Abstract:
Purine-arabinosides and a method for making purine-arabinosides are disclosed. The method comprises contacting an arabinose donor and a purine source in the presence of an effective amount of enzyme produced by a bacterium and capable of transarabinosylation from the arabinose donor to the purine source, whereby a 9-(β-D-arabinofuranosyl)-purine is produced.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a method for producing purine-arabinosides, particularly by an enzymatic process. 
     2. Description of the Prior Art 
     Purine-arabinosides (9-(β-D-arabinofuranosyl)-purines) have potential utility as agricultural chemicals or medicinal agents. For example, it has been reported that adenine arabinoside, one of the purine arabinosides has been used successfully to treat several diseases caused by the herpes virus including chickenpox and shingles. 
     As to known methods for producing the purine arabinosides, several chemically synthetic methods have been proposed, (J. Org. Chem. 27, 3274, (1962); J. Org. Chem. 28, 3004 (1963); J. Org. Chem, 32, (1976); Tetrahedron Letters 1970, 4673; and Japanese Published Exmined Patent Application No. 7271/1972). It is further reported that adenine arabinoside is produced when Streptomyces antibioticus is cultured in conventional culture media (Japanese Published Examined Patent Application No. 41558/1972). 
     SUMMARY OF THE INVENTION 
     It has been found that purine arabinosides are produced in aqueous reaction media from an arabinose donor such as uracil arabinoside or D-arabinofuranose-1-phosphate and a purine-source such as adenine, hypoxanthine, adenosine and adenosine-5&#39;-monophosphate by the action of an enzyme produced by various bacteria. 
     A commercially applicable method for producing purine arabinosides has now been provided by 
     (a) holding at a temperature in the range from 40° to 70° C. in an aqueous medium an arabinose donor selected from the group consisting of D-arabinofuranose-1-phosphate, and the compound having Formula I on a nucleotide thereof; and a purine source selected from the group consisting of unsubstituted or 2,6 and/or 8-substituted purine and its ribofuranoside, ribofuranotide, deoxyribofuranoside or deoxyribofuranotide, in the presence of an effective amount of an enzyme produced by a bacterium and capable of transarabinosylation from the arabinose donor to the unsubstituted or 2,6 and/or 8-substituted purine of the purine source, whereby the β-D-arabinofuranosyl radical is attached to the 9-position of the unsubstituted or 2,6 and/or 8-substituted purine; and 
     (b) recovering the produced 9-(β-D-arabinofuranosyl)-unsubstituted or 2,6 and/or 8-substituted purine. ##STR1## X represents O, S or NH; Y represents OH, NH 2 , SH or SR(R is a lower alkyl group); and 
     Z represents H, halogen, NO 2 , CH 3  or CH 2  OH. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein: 
     FIG. 1 is the NMR spectrum of the crystalline product obtained in Example 5. 
     FIG. 2 is the ultra-violet spectrum of the product obtained in Example 5. 
     FIG. 3 is the IR spectrum of the product obtained in Example 5. 
     FIG. 4 is the NMR spectrum of the crystalline product obtained from Example 6. 
     FIG. 5 is the UV spectrum of the product obtained from Example 6. 
     FIG. 6 is the IR spectrum of the product obtained from Example 6. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The arabinose donors of this invention are D-arabinofuranose-1-phosphates of, the compounds of formula I, or the phosphate of the compound showing formula I. The specimens of the arabinose donors are shown in the Examples of this invention. 
     The purine sources of this invention are unsubstituted or 2,6 and/or 8-substituted purine and its ribofuranoside, deoxyribofuranoside or deoxyribofuranotide. The 2,6 and/or 8-substituted purine used in this invention as the purine source can be prepared by the following method: the Ribofuranoside of a 2,6 and/or 8-substituted purine is held with the enzyme of this invention in an aqueous medium containing 0.1 M KH 2  PO 4  at 60° C. for 24 hours. When the 2,6 and/or 8 substituted purine of the originally used 2,6 and/or 8-substituted purine ribofuranoside, and D-ribofuranose-1-phosphate or D-ribose derived from the above ribofuranoside are produced in the aqueous medium, the 2,6 and/or 8-substituted purine can then be used as the purine source. 
     The substituents of the 2,6 and/or 8-substituted purines are, for example, halogen, hydroxyl, amino, lower alkyl, alkoxyl, aryl, aralkyl, mercapto, alkylamino, alkylmercapto, alkylsulfonyl, alkylsulfenyl, carboxyl, alkoxycarbonyl, cyano, and nitro radicals. 
     The D-arabinofuranose of the arabinose donor is enzymatically transferred to and attached to 9-position of the unsubstituted or 2,6/or 8-substituted purine of the purine source. Thus, the product of this invention is 9-(β-D-arabinofuranosyl)-unsubstituted or 2,6 and/or 8-substituted purine. 
     The bacterial enzyme capable of transarabinosylation from the arabinose donor to unsubstituted or 2,6 and/or 8-substituted purine of the purine source is produced mainly in the bacterial cells and is present to a small extent in the supernatant of the culture liquids. The bacteria capable of producing the enzyme belong, as found so far, to the genera Pseudomonas, Flavobacterium, Achromobacter, Salmonella, Citrobacter, Escherichia, Klebsiella, Enterobacter, Aeromonas, Serratia, Erwinia, Proteus, Xanthomonas, and Bacterium. 
     Specimens of the bacteria are: 
     
         ______________________________________Pseudomonas stutzeri           NRRL B-11346 (FERM-P 4170),Flavobacterium rhenanum           NRRL B-11343 (CCM 298),Flavobacterium acidoficum           ATCC 8366,Flavobacterium proteus           ATCC 12841,Achromobacter lacticum           NRRL B-11340 (CCM 69),Salmonella typhimirim           NRRL B-11347 (FERM-P3735),Citrobacter freundii           ATCC 8090,Citrobacter freundii           ATCC 6750,(Citrobacter intermedium)Escherichia coli           ATCC 9637,Escherichia aurescens           ATCC 12814,Klebsiella pneumoniae           ATCC 9621,(Enterobacter aerogenes)Serratia liquefaciens           ATCC 14460,(Enterobacter liquefaciens)Enterobacter aerogenes           ATCC 13048,Aeromonas punctata           ATCC 11163,Aeromonas salmonicida           ATCC 14174,Serratia marcescens           IFO 3048,Erwinia carotovora           NRRL B-11342 (CCM 872),Erwinia amylovara           NRRL B-11341 (CCM 1017),Erwinia herbicola           ATCC 14537,Proteus vulgaris           NRRL B-11345 (FERM-P3394),Proteus rettgeri           NRRL B-11344 (FERM-P3395),Bacterium cadaveris           IFO 3731, andXanthomonas citri           NRRL B-11348 (FERM-P3396).______________________________________ 
    
     In order to produce the enzyme using the bacteria as mentioned above, the bacteria are cultured in or on conventional culture media. The culture media contain conventional carbon sources, nitrogen sources, inorganic ions, and when required minor organic nutrients such as vitamins and amino acid. Usual manner can be applied to culture the bacteria in the conventional media, that is, the bacteria are cultured aerobically preferably at a pH of a range from 4 to 9 and a temperature of a range from 25° to 40° C. 
     As the enzyme source, intact cells, culture liquids containing the cells are used preferably. Additionally, cells dried with acetone, freeze-dried cells, homogenized cells, cells treated with supersonic waves, cells treated with toluene, surfactants or lysozyme are employed giving desirable results. Moreover protein fractions having the enzyme activity capable of transarabinosylation from the arabinose donor to unsubstituted or 2,6 and/or 8-substituted purine of the purine source can be used preferably as the enzyme source. It is expected that there is more than one enzyme participating in the production of the purine arabinosides. 
     The production of the purine arabinosides can be carried out by holding in the culture media of the bacteria the purine source and the arabinose donor. In this case, the arabinose donor and purine source are added into the culture media after the bacteria has grown sufficiently, and thereafter the temperature is maintained at 40° C. to 70° C. The production of the purine arabinoside can be also carried out by contacting the purine source and arabinose-donor with the cells or the enzyme sources as mentioned above in aqueous reaction media other than culture media. Thus, in this invention, &#34;aqueous medium&#34; means culture medium or reaction medium (reaction mixture). The reaction media are maintained preferably at a temperature from 40° C. to 70° C., and at a pH of 4 to 10 for 5 to 100 hours. 
     The reaction temperature (40° C. to 70° C.) of this invention is specific in the point that the temperature is higher than the ordinarily enzyme reaction temperature, and it is critical. 
     The purine arabinosides produced in the culture media or the reaction media can be recovered by conventional manners such as ion exchange method or crystallization technique. 
     Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified. 
     EXAMPLE 1 
     An aqueous culture medium of pH 7.2 was prepared which contained, per deciliter, 0.5 g yeast extract, 1.0 g peptone, 0.5 g bouillon, and 0.5 g NaCl. Five ml batches of the aqueous culture medium were placed in test tubes, and heated to sterilize. Each one loopful inocculum of the bacteria listed in Table 1 was transferred into each batch of the aqueous culture medium. Cultivation was carried out at 30° C. for 36 hours with shaking. The cells produced in the culture liquid were collected by centrifugation and washed with physiological saline. The cells thus obtained (50 mg(wet)/ml) were suspended in samples of 0.05 M phosphate buffer of pH 7.0, and 0.5 ml of the suspension of the cells was mixed with 0.5 ml of reaction mixture of pH 7.5 containing 0.5 g/dl uracil arabinoside, 0.2 g/dl hypoxanthine and 50 mg/dl KH 2  PO 4 . Each mixture was held at 60° C. for 15 hours, and thereafter heated to 100° C. for 5 minutes. 
     Each product in the reaction mixture was identified as 9-β-D-arabinofuranosylhypoxanthine (hypoxanthine arabinoside) by high speed liquid chromatography, and the amounts of the hypoxanthine arabinoside in the reaction mixture were determined by high speed liquid chromatography, and are shown in Table 1. 
     
                       TABLE 1______________________________________microorganism hypoxanthine arabinosideused          accumulated mg/dl______________________________________NRRL B-11343  3.7ATCC 8366     6.6ATCC 12841    6.7NRRL B-11340  5.7NRRL B-11347  7.5ATCC 8090     11.3ATCC 6750     13.2ATCC 9637     10.5ATCC 12814    17.0ATCC 9621     126.0ATCC 14460    17.0ATCC 14174    36.0ATCC 11163    4.1IFO 3048      23.0NRRL B-11342  14.0NRRL B-11341  18.0ATCC 14537    21.0NRRL B-11345  9.6NRRL B-11344  2.4NRRL B-11348  11.0IFO 3731      12.0NRRL B-11346  7.5ATCC 13048    55.7______________________________________ 
    
     EXAMPLE 2 
     In the method shown in Example 1, adenine was substituted for hypoxanthine, and the amounts of adenine arabinoside shown in Table 2 were produced in the reaction mixture. 
     EXAMPLE 3 
     In the method shown in Example 1, cytosine arabinoside was substituted for uracil arabinoside, and the amounts of hypoxanthine arabinoside shown in Table 3 were produced in the reaction mixture. 
     EXAMPLE 4 
     In the method shown in Example 1, adenine riboside-5&#39;-monophosphate was substituted for hypoxanthine, and the amounts of adenine arabinoside shown in Table 4 were accumulated in the reaction mixture. 
     
                       TABLE 2______________________________________microorganism  adenine arabinosideused           accumulated mg/dl______________________________________ NRRL B-11343  4.5ATCC 8366      8.2ATCC 12841     8.0NRRL B-11340   6.5NRRL B-11347   8.6ATCC 8090      13.3ATCC 6750      15.0ATCC 9637      10.6ATCC 12814     18.8ATCC 9621      132.0ATCC 14460     26.0ATCC 14174     41.0ATCC 11163     18.5IFO 3048       32.6NRRL B-11342   20.5NRRL B-11341   22.5ATCC 14537     31.5NRRL B-11345   26.3NRRL B-11344   28.6NRRL B-11348   13.5IFO 3731       21.2NRRL B-11346   8.6ATCC 13048     71.8______________________________________ 
    
     
                       TABLE 3______________________________________microorganism hypoxanthine arabinosideused          accumulated mg/dl______________________________________NRRL B-11343  4.2ATCC 8366     5.5ATCC 12841    8.2NRRL B-11340  2.6NRRL B-11347  4.8ATCC 8090     6.5ATCC 6750     10.3ATCC 9637     6.3ATCC 12814    3.6ATCC 9621     82.1ATCC 14460    15.0ATCC 14174    20.5ATCC 11163    0.8IFO 3048      13.6NRRL B-11342  2.6NRRL B-11341  8.7ATCC 14537    15.0NRRL B-11345  8.1NRRL B-11344  0.5NRRL B-11348  0.8IFO 3731      10.6NRRL B-11346  3.2ATCC 13048    40.2______________________________________ 
    
     
                       TABLE 4______________________________________microorganism  adenine arabinosideused           accumulated mg/dl______________________________________NRRL B-11343   3.8ATCC 8366      5.6ATCC 12841     7.2NRRL B-11340   3.5NRRL B-11347   8.3ATCC 8090      10.2ATCC 6750      8.6ATCC 9637      5.5ATCC 12814     6.9ATCC 9621      82.3ATCC 14460     13.5ATCC 14174     25.5ATCC 11163     9.6IFO 3048       21.5NRRL B-11342   15.5NRRL B-11341   11.5ATCC 14537     18.3NRRL B-11345   12.6NRRL B-11344   15.8NRRL B-11348   8.3IFO 3731       14.5NRRL B-11346   8.5ATCC 13048     49.6______________________________________ 
    
     EXAMPLE 5 
     A hundred ml batches of the aqueous culture medium shown in Example 1 were placed in a 500 ml shaking flask and heated to sterilize. Klebsiella pneumoniae ATCC 9621 was inocculated in the aqueous culture medium and cultured at 30° C. for 36 hours with shaking. Cells produced in the resultant culture liquid were collected by centrifugation, and 30 g (wet) of the cells was put into 1 l of the reaction mixture of pH 7.0 containing 1.5 g 2-methylhypoxanthine, 7.3 g uracil arabinoside and 3.4 g KH 2  PO 4 . The reaction mixture was held at 60° C. for 36 hours. 
     Cells were removed from the reaction mixture by centrifugation, the supernatant was passed through cation exchange resin (&#34;Amberlite CG-120&#34;), and the resin was washed with 0.1 N ammonium acetate (pH 6.8). After eluting with 0.1 N ammonium hydroxide, the eluate was evaporated and cooled, and 710 mg crystals were obtained. 
     The crystalline product was determined as 9-(β-D-arabinofuranosyl)-2-methylhypoxanthine(2-methylhypoxanthine arabinoside) by its NMR spectrum, UV spectrum, IR spectrum, and elemental analysis. 
     Elemental analysis: Calculated; C:46.8%, H:5.0%, N:19.8%. Found; C:46.5%, H:5.1%, N:19.5%. 
     NMR spectrum: shown in FIG. 1. 
     UV spectrum: shown in FIG. 2. 
     IR spectrum: shown in FIG. 3. 
     EXAMPLE 6 
     Thirty grams of the cells obtained in Example 4 were put into 1 l of reaction mixture containing 1.7 g 2-chloro-hypoxanthine, 7.3 g uracil arabinoside, and 3.4 g KH 2  PO 4 , and the reaction mixture was held at 60° C. for 36 hours. After removing the cells from the reaction mixture, the supernatant was passed through anion exchange resin (&#34;Dowex IX4&#34;), and the resin was washed with 0.1 N ammonium acetate of pH 6.8. After eluting with 0.1 N ammonium acetate of pH 4.0, the eluate was evaporated, and charged on &#34;Sephadex G-10&#34;, and developed with water. The eluate portions showing the first of two peaks peak of UV absorption of the two was collected, evaporated and cooled. Then, 326 mg crystals were obtained. 
     The crystalline product was determined as 9-(β-D-arabinofuranosy)-2-chlorohypoxanthine(2-chlorohypoxanthine arabinoside) by its NMR spectrum, UV spectrum, IR spectrum, elemental analysis and Beilstein test. 
     Elemental analysis: Calculated; C:39.68, H:3.66, N:18.51. Found; C:39.42, H:3.72, N:18.25. 
     NMR spectrum: shown in FIG. 4 
     UV spectrum: shown in FIG. 5 
     IR spectrum: shown in FIG. 6 
     Beilstein test: positive (green) 
     EXAMPLE 7 
     In the method shown in Example 1, 2-methylhypoxanthine or 2-chlorohypoxanthine was substituted for hypoxanthine, and the amounts of 2-methylhypoxanthine arabinoside or 2-chlorohypoxanthine arabinoside shown in Table 5 were accumulated in the reaction mixture. 
     EXAMPLE 8 
     In the method shown in Example 1, 0.2 g/dl hypoxanthine was replaced with 0.4 g/dl inosine, and the amounts of hypoxanthine arabinoside shown in Table 6 were produced in the reaction mixture. 
     EXAMPLE 9 
     A hundred ml of the aqueous culture medium shown in Example 1 was placed in a 500 ml shaking flask, heated to sterilize, and inoculated with Aeromonas salmonicida ATCC 14174. Cultivation was carried out at 30° C. for 36 hours with shaking. 
     Cells produced in the resultant culture liquid were collected by centrifiguation, and 2.0 g (wet weight) of the cells were put into 100 ml reaction mixture of pH 7.5 containing 100 mg hypoxanthine, 300 mg uracil arabinoside and 50 mg KH 2  PO 4 . The reaction mixture was then held at 60° C. for 15 hours. 
     Twenty five mg of crystals of hypoxanthine arabinoside were obtained from the reaction mixture. 
     
                       TABLE 5______________________________________                      2-chlorohypo-         2-methylhypoxan-                      xanthine         thine arabinoside                      arabinoside         accumulated  accumulatedmicroorganism mg/dl        mg/dl______________________________________NRRL B-11343  2.1          0.5ATCC 8366     3.4          0.8ATCC 12841    4.0          2.1NRRL B-11340  5.5          2.5NRRL B-11347  4.8          2.8ATCC 8090     8.7          3.6ATCC 6750     9.5          8.2ATCC 9637     4.7          5.1ATCC 12814    12.0         10.5ATCC 9621     80.5         51.6ATCC 14460    18.5         11.3ATCC 14174    21.6         10.0ATCC 11163    0.8          0.05IFO 3048      15.4         10.8NRRL B-11342  2.5          0.1NRRL B-11341  12.0         10.5ATCC 14537    15.5         12.1NRRL B-11345  0.6          0.05NRRL B-11344  8.2          0.5NRRL B-11348  12.5         0.8IFO 3731      21.6         2.1NRRL B-11346  15.3         10.3ATCC 13048    40.2         28.7______________________________________ 
    
     
                       TABLE 6______________________________________          hypoxanthine arabinosidemicroorganism used          accumulated mg/dl______________________________________NRRL B-11343   2.8ATCC 8366      3.6ATCC 12841     5.5NRRL B-11340   4.3NRRL B-11347   6.2ATCC 8090      8.8ATCC 6750      7.4ATCC 9637      1.6ATCC 12814     13.6ATCC 9621      83.3ATCC 14460     6.2ATCC 14174     16.8ATCC 11163     0.9IFO 3048       15.3NRRL B-11342   6.8NRRL B-11341   10.2ATCC 14537     8.9NRRL B-11345   8.5NRRL B-11344   0.8NRRL B-11348   7.2IFO 3731       5.8NRRL B-11346   3.3ATCC 13048     40.4______________________________________ 
    
     EXAMPLE 10 
     Klebsiella pneumoniae ATCC 9621 was cultured in the manner shown in Example 9. Cells in the resultant culture liquid were collected by centrifugation, and 2 g (wet weight) of the cells were suspended in 100 ml reaction mixture of pH 7.5 containing 100 mg hypoxanthine, 300 mg cytosine arabinoside, 50 mg KH 2  PO 4 , and the reaction mixture was held at 60° C. for 15 hours. 
     The cells in the reaction mixture were removed by centrifugation, and a concentrate of the supernatant was passed through anion exchange resin (&#34;Dowex-1&#34; OH form, pH 6.8). After eluting with 0.1 N formic acid of pH 4.0, the eluate was passed through &#34;Sephadex G-10&#34;. Eluate (250 ml) obtained by eluting with water was concentrated and the concentrate was added with methanol and cooled to form crystals of the product. After re-crystallization with water, 35 mg purified crystals were obtained. 
     The crystalline product was identified with authentic hypoxanthine arabinoside by its NMR spectrum, IR spectrum and UV spectrum. 
     EXAMPLE 11 
     Klebsiella pneumoniae ATCC 9621 was cultured by the same manner as in Example 9, and cells were collected by centrifugation. 
     Hypoxanthine in the reaction mixture in Example 9 was replaced with adenine, and the reaction mixture was held at 60° C. for 15 hours. The supernatant of the reaction mixture was concentrated to 20 ml. Upon cooling the concentrate, 80 mg crystals were obtained. 
     The crystalline product was identified with authentic adenine arabinoside by its NMR spectrum, IR spectrum, and UV spectrum. 
     EXAMPLE 12 
     Erwinia hervicola ATCC 14537 was cultured by the same manner as in Example 9, and the cells produced were collected by centrifugation. 
     The cells thus obtained (2 g (wet weight)/dl) were suspended in 100 ml of a reaction mixture of pH 7.5 containing 100 mg/dl adenine, 300 mg/dl cytosine arabinoside and 50 mg KH 2  PO 4 , and held at 60° C. for 15 hours. 
     After removing the cells from the reaction mixture, the reaction mixture was concentrated to 20 ml, and cooled. The crystals thus obtained were recrystallized with water and 55 mg purified crystals were obtained. The crystalline product was identified with adenine arabinoside by its NMR spectrum, IR spectrum and UV spectrum. 
     EXAMPLE 13 
     Cells (5 g (wet)/dl) of Aeromonas salmonicida ATCC 14174 were suspended in 100 ml batches of a reaction mixture containing 30 mM cytosine arabinoside, 25 mM KH 2  PO 4 , and 10 mM of one of the purines shown in Table 7. The reaction mixtures were placed in test tubes and held at 60° C. for 15 hours. 
     Newly formed product having UV absorption in the resultant reaction mixture was separated by liquid chromatography. The eluate of the chromatography was concentrated and added with ethanol, whereby crystals were formed in the eluate. 
     From NMR spectra and UV spectra of the purified crystalline products, the products were ascertained as the arabinosides of the respective purines used as the starting materials. 
     Conversion ratio of the purine arabinosides from purine source were determined by measuring molecular extinction coefficient, and are shown in Table 7. 
     
                       TABLE 7______________________________________                          Conversion                          ratioStarting material        Product           (%)______________________________________xanthine     xanthine arabinoside                          15guanine      guanine arabinoside                          8purine       purine arabinoside                          236-mercaptopurine        6-mercaptopurine arabinoside                          82,6-diaminopurine        2,6-diaminopurine arabino-                          38        side6-mercaptoguanine        6-mereaptoguanine arabino-                          7        side2-methylhypoxanthine        2-methylhypoxanthine                          35        arabinoside2-chlorohypoxanthine        2-chlorohypoxanthine                          18        arabinoside______________________________________ 
    
     EXAMPLE 14 
     Cells (5 g (wet)/dl) of Klebsiella pneumoniae ATCC 9621 were suspended in 100 ml batches of a reaction mixture placed in test tubes, containing 30 mM uracil arabinoside, 25 mM KH 2  PO 4 , and one of the purine sources (10 mM) listed in Table 8, and the reaction mixture was held at 60° C. for 15 hours. 
     Newly formed product having UV-absorption in the resultant reaction mixture was separated by liquid chromatography. The eluate of the chromatography was concentrated and added to ethanol, whereby crystals were formed in the eluate. 
     From NMR spectra of the purified crystalline products, the products were ascertained as the arabinoside of the respective purine sources used as starting materials. 
     Conversion rate of purine arabinosides from the purine sources used was determined by measuring the molecular extinction, coefficient, and are shown in Table 8. 
     
                       TABLE 8______________________________________                          Conversion                          ratioStarting material        Product           (%)______________________________________xanthine     xanthine arabinoside                          65guanine      guanine arabinoside                          20purine       purine arabinoside                          366-mercaptopurine        6-mercaptopurine arabinoside                          82,6-diaminopurine        2,6-diaminopurine arabino-                          52        side6-mercaptoguanine        6-mercaptoguanine arabino-                          5        side______________________________________ 
    
     EXAMPLE 15 
     In the method shown in Example 5, 2-methylhypoxanthine was replaced with 2-ethylhypoxanthine. The resultant reaction mixture was charged on thin-layer silica-gel, and the chromatogram was developed with water-saturated butanol. The part of Rf 0.4 having absorption at 260 nm on the thin-layer was collected, and suspended in 0.1 NHCl, and silica-gel was removed from the suspension. 
     When the supernatant of the suspension was made 6 N with HCl and boiled for 10 minutes, orcinol-ferric chloride reaction of the boiled suspension became positive, and 2-ethylhypoxanthine was found in the boiled suspension by paper-chromatography. Thus, it is suggested that 2-ethylhypoxanthine arabinoside was produced in the reaction mixture. 
     EXAMPLE 16 
     Cells of Klebsiella pneumoniae ATCC 9621 were obtained by the same manner as in Example 9, suspended in 0.5 M phosphate buffer of pH 7.5 to obtain 100 g (wet)/l, and treated with super supersonic. 
     A hundred ml of a reaction mixture, of pH 7.5 containing 50 ml/dl the supernatant, 500 mg/dl uracil arabinoside-5-monophosphate, 100 mg/dl hypoxanthine and 30 mg/dl KH 2  PO 4 , was held at 60° C. for 15 hours. Then the reaction mixture was centrifuged to remove precipitates, and the supernatant was passed through cation exchange resin (&#34;Chromobead C-2&#34;). 
     Elution was made with 0.3 N formic acid, and the eluate was charged on anion exchange resin (&#34;Dowex 1×4&#34;). Hypoxanthine arabinoside was eluted by gradient elution with ammonium formate of pH 9 to 3 and 8 mg of crystals were obtained from the eluate. 
     EXAMPLE 17 
     One ml of a reaction mixture containing, per milliliter, 0.2 ml of the supernatant shown in Example 16. 10 mg uracil arabinoside, 2 mg KH 2  PO 4 , 2 mg of one of the purine sources shown below was held at 60° C. in a test tube for 15 hours, and heated at 100° C. for 5 minutes. 
     After removing precipitates in the reaction mixture, the reaction mixture was subjected to paper chromatography, and the spot having UV-absorption and having a Rf value different from that of the purine sources used as the starting material was cut, and put into 0.1 N HCl. Then the 0.1 N HCl was made 6 N by adding concentrated HCl after removing filter paper, and boiled for 10 minutes, arabinose was found by an ferric chloride reaction in the boiled 6 NHCl. Thus, it is expected that arabinosides of the following sources used as the starting materials were produced in the reaction mixtures: 
     6-chloropurine 
     2-chlorohypoxanthine 
     2-aminopurine 
     2-methylthiohypoxanthine 
     8-chloroadenine 
     6-mercaptopurine 
     6-methylthiopurine 
     2-amino-6-mercaptopurine 
     6-carboxypurine 
     8-bromoadenine 
     EXAMPLE 18 
     In the method shown in Example 16, uracil arabinoside-5&#39;-monophosphate was replaced with cytocine arabinoside-5&#39;-monophosphate. In the resultant reaction mixture, hypoxanthine arabinoside was found. 
     
                       TABLE 9______________________________________microorganism hypoxanthine arabinosideused          accumulated mg/dl______________________________________NRRL B-11343  2.8ATCC 8366     5.5ATCC 12841    6.3NRRL B-11340  6.0NRRL B-11347  5.2ATCC 8090     8.8ATCC 6750     10.6ATCC 9637     8.5ATCC 12814    12.3ATCC 9621     103.6ATCC 14460    12.5ATCC 14174    29.3ATCC 11163    4.0IFO 3048      24.0NRRL B-11342  15.2NRRL B-11341  17.6ATCC 14537    22.3NRRL B-11345  15.6NRRL B-11344  3.2NRRL B-11348  10.6IFO 3731      18.3NRRL B-11346  8.2ATCC 13048    48.5______________________________________ 
    
     EXAMPLE 19 
     In the method shown in Example 1, D-arabinofuranose-1-phosphate was substituted for uracil arabinoside, and the amounts of hypoxanthine arabinoside shown in Table 9 were accumlated in the reaction mixture. 
     EXAMPLE 20 
     In the method shown in Example 19, one of the purine sources listed in Table 10 was substituted for hypoxanthine, and newly formed product having UV-absorption in the resultant reaction mixture was separated by preparative high speed liquid chromatography. The eluate of the chromatography was concentrated and added to ethanol, whereby crystals were formed in the eluate. From NMR spectra and UV spectra of the crystalline products, the products were ascertained as the arabinosides of the respective purine sources used as the starting materials. 
     The conversion ratio of the purine sources used to the purine arabinosides was determined by measuring the molecular extention coefficient and shown in Table 10. 
     
                       TABLE 10______________________________________                          Conversion                          ratioStarting material        product           (%)______________________________________xanthine     xanthine arabinoside                          15guanine      guanine arabinoside                          8purine       purine arabinoside                          236-mercaptopurine        6-mercaptopurine arabinoside                          82,6-diaminopurine        2,6-diaminopurine arabino-                          38        side6-mercaptoguanine        6-mercaptoguanine 7        arabinoside2-methylhypoxanthine        2-methylhypoxanthine                          35        arabinoside2-chlorohypoxanthine        2-chlorohypoxanthine                          18        arabinoside______________________________________ 
    
     EXAMPLE 21 
     Klebsiella pneumoniae ATCC 9621 was cultured by the same manner as in Example 9, and the cells produced were collected by centrifugation. 
     20 mg of the cells obtained were suspended in 1 ml of a reaction mixture of pH 7.0 containing 1.5 mg of adenine. 10 mg of one of the pyrimidine arabinosides listed in Table 11, and 3.4 mg of KH 2  PO 4 , and the reaction mixture was held at 60° C. for 15 hours. 
     Cells were removed from the reaction mixture by centrifugation. Adenine arabinoside accumulated was identified by high speed liquid chromatography. 
     
                       TABLE 11______________________________________  arabinose donor______________________________________4-thiouracil arabinofuranoside4-(S-methyl-)thiouracil arabinofuranoside2-thiouracil arabinofuranoside5-nitrouracil arabinofuranoside5-hydroxymethyluracil arabinofuranosideisocytosine arabinofuranoside5-fluorouracil arabinofuranoside5-bromouracil arabinofuranoside5-Iodouracil arabinofuranosidethymine arabinofuranoside______________________________________ 
    
     EXAMPLE 22 
     In the method shown in Example 11, adenine in the reaction mixture was replaced with 200 mg adenylic acid, and the reaction mixture was held at 60° C. for 15 hours. The supernatant of the reaction mixture was concentrated to 30 ml. Upon cooling the concentrate, 48 mg crystals were obtained. The crystalline product was identified with authentic adenine arabinoside by its NMR spectrum IR spectrum, and UV spectrum. 
     EXAMPLE 23 
     In the method shown in Example 11, adenine in the reaction mixture was replaced with 150 mg guanosine, and the reaction mixture was held at 60° C. for 15 hours. The crystals of 2-(β-D-arabinofuranosyl)guanine (guanine arabinoside) (28 mg) were obtained from the supernatant of the resulted reaction mixture. 
     EXAMPLE 24 
     In the method shown in Example 13, adenosine, deoxyadenosine, deoxyadenylic acid, guanylic acid, deoxyguanylic acid, xanthosine, deoxyxanthosine, deoxyinosine or deoxyinosinic acid were used in place of hypoxanthine as the purine source. From the above adenine source, adenine arabinoside was formed in the reaction mixture and separated by the usual manner. From the above guanine source, guanine arabinoside was formed. From the above xanthine source, the xanthine arabinoside was formed. From the hypoxanthine source, hypoxanthine arabinoside was formed. 
     Having now fully described this invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention set forth herein.