. The essence is that the immobilized component of the biocatalyst alginate gel-based cells form cells bacteria of Escherichia coli SPT-. Said the biocatalyst is preferably used for the preparation of 9- (2-deoxy-beta-D-ribofuranosyl) 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-ribonuclease.

Biocatalysts based on alginate gels containing bound intact cells of microorganisms / e.g. bacteria, yeast or inferior fungi are used in modern biotechnological processes for the production of a wide variety of compounds. They are based on the fact that some enzymes retain the activity as in feeder cells (higher than the isolated enzymes) when immobilized and therefore can very effectively catalyze the biotransformation 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 in the most mobilized growing cells) and furthermore easy to carry out the reaction (and especially the isolation) of the products.

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

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

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

For the preparation of natural 2-deoxyribonucleosides, the most suitable source is deoxyribonucleic acid (DNA), most preferably obtained from fish roe. DNA cleavage must be carried out by the action of enzymes and the cost of the materials thus obtained is high. Another possibility is to convert from more readily available beta-D-ribonucleosides derived 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 suitable heterocyclic base derivatives 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 which, in special cases, allow 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. Seel and A. Kehne: Liebigs Ann. Chem. 1983, 876 ) or by reacting the sodium salts of the purine bases with 3,5-disubstituted 2-deoxy-D-ribofuranosyl chloride (Kazimierczuk Z., Cottam HB, Revankar GR Robins RK: J. Amer. Chem. Soc. 106, 6 379 (1984)).

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

In contrast, synthesis of 2-deoxy-beta-D-ribonucleosides can be accomplished by enzymatic catalysed reaction of both natural and modified heterocyclic bases (purine, pyrimidine) with 2-deoxy-beta-D-ribonucleosides in the presence of deoxyribosyltransferase enzyme. Some microorganisms, such as the genus Lactobacillus (Cardinaud R., Methods Enzymol. 51, 446 (1978)) are a particularly rich source of this enzyme, and this reaction, carried out in an aqueous environment under near neutral conditions, transfers the sugar residue from the nucleoside These reactions have already been used to prepare both pyrimidine and purine deoxyribonucleosides (Holguin J., Cardinaud R .; Eur. J. Biochem. 54, 505 (1975); Holguin J., Cardinaud R. Salemink CA: Eur J Biochem 54, 515 (1975);

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

222, 133 (1983); Carson D.A., Wasson D.B., Kaye J., Ullman B., Martin Jr. D.W., Robins R.K.,

Montgomery J.A .: Why. Nati. Acad. Sci. U.S. 7, 6865 (1980)). The advantage of the process is the exclusive formation of the beta-anomer and the possibility of partial regeneration of the donor and unused base. A disadvantage of the prior art is the necessity to at least partially purify the required enzyme, thereby limiting the practical application of the method.

Among the purine deoxyribonucleosides besides the cytostatic 9- (2-deoxy-beta-D-ribofuranosyl) -6-thioguanine 9- (2) deoxy-beta-D-ribofuranosyl) derivatives of 2,6-diaminopurine, 2-fluoradenine and especially 9- (2-deoxy-beta-D-ribofuranosyl) -6-methylpurine. It exhibits high and selective toxicity against mycoplasma infected cells. (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 hitherto been prepared by difficult chemical synthesis (Robins M.J., Robins R.K .: J. Amer. Chem. Soc.

87, 4,934 (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 furthermore 2-deoxyadenosine and 2'-deoxyguanosine required for chemical gene synthesis. Their preparation by chemical synthesis (Kazimierczuk et al., Supra) or by 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 less advantageous. For the production of 2'-deoxyadenosine by enzyme transdeoxyribosylation, the main drawback is the poor solubility of adenine, as well as the need to rid said enzyme of contaminating adenosine aminase, which easily degrades the resulting product. A major 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 overcome by the present invention of a biocatalyst for the preparation of purine 2'-deoxyribonucleosides consisting of an alginate gel containing mobilized cells of Escherichia coli SPT, of the deoxy (beta) -D-ribofuranosyl) purines of formula (I)

wherein R ¹ is hydrogen, methyl, amino, alkylamino, dialkylamino, wherein the alkyl is a chain of 1 to 3 carbons, hydroxy, dimethylaminomethylene amino, methylthio 2 or halogen, R is hydrogen, methyl, amino, dimethylaminomet, yleneamino, methylthio 3 " or halogen, and R is hydrogen or amino. Its use consists in the solution or suspension of the purine base of the general formula II (11)

Where R to R have the same meaning as in formula I at a final concentration of 10 moles to 10 1 moles of 1 liter in an aqueous buffer having a pH of 5.0 to 8.0, preferably ammonium acetate or TRIS hydrochloride, which comprises the 2'-deoxy-beta-D-ribonucleoside of formula III

wherein J _is hydrogen or methyl group, preferably a 2'-deoxyuridine, in a molar ratio of 1: 1 to 1:10 relative to the compound of formula II is mixed with the biocatalyst in a volume ratio of 100: 1 to 1: 1 on a biocatalyst or passed through column of the biocatalyst at 20 ° C to 45 ° C, and the compound of 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 the compounds of the formula I,

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

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

OH in that 6-methylpurine of formula V is OH

CH ₃

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

The use of a biocatalyst for the preparation of 2'-deoxyadenosine VI is also preferred

NH,

° A>

(VI)

OH in that N-dimethylaminomethylenadenine of formula VII n = ch-n (ch ₃ ) ₂

H (VII) is reacted with a compound of formula III in the presence of a biocatalyst, after filtration the reaction mixture is diluted with an equal volume of concentrated aqueous ammonia, left at 20 ° C to 50 ° C for 2 to 24 hours, evaporated under reduced pressure and 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

O h ₂ n

HIM

HN

Me, dt;>

(VIII)

OH comprising N-dimethylaminomethyleneguanine of formula IX

HN | CH ₃ ) ₂ n-ch =

O do

H (IX) reacts with a compound of formula III and a biocatalyst, isolating the compound of formula X with octadecyl-silica gel chromatography

(X) which is reacted with an aqueous solution containing from 10 to 25% by weight of ammonia at temperatures between 20 ° C and 50 ° C and the compound of formula VIII is isolated by crystallization after evaporation under reduced pressure.

The invention is based on the discovery that a high level of deoxyribsyltransferase is present in the auxotrophic mutant of a thymine-dependent strain of Escherichia coli (SPT). This bacterial strain has already been used for the enzymatic synthesis of radiolabeled 5'-deoxyribonucleotides of thymine (Cs. Aut. Os. 172 684): a cell-free extract from the cells of this mutant catalyses the transformation of thymine to 2'-deoxythymidine-5'-monophosphate. Using 2-deoxyribose-1-phosphate (or 2'-deoxyadenosine) as a source of deoxyribosyl function, first transdeoxyribosylation of thymine occurs to form 2'-deoxythymidine and its subsequent phosphorylation by other enzymes present in this extract. Although the present invention utilizes the same strain of SPT, it differs from this use in the following essential respects: (a) the biocatalyst of the invention utilizes whole bacterial cells which are immobilized in the gel material, (b) Under these conditions exclusively transdeoxyribosylation reactions (o) The donor of the deoxyribosyl moiety is the pyrimidine 2'-deoxyribonucleoside. The novelty of the present invention is that it utilizes other enzyme activities contained in these cells which, using pyrimidine deoxyribonucleosides as donors, make it possible to prepare purine 2-deoxyribonucleosides from heterocyclic bases.

Under the conditions according to the invention, the synthesis of the compounds is carried out specifically, so that the relevant enzyme is not isolated or purified and the isolation of the reaction products of the formula I is considerably simplified.

The preparation of the biocatalyst is very simple: the cell suspension of Escherichia coli SPT is introduced into an alginate sol in water (resulting in a content of 10-25% by weight of biomass) and the resulting salt is gelled by dropwise addition to an aqueous solution of salts such as calcium chloride. The biocatalyst thus prepared is sufficiently stable and does not lose enzyme activity after several weeks at temperatures slightly above freezing. Its recovery after the reaction is not expedient, although it can be reused for the reaction.

The enzyme reaction takes place in a heterogeneous mixture of biocatalyst and aqueous buffered solutions of both reaction substrates. A pH of 5.2 to 7.0 and a low ionic strength of the buffer have proven to be optimal. For preparative purposes, either a volatile buffer (e.g., triethylammonium hydrogen carbonate) or preferably 0.05 mol 1 ammonium acetate pH 5.8-5.9 can be used. Other buffer solutions may 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 pumping the solution through the biocatalyst column. The heterocyclic purine bases of formula II are in some cases poorly soluble in water, but the reaction can also be successfully carried out with the substance suspended in the buffer solution, since the resulting product of formula I is usually well soluble in water. As the deoxyribosyl residue donor, e.g.

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

The reaction proceeds 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 to the reactans, the ratio of the reactants to each other, their concentrations and the reaction time. For optimum results and maximum efficiency, a molar ratio of II (acceptor) to 2'-deoxyuridine (donor) of 1: 2 to 1: 3 has proven to be successful. For purposes preparativnl can be preferably used solutions of the resulting concentration to 0.2 mol l ^-1 at donor and 0.01 mol of 1 ^- ^ to an acceptor, i.e. with just five times the salt concentration of the buffer solution. The amount of biocatalyst used depends on the grain size: in the standard design (5 to 6 mm diameter geospherical beads), preferably 10 to 100 biocatalyst beads per 10 ml reaction mixture are used. This amount depends on the quality of the cell material, the relative amount of immobilized cells (based on the biocatalyst volume / i on the acceptor chemical properties). The reaction rate can be increased by the addition of another biocatalyst.

To interrupt the reaction, the incubation mixture is decanted from the bio-catalyst 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 (respectively thymine and 2'-deoxythymidine) in a ratio of about 1: 1, the starting heterocyclic base and the reaction product. The conversion to the compound of formula I is usually 70 to 100%. The chemical properties of these components allow in most cases easy isolation of the product and recovery of the other components of the mixture. For separation, for example, ion exchange chromatography on cation exchange resin, paper separation chromatography or chromatography on hydrophobised silica gel (e.g. octadecyl-silica gel) in an aqueous medium can be used. In this procedure, the reaction mixture can be directly applied to the sorbent column after ultrafiltration without prior concentration.

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

The reaction proceeds with compounds substituted in both the 2,6 and 8 positions of the purine ring and is not accompanied by side reactions. For example, 6-methylpurin-2'-deoxyribonucleoside (IV) (see above) can be prepared in good preparative yield. In the case of adenine, a 2'-deoxyinosulfide may be formed as a product of deamination of the reaction product over an extended reaction time. Both substances are easy to separate; however, for the economical preparation of the important 2'-deoxyadenosine (VI), a variant in which adenine is replaced by N, N-dimethylaminomethylenadenine of formula VII, readily available from adenine (Okumura K. et al., J. Org. Chem. 36, 1573 (1971)). The resulting transdeoxyribosylation product is not deaminated by adenosine deaminase. Treatment with a weakly alkaline medium, such as aqueous ammonia solutions, yields pure 2'-deoxyadenosine. Since the starting base VII is well soluble in water, this variant can be used for large-scale preparation of 2'-deoxyadenosine.

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

The biocatalyst according to the invention is also useful for the preparation of purine 2'-deoxyribonucleosides labeled with hydrogen, carbon and other atoms nuclides (radicuclides). Depending on the circumstances, either a labeled acceptor or donor or both labeled components (2'-deoxyuridine labeled in the sugar moiety) can be used to obtain labeled products on one or both moieties of the molecule. The procedure allows easy recovery of the labeled precursors.

Thus, a process for the synthesis of purine 2'-deoxyribonucleosides, based on the use of a biocatalyst, enables efficient preparation of these important substances in high yields from available raw materials, with low equipment and energy demands, and with easy product isolation and recovery of feedstocks.

In the following, the invention is illustrated by, but not limited to, the preparation of the subject biocatalyst and its use for the preparation of purine 2'-deoxyrobonucleosides.

He did

Preparation of Escherichia ooli SPT biocatalyst. A mixture of 0.75 g of alginate (e.g. Protanal LF, manufactured by Protan A / S.P.O., Drammen, Norway) and 15 ml of water is stirred until solo formation is obtained. 2.5 g wet weight of Escherichia coli SPT bacterial cell biomass (prepared according to Cs. Aut. Ref. 172 684) pre-suspended in 10 ml of 0.9% aqueous potassium chloride solution are gradually mixed into this sol. The resulting homogeneous mixture was dropped from a glass column having an internal diameter of 2.4 cm into a constantly stirred 2% calcium chloride solution in water (100 mL) at 20-25 ° C. Gel beads are formed in the calcium chloride solution which are sufficiently mechanically strong after 6 hours. The solution was decanted off and the gel beads were washed by decanting with 0.05 mol 1 ammonium acetate. This material was used as a biocatalyst in other examples.

Example 2

General procedure for the preparation of 2'-deoxyribonucleosides of formula I. To a solution of 25 µmoles of the heterocyclic base of formula II and 11.4 mg (100 µmoles) of 2'-deoxyuridine in 5 ml of 0.05 mol / ^l ammonium acetate pH 5.8, Add 7 beads of the biocatalyst according to example 1 and stir the mixture by slowly rocking at 37 ° C in a bath. of a mixture of 0.05 mol ¹⁷ triethylammonium acetate and methanol according to the data in Table 2. Elution rate 0.6 ml / min, detection at 254 nm. The peak halves of I and II are integrated and the conversion to I is calculated. These data are shown in Table 1.

Example 3

Comparison of the DNA efficiency of 2'-deoxyuridine and 2'-deoxythymidine. The reaction was carried out according to Example 2 using 25 µmole of 6-methylpurine and 50 µmole of 2'-deoxyuridine in 5 ml of buffer or using 25 µmole of 6-methylpurine and 50 µmole of 2'-deoxythymidine in 5 ml of buffer. 7 beads of the biocatalyst according to Example 1 were added to each mixture and stirred by rocking at 37 ° C. After 4 hours, aliquots of 10 µl of the mixtures were analyzed according to Example 2. When reacting with 2'-deoxyuridine, the conversion of 6-methylpurine to 9- (2-deoxy-beta-ribofuranosyl) -6-methylpurine was 54.5% when reacted with 2'-deoxyuridine. 2'-deoxythymidine 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 of Sorensen pH 6.9 phosphate buffer was added 150 beads of the biocatalyst according to Example 1 and the mixture was rocked at 37 ° C. hours. Decant the solution and wash the beads with 10 ml of water. This solution was filtered through a membrane filter (Millipore 0.4 / u) and loaded onto a column of 190 ml octadecyl-silica gel (30 / u) equilibrated with water. The column is eluted with water at a flow rate of 2 ml / min and the elution progress is monitored on a Uvicord LKB (Uppsala, Sweden) at 254 nm. The uracil and 2'-deoxyuridine were eluted sequentially, the fraction quality (10 min-intervals) was checked by the analysis of Example 2 in 0.05 moles of ^1-7 triethylammonium acetate. The 2'-deoxyuridine fractions were combined, evaporated at 40 ° C / 2 kPa and the residue crystallized from ethanol-ether. 128 mg of 2'-deoxyuridine are recovered.

Further elution with water gave 6-methylpurine (90 mg after crystallization from ethanol-petroleum ether). Then the column is washed under the same conditions with a 1: 9 mixture of methanol and water and the main fractions absorbing in ultraviolet light are 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 are obtained, m.p. 161 ° C. For (250.3) calculated: C 52.79, H 5.64, N 22.39;

Found: 52.85% C, 5.56% H, 22.14% N. Mass Spectrum: M ⁺ 250. UV Spectrum: _λmax ^{265 nm (£} max ^{7000) at pH 7} 'Anax ^{263 nm (} max max) ^{7,900) at pH 12} '

Example 5

Preparation of 2'-deoxyadenosine. To a solution of 76.4 mg (0.4 mmol) of N, 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 was added 100 beads. of the biocatalyst of Example 1 and the mixture was incubated at 37 ° C with slow rocking for 18 hours. The solution is decanted off, replaced with a fresh mixture (see above) and incubated for 3 hours under the same conditions. The same procedure is repeated once more. The combined decanted solutions (30 ml) were filtered according to Example 3 and the filtrate was mixed with an equal volume of conc. aqueous ammonia. The mixture was allowed to stand at room temperature for 18 h and evaporated at 40 ° C / 2 kPa.

The residue in 10 ml of water is added to a column of 100 ml of Amberlite IRC 50 (H ⁺ form) and the column is washed with water until loss of UV-absorption. Thereafter, a dilute (1:10) aqueous ammonia elutes the UV-absorbing fraction of the adenine compounds, which is evaporated under the same conditions. This residue was applied to a 90 ml column of octadecyl-silica gel (30 µl) in 5 ml of water and the column was eluted with water (2 ml / min). After the elution of the adenine is complete, it is further eluted with a 1: 9 mixture of methanol and water. Ultraviolet absorption refers to the elution of the product. This was combined, evaporated under the above conditions and crystallized from ethanol-ether. 138 mg (62%) of 2'-deoxyadenosine, identical to authentic material (Calbiochem, USA) were obtained by 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 was added 15 ml of dineopentylacetate of dimethylformamide, and the mixture was stirred at 110 ° C under a chloro-calcium cap to form a clear solution. Evaporate at 60 ° C / 13 Pa, add 50 ml of ethanol and ca 2 g of solid carbon dioxide. After dissolution, the mixture was re-evaporated at 40 ° C / 2 kPa and the residue crystallized from water. 3.5 g (85%) of N ² -dimethylaminomethyleneguanine are obtained, melting at 260 ° C. H, 4.89;% H, 4.89;% H, 4.89;

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

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

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

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

' _M ' max max nax

Example 7

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

Example 8

Preparation of [ ¹ ', 4'-C-guanine] -2'-deoxyguanosine. In a 10 ml vial, 1.85 MBq of [.alpha.] @ 1 H -guanine (specific activity 6.5 GBq / mmol) is evaporated under vacuum, 2 ml of 0.05 mol. 1 ammonium acetate pH 5.8, 3 balls of biocatalyst are added. according to Example 1 and 1 ml of a solution of 60 µg of 2-deoxyuridine in water. The mixture is incubated for 60 min at 37 ° C, transferred quantitatively to another 10 ml vial and concentrated in vacuo (2 kPa) over phosphorus pentoxide to a volume of ca 50 µl. This concentrate is applied in three portions to a Silasorb column (see Example 2) and chromatography is carried out in 0.05 mol 1 of triethylammonium acetate. The 2'-deoxyguanosine fractions obtained are evaporated at 40 ° C / 2 kPa and made up to 1 ml with water. The 2'-deoxyguanosine content, determined by measuring the redioactivity of the aliquot, is 0.74 MBq (40% yield).

Example 9

Determination of donor: acceptor ratio. Weigh 5 mg of 6-methylpurine into four 25 ml flasks, followed by 8.75 mg, 17.5 mg, 26.25 mg and 35 mg of 2'-deoxyuridine respectively. To the flasks was added 5 mL of 0.05 L of 1 ^L ammonium acetate pH 5.8 and 5 balls of the biocatalyst each of Example 1.

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

He did

Determination of the influence of pH on the synthesis. Weigh 5 ml of 6-methylpurine and 17.5 mg of 2'-deoxyuridine into 25 ml flasks and add 5 ml of 0.05 mol 1 ^{l of} buffer (ammonium acetate pH 5.2 to 6.2 or Sorensen phosphate pH 6.9). The flasks were incubated for 90 min at 37 ° C after addition of 5 beads of the biocatalyst according to Example 1 and the conversion was determined by the method of Example 2. The values obtained are shown in Table 4. The optimum pH for this synthesis was 5.8.

Table 1

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

Conversions i

R

^CH 3 H H 64.5 nh ₂ H H 63,5 ¹ CH ₃ NH H H 64.5 (ch ₂ ) ₂ h H H 70.0 (CH ₃ ) ₂ N-CH = N H H 75.0 NH ₂ ^CH 3 H 67.0 ^NH 2 nh ₂ H 46.0 ™ 2 H NH ₂ 21.0 Cl H H 63.5 Cl Cl H 33.0 II nh ₂ H 46.0 ^NH 2 SCK ₃ H 72.0 OH H H 38.5 OH NH ₂ H 34.0 OH (CH ₃ ) ₂ n-ch = n H 58.0 sch ₃ H H 63.0 ^and See Example 2; thereof 11% 2'-deoxyinosine incurred deamination

·.

Table 2

Liquid chromatography of compounds of formulas I and II to ^a

R R ‘ R ^J Sl S2 S3 S4 S5 ^NH 2 H Η I 6.59 3.59 II 3.10 2.46 ch ₃ nh H Η I 6.36 II 4.55 (ch ₃ ) ₂ n H Η I 4.69 II 4.03 (CH ₃ ) ₂ N-CH = N H Η I 2.05 II 1.69 ^NH 2 ^CH 3 Η I 4.61 II 2.79 ^NH 2 ^NH 2 Η I 1.84 II 1,28 ^NH 2 ^SCÍ1 3 Η I 3.90 II 3.45 ^NH 2 H NH _O I 3.17 ² II 1.66 H nh ₂ Η I 1.89 II 1.08 ^CH 3 H Η I 2.90 II 1.75 Cl H Η I 3.55 II 2.18 Cl Cl Η I 2.79 II 1.69 OH H Η I 1.88 1.09 II 0.77 0.54 OH ^NH 2 Η I 1,02 II 0.17 OH (ch ₃ ) _O N-CH = N Η I 3.93 ² II 2.26 ^SCH 3 H Η I 2.31 II 2.26 Uráčil 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

2'-Deoxyuridine: 6-methylpurine molar ratio Conversion of 6-methylpurine,% ¹ See Example 9.

Table 4

Effect of pH on 9- (2-deoxy-beta-D-ribofuranosyl) -6-methylpurine enzyme synthesis ^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 '.