CA1120876A - Method for producing purine arabinosides - Google Patents
Method for producing purine arabinosidesInfo
- Publication number
- CA1120876A CA1120876A CA000309031A CA309031A CA1120876A CA 1120876 A CA1120876 A CA 1120876A CA 000309031 A CA000309031 A CA 000309031A CA 309031 A CA309031 A CA 309031A CA 1120876 A CA1120876 A CA 1120876A
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- Prior art keywords
- atcc
- purine
- nrrl
- arabinoside
- group
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
Abstract
ABSTRACT
Purine arabinosides are produced in an aqueous medium at a temperature in the range of 40° to 70°C from an arabinose donor and a purine source by the action of an enzyme of bacterial origin; the purine arabinosides may be used as agricultural chemicals or or medicinals.
Purine arabinosides are produced in an aqueous medium at a temperature in the range of 40° to 70°C from an arabinose donor and a purine source by the action of an enzyme of bacterial origin; the purine arabinosides may be used as agricultural chemicals or or medicinals.
Description
This invention relates to a method for producing puri~e-arabinosides, particularly by an enzymatic process.
Purine-arabinosides (9-(~-D-arabinofuranosyl)-purines) have potential utility as agricultural chemicals or medici-nal agents. For example, it has been reported that adenine arabinoside, one o~ 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 arabino-side~, 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 Patent Application ~o.
7271/1972). It is further reported that adenine arabinoside is produced when Streptomyces antibioticus is cultured in conventional culture media (Japanese Published Patent Application ~o. 41558/1972).
It has been found that purine arabino~ides are pro-duced in aqueous reaction media from arabinose donor, for example uracil arabinoside or D-arabinofuranose-l-phosphate and purine-source such as adenine, hypoxanthine, adenosine and adenosine-5'-monophosphate by the action of an enzyme produced by various bacteria, and this represents a simple method of manufacture.
~12~876 The present invention provides a method for pro-ducing purine arabinosides, which method is susceptible to commercial exploitation.
According to the invention there is provided a method of producing a purine arabinoside which comprises:
(a) holding at a temperature in the range from 40 to 70C, in an aqueous medium, an arabinose donor selected from the group consisting of D-arabinofuranose-l-phosphate, the compound having Formula I, as defined below, and the nucleotide of the compound ~I), and a purine source selected from the group consisting of purine, purine substituted at one or more of the 2-, 6- or.8- positions and ribofuranosides, ribofuranotides, deoxyribofuranosides and deoxyribofuranotides thereof, 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 substituted purine of the purine source, whereby ~-D-arabinofuranosyl radical is attached to the 9-position of the unsubstituted or substltuted purine of the purine source, and (b) recovering the produced 9-(~-D-arabinofuranosyl)-unsubstituted or substituted purine.
Formula I
N'~"~ ~Z
l ~ X represents O, S or NH, ~ ~ , : X Y represents OH, ~H~; SH or OH SR(R is lower alkyl _ ~ O~ group), and Z represents H, halogen, NO2, ] IO CH3 or CH2OH.
In another aspect of the invention there is provided novel purine arabinosides.
)876 The arabinose donors of this invention are D-arabino-furanose-l-phosphate, the compound shown in formula I, or the phosphate of the compound shown in formula I.
The specimens of the arabinose donors are shown in Examples of this invention.
The purine sources of this invention include purine, purine substituted at one or more of the 2-, 6- and 8- positions and their ribofuranosides, ribofuranotides -deoxyribofuranosides and deoxyribofuranotides. The substituted purine used in this invention as the purine source can be detected by the follow-ing method: Ribofuranoside substituted at one or more of the
Purine-arabinosides (9-(~-D-arabinofuranosyl)-purines) have potential utility as agricultural chemicals or medici-nal agents. For example, it has been reported that adenine arabinoside, one o~ 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 arabino-side~, 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 Patent Application ~o.
7271/1972). It is further reported that adenine arabinoside is produced when Streptomyces antibioticus is cultured in conventional culture media (Japanese Published Patent Application ~o. 41558/1972).
It has been found that purine arabino~ides are pro-duced in aqueous reaction media from arabinose donor, for example uracil arabinoside or D-arabinofuranose-l-phosphate and purine-source such as adenine, hypoxanthine, adenosine and adenosine-5'-monophosphate by the action of an enzyme produced by various bacteria, and this represents a simple method of manufacture.
~12~876 The present invention provides a method for pro-ducing purine arabinosides, which method is susceptible to commercial exploitation.
According to the invention there is provided a method of producing a purine arabinoside which comprises:
(a) holding at a temperature in the range from 40 to 70C, in an aqueous medium, an arabinose donor selected from the group consisting of D-arabinofuranose-l-phosphate, the compound having Formula I, as defined below, and the nucleotide of the compound ~I), and a purine source selected from the group consisting of purine, purine substituted at one or more of the 2-, 6- or.8- positions and ribofuranosides, ribofuranotides, deoxyribofuranosides and deoxyribofuranotides thereof, 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 substituted purine of the purine source, whereby ~-D-arabinofuranosyl radical is attached to the 9-position of the unsubstituted or substltuted purine of the purine source, and (b) recovering the produced 9-(~-D-arabinofuranosyl)-unsubstituted or substituted purine.
Formula I
N'~"~ ~Z
l ~ X represents O, S or NH, ~ ~ , : X Y represents OH, ~H~; SH or OH SR(R is lower alkyl _ ~ O~ group), and Z represents H, halogen, NO2, ] IO CH3 or CH2OH.
In another aspect of the invention there is provided novel purine arabinosides.
)876 The arabinose donors of this invention are D-arabino-furanose-l-phosphate, the compound shown in formula I, or the phosphate of the compound shown in formula I.
The specimens of the arabinose donors are shown in Examples of this invention.
The purine sources of this invention include purine, purine substituted at one or more of the 2-, 6- and 8- positions and their ribofuranosides, ribofuranotides -deoxyribofuranosides and deoxyribofuranotides. The substituted purine used in this invention as the purine source can be detected by the follow-ing method: Ribofuranoside substituted at one or more of the
2-, 6- and 8- positions is held with the enzyme of this invention in an aqueous medium containing O.IM KH2P04 at 60C for 24 hours. When the substituted purine of the originally used substituted purine ribofuranoside, and D-ribofuranose-l-phosphate or D-ribose derived from the above ribofuranoside are produced in the aqueous medium, the substituted purine can be used as the purine source.
The substituents at the 2-, 6- and 8- positions, when present, may be the same or different and may be 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 en-~ymatically transferred to the attached to 9-position of the unsubstituted or substituted purine of the purine source~
Thus, the product of this invention is 9-(~-D-arabinofuranosyl)-unsubstituted or substituted purine, with the substituent(s) being at one or more of the 2-, 6- and 8- positions.
The substituents at the 2-, 6- and 8- positions, when present, may be the same or different and may be 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 en-~ymatically transferred to the attached to 9-position of the unsubstituted or substituted purine of the purine source~
Thus, the product of this invention is 9-(~-D-arabinofuranosyl)-unsubstituted or substituted purine, with the substituent(s) being at one or more of the 2-, 6- and 8- positions.
- 3 -llZ~876 The bacterial enz~me capable of transarabinosylation from the arabinose donor to unsubstituted or 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 is suitably from the genera Pseudomonas, Flavobacter-ium,Achromobacter,Salmonella, Citrobacter, Escherichia, Klebsiella, Enterobacter, Aeromonas, Serratia, Erwinia, Proteus, Xanthomona~, or Bacterium.
Suitable bacteria include, by way of example:
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 ~RRL B-11340 (CCM 69), Salmonella typhimurum ~RRL B-11347 (FERM-P3735), Citrobacter freundii ATCC 8090, Citrobacter freundii ATCC 6750, (Citrobacter intermedium) Escherichia coli ATCC 9637, Escherichia aurescens ATCC 12814, ~}ebsiella pneumoniae ATCC 9621, (Enterobacter aerogenes) Serratia liquefaciens ATCC 14460, (Enterobacter liquefaciens) Enterobacter aerogenes ATCC 13048, Aeromonas punctata ATCC 11163, ! ' 8~76 Aeromonas salmonicida ATCC 14174, Serratia marcescens IFO 3048, Erwinia carotovora NRRL B-11342~CCM 872), Erwinia amylovara NRRL g-11341(CCM 1017), Erwinia herbicola ATCC 14537, Proteus vulgaris NRRL B-11345(FERM-P3394), Proteus rettgeri NRRL g-11344(FERM-P3395), Bacterium cadaveris IFO 3731, and Xanthomonas 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 conven-tional culture media. The culture media contain conven-tional carbon sources, nitrogen sources, inorganic ions, and when required minor organic nutrients such as vitamines and amino acids. 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 40C.
As the enzyme source, intact cells, culture liquids containing the cells are used preferably. Additionally, cells dried with acetone, frçeze-dried cells, homogenated cells, cells treated with super sonic waves, cells treated with toluene, surfactants or lysozyme are employed giving deslrable results. Moreover protein fractions having the enzyme activity capable of transarabinosylation from the arabinose donor to unsubstituted or substitut-ed purine of the purine source can be used preferably as , 112~876 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 have grown sufficiently, and thereafter the temprerature is maintained at 40C to 70C.
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, "aqueous mediuml' means culture medium or reaction medium (reaction mixture). The reaction media are maintained preferably at a temperature from 40C to 70C, and at a pH of 4 to 10 for 5 to 100 hours.
The reaction temperature (40C to 70C) of this invention is specific in the point that the temperature is higher than the ordinarly enzyme reaction temperature, and 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~
... , _ . .. .. . ... .
~Z~)876 The invention i9 illustrated by reference to the drawings in which:
Figure 1 is an ~MR spectrum of the crystalline product produced by the process of the invention in Example 5;
Figure 2 is an W spectrum of the product of Example 5, Figure 3 is an IR spectrum of the product of Example 5;
Figure 4 is an NMR spectrum of the crystalline product produced by the process of the invention in Example 6, Figure 5 is an W spectrum of the product:of Example 6,.and : Figure 6 is an IR spectrum of the product of Example 6.
:~
: ~ , ' - 6a -Example 1 l~Z'J876 An aqueous culture medium of pH 7.2 was prepared which contained, per deciliter, 0.5g yeast extract, l.Og peptone, 0.5g bouillon, and 0.5g NaCl. Five ml batches of the aqueous culture medium were placed in test tubes, and heated to sterilize. Each one loopful inoculum of the bacteria listed in Table 1 was transferred into each batch of the aqueous culture medium. Cultivation was carried out at 30C for 36 hours with shaking. The cells produced in the culture liquid were collected by centri-fugation and washed with physiological saline. The cells thus obtained (50mg(wet)/ml) were suspended in samples of 0.05M phosphate buffer of pH7.0, and 0.5ml of the suspen-sion of the cells was mixed with 0.5ml of reaction mixture of pH7.5 containing 0.5g/dl uracil arabinoside, 0.2g/dl hypoxanthine and 50mg/dl KHlPO4. Each mixture was held at 60C for 15 hours, and thereafter heated to 100~ for 5 minutes.
Each product in the reaction mixture was identified as 9-~-D-arabinofuranosylhypoxanthine(hypoxanthine arabino-side) by high speed liquid chromatography, and the amounts of the hypoxanthine arabinoside in the reaction mixture were determined by the high speed liquid chromatography, and are shown in Table 1.
)8~76 Table 1 microorganismhypoxanthine arabinoside used accumulated mg/dl NRRL B-11343 3.7 ATCC 8366 6.6 ATCC 12841 6.7 NRRL B-11340 5.7 NRRL B-11347 7.5 ATCC 8090 11.3 A TCC 6750 13.2 ATCC 9637 10.5 ATCC 12814 17.0 ATCC 9621 126.0 ATCC 14460 17.0 A TCC 14174 36.0 ATCC 11163 4 .1 IF 0 3048 23.0 NRR L B- 11342 14.0 NRRL B-11341 18.0 ATCC 14537 21.0 NRRL B-11345 9.6 NRRL B-11344 2.4 NRRL B-ll348 11.0 IF0 3731 12.0 NRRL B-11346 7.5 ATCC 13048 55.7 llZ~876 Example 2 In the method shown in Example 1, adenine was sub-stituted 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 l,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 : -S'-monophosphate was substituted for hypoxanthine, and the amounts of adenine arabinoside shown in Table 4 were accumulated in the reaction mixture.
:~: :
, ' Table 2 microorganismadenine arabinoside usedaccumulated mg/dl NRRL B-113434.5 ATCC 8366 8.2 ATCC 12841 8.0 NRRL B-113406.5 NRRL B-113478.6 ATCC 8090 13.3 ATCC 6750 15.0 ATCC 9637 10.6 ATCC 12814 18.8 A TCC 9621L32.0 ATCC 14460 26.0 A TCC 1417441.0 ATCC 1116318.5 IFO 3048 32.6 NRRL B-1134220.5 NRRL B-1134122.5 ATCC 1453731.5 NRRL B-1134526.3 NRRL B-1134428.6 NRRL B-1134813.5 IF0 3731 21.2 NRRL B-113468.6 A TCC 1304871.8 . . . _ . _ ~ _ _ . . .
~lZr~876 Table 3 .
micr~organismhypoxanthine arab~noside used accumulated mg/dl NRRL B-11343 4.2 ATCC 8366 5.5 ATCC 12841 8.2 NRRL B-11340 2.6 NRRL B-11347 4.8 ATCC 8090 6.5 ATCC 6750 10.3 ATCC 9637 6.3 ATCC 12814 3.6 ATCC 9621 82.1 ATCC 14460 15.0 ATCC 14174 20.5 ATCC 11163 0.8 IF0 3048 13.6 NRRLB~11342 2.6 NRRL B- 11341 8.7 A TCC 14537 15.0 NRRLB--11345 8.1 NRRLB-11344 0.5 NRRLB--11348 0.8 IF0 3731 10.6 NRRLB~11346 3.2 ATCC 13048 40.2 _ .
-11~
:112~8~76 Table 4 microorganismadenine arabinoside usedaccumulated mg/dl _ NRRL B-11343 3.8 ATCC 8366 5.6 ATCC 12841 7.2 NRRL B-11340 3.5 . NRRL B-11347 8.3 ATCC 8090 10.2 ATCC 6750 8.6 ATCC 9637 5.5 ATCC 12814 6.9 ATCC 9621 82.3 ATCC 14460 13.5 ATCC 14174 25.5 IF0 3048 21.5 :: NRRL B-11342 15.5 ` NRRL B-11341 11.5 ATCC 14537 18.3 NRRL B-11345 12.6 NRRL B-11344 15.8 NPRL B-11348 8.3 IFO 3731 14.5 NRRL B-11346 8.5 .
. _ .. . .
Example 5 llZ~876 A hundred ml batches of the aqueous culture medium shown in Example 1 were placed in 500ml shaking flask and heated to sterilize. Klebsiella pneumoniae ATCC 9621 was inoculated in the aqueous culture medium and cultured at 30C for 36 hours with shaking. Cells produced in the resultant culture liquid were collected by centrifugation, and 30g twet) of the cells was put into 1~ of the reaction mixture of pH 7.0 containing 1.5g 2-methylhypoxanthine, 7.3g uracil arabinoside and 3.4g KH2PO~. The reaction mixture was held at 60C for 36 hours.
Cells were removed from the reaction mixture by centrifugation, the supernatant was passed through cation exchange resin("Amberlite CG-120"), and the resin was washed with O.lN ammonium acetate(pH 6.8). After eluting with O.lN ammonium hydroxide, the eluate was evaporated and cooled, and 710mg crystals were obtained.
The crystalline product was dertermined as 9~ D-arabinofuranosyl)-2-methylhypoxanthine(2-methylhypoxanthine arabinoside) by NMR spectrum, W specturm, 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 Figure 1.
UV spectrum : shown in Figure 2.
IR spectrum : shown in Figure 3.
* trade mark -13-Example 6 11~876 Thirty grams o~ the cells obtained in Example 4 was put into lQ of reaction mixture containing 1.7g 2-chloro -hypoxanthine, 7.3g uracil arabinoside, and 3.4g KH7PO4, and the reaction mixture was held at 60C for 36 hours.
After removing the cells from the reaction mixture, the supernatant was passed through anion exchange resin ("Dowex IX4"), and the resin was washed with O.lN ammonium acetate of pH6.8. After eluting with O.lN ammonium acetate of pH4.0, the eluate was evaporated, and charged on *
"Sephadex G-10", and developed with water. The eluate portions showing first peak of UV absorption of the two were collected, evaporated and cooled. Then, 326mg crystals were obtained.
The crystalline product was determined as 9-(~-D-arabinofuranosyl~-2-chlorohypoxanthine(2-chlorohypoxanthine arabinoside) by 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 Figure 4 UV spectrum : shown in Figure 5 IR spectrum : shown in Figure 6 Beilstein test : positive (green) * trade mark -14-..... . _ 1~2~876 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, 0.2g/dl hypoxanthine was replaced with 0.4g/dl inosine, and the amounts of hypo-xanthine 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 500ml shaking flask,heated to sterilize, and inoculated with Aeromonas salmonicida ATCC
14174. cultivation was carried out at 30C for 36 hours with shaking.
Ce~ls produced in the resultant culture liquid were collected by centrifiguation, and 2.0g (wet weight) of the cells were put into IOOml reaction mixture of pH 7.5 containing lOOmg hypoxanthine, 300mg uracil arabinoside and SOmg KH~P04. The reaction mixture was then held at 60OC for 15 hours.
Twenty five m~j crystals of hypoxanthine arabinoside were obtained from the reaction mixture.
`
.
Table 5 m;.cro~rganism 2-methylhypoxan- 2-chlorohypo-thine arabinoside xanthine accumulated arabinoside mg/dl accumulated _ . m~ l NRRL B-11343 2.1 0.5 ATCC 8366 3.4 0.8 ATCC 12841 -4.0 2.1 NRRL B-11340 5.5 2.5 NRRL B-11347 4.8 2.8 ATCC 8090 8.7 3.6 ATCC 6750 9.5 8.2 ATCC 9637 4.7 5.1 ATCC 12814 12.0 10.5 ATCC 9621 80.5 51.6 ATCC 14460 18.5 11.3 A TCC 14174 21.6 10.0 ATCC 11163 0.8 0.05 IF0 3048 15.4 10.8 NRRL B-1134 22. 5 0.1 NRRL B-11341 12.0 10.5 ATCC 14537 15.5 12.1 NRRL B-11345 0.6 0.05 NRRL B-11344 8. 2 O. 5 NRR L B- 1134 8 12. 5 0.8 IFO 3731 21.6 2.1 NRRL B- 11346 15.3 10.3 ATCC 13048 40.2 28.7 . -16-Table 6 microorganism used hypoxanthine arabinoside .accumulated mg/dl NRRL B-11343 2.8 ATCC 8366 3.6 ATCC 12841 5.5 NRRL B-11340 4.3 NRRL B-11347 6.2 ATCC 8090 8.8 ATCC 6750 7.4 ATCC 9637 1.6 ATCC 12814 13.6 ATCC 9621 83.3 ATCC 14460 6.2 ATCC 14174 16.8 ATCC 11163 0.9 IF0 3048 15.3 NRR L B-11342 6.B
NRR L B-11341 10.2 ATCC 14537 8.9 WRR L B- 11345 8.5 NRRL B-11344 0.8 NRRL B-11348 7.2 IF O 3731 5.8 NRRL B-11346 3.3 ATCC 13048 40.4 ~ - .' Example 10 ~V876 Klebsiella pneumoniae ATCC 9621 was cultured by the manner shown in Example 9. Cells in the resultant culture liquid were collected by centrifugation, and 2g (wet weight) of the cells were suspended in 100ml reaction mixture of pH7.5 containing 100mg hypoxanthine, 300mg cytosine arabino-side, 50mg XH~PO4, and the reaction mixture was held at 60C
for 15 hours.
The cells in the reaction mexture were removed by cen-trifugation, and a concentrate of the supernatant was passed through anion exchange resin ("Dowex-~" OH form,pH6.8).
After eluting with 0.lN formic acid of pH4.0, the eluate was passed through "Sephadex G-l~". Eluate (250ml) 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, 35mg purified crystals were ob~ained.
The crystalline product was identified with authentic hypoxanthine arabinoside by NMR spectrum, IR spectrum and UV spectrum.
* trade mark -18-Example 11 112~876 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 60C 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 with 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 collect-ed by centrifugation.
The cells thus obtained (2g (wet weight)/dl)were suspended in lOOml of a reaction mixture of pH7.5 containing lOOmg/dl adenine, 300mg/dl cytosine arabinoside and 50mg ::
KH~PO4, and held at 60C for 15 hours.
After removing the cells from the reaction mixture, the reaction mixture was concentrated to 20ml, and cooled.
The crystals thus obtained were recrystallized with water and 55mg purified crystals were obtained. The crystalline product was identified with adenine arabinoside by NMR
spectrum, IR spectrum and W spectrum.
Exa~ple 13 i~2~876 Cells (5g (wet)~dl) of Aeromonas salmonicida ATCC
14174 were suspended in lOOml batches of a reaction mixture containing 30mM cytosine arabinoside, 25mM KHlP04, and lOmM
of one of the purines shown in Table 7. The reaction mixtures were placed in test tubes and held at 60C for 15 hours.
Newly formed product having UV absorption in the resal-tant reaction mixture was separated by liquid chromatography The eluate of the chromatography was concentrated and added with ethano], whereby crystals were formed in the eluate.
From NMR spectra and UV spectra of the purified cry-stalline products, the products were ascertained as the arabinosides of the respective purines used as the starting materials.
Conversionratio of the purine arabinosides from purine source were determined by measuring molecular extinction co-efficient, and are shown in Table 7.
Table 7 Starting material Product Conversion ratio (~) xanthine xanthine arabinoside 15 guanine guanine arabinoside 8 purine purine arabinoside 23 6-mercaptopurine 6-mercaptopurine arabinoside 8 2~6-diaminopurine 2~6-diaminopurine arabinoside38 6-mercaptoguanine 6-mereaptoguanine arabinoside 7 2-methylhypoxanthine 2-metlylhypoxanthine arabinoside 35 -chlorohypoxanthine 2-chlorohypoxanthi~,e arabinoside,,, 18 . .
Example 14 llZ~876 Cells t5g twet)/dl) of Klebsiella pneumoniae ATCC 9621were suspended in lOOml batches of a reaction mixture placed in test tubes, containing 30mM uracil arabinoside, 25mM
K~I~Po4, and one of the purine sources (19 mM) listed inTable 8, and the reaction mixture was held at 60OC for 15 hours.
Newly formed product having UV-absorption in the resul-tant reaction mixture was separated by liquid chromato-graphy. The eluate of the chromatography was concentrated and added with 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 molecular extinction.
coefficient, and are shown in Table 8.
Table 8 5tarting material Product Conversion ratio(%) xanthine xanthine arabinoside 65 guanine guanine arabinoside 20 purine purine arabinoside 36 6-mercaptopurine 6-mercaptopurine arabinoside 8 2~6-diaminopurine 276-diaminopurine arabinoside 52 6-mercaptoguanine 6-mercaptoguanine arabinoside 5 _ _ .
Example 15 In the method shown in Example 5, 2-methylhypoxanthine was replaced with 2-ethylhypoxanthine. The resultant reaction mixture was charged on silica-gel thin-layer, 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 O.lNHCl, and sillca-gel was removed from the suspension.
When the supernatant of the suspension was made 6N 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-ethyl-hypoxanthine 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.~5M
phosphate buffer of pH 7.5 to obtain 100 g (wet)/l, and treated with super sonic waves, A hundred ml of a reaction mixture, of pH 7.5 contain-ing 50ml/dl the supernatant, 500m~/dl uracil arabinoside -5-monophosphate, 100 mg/dl hypoxanthine and 30 mg/dl KHjPO~, was held at 60C for 15 hours. Then the reaction mixture was centrifiuged to remove precipitates, and the supernatant was passed through cation exchange resin ("Chromobead C-2'~).
* trade mark -22-'376 Elution was made with 0.3N formic acid, and the eiuate was charged on anion exchange resin ("Dowex lX4~
Hypox~nthine ~rabinoside was eluted by gr~dient elution with ammonium formate of pH 9 to ~ and 8 m~ of crystals were obtained from the eluate.
Example 17 One ml of a reaction mixture containing, per millili-ter, 0.2 ml of the supernatant shown in Example 16, 10 mg uracil arabinoside, 2 mg KH2PO~, ~ mg of one of the purine sources shown below was held at 600C in a test tube for L5 hours, and heated at 100C 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 cutted, and put into O.lN HCl.
/Then the O lN HCl was made 6N by adding concentrated HCl after removing filter paper, and boiled for 10 minutes, arabinose was found by orcinol-ferric chloride reaction in the boiled 6NHCl.
Thus, it is expected that arabinosides of the purine sources used as the starting materials were produced in the reaction mixtures 6-chloropurine 6-mercaptopurine ~-~hlorohypoxanthine 6-methylthiopurine 2-aminopurine 2-amino-6-mercaptopurin~
~-methylthiohypoxanthine 6-carboxypurine 8-chloroadenine 8-bromoadenine * trade mark -23-- ~z~76 Example 18 In the method shown in Example 16, uracil arabino-side-5'-monophosphate was replaced with cytocine arabino-side-5'-monophosphate. In the resultant reaction mixture, hypoxanthine arabinoside was found.
:
: :
:~ :
;~ -24-llZ~)876 Table 9 microorganism hypoxanthine arabinoside used accumulated mg/dl NRRL B-11343 2.8 ATCC 8366 5.5 ATCC 12841 6.3 NRR L B- 11340 6.0 NRRL B-11347 5.2 ATCC 8090 8.8 ATCC 6750 10.6 ATCC 9637 8.5 ATCC 12814 12.3 ATCC 9621 103.6 ATCC 14460 12.5 ATCC 14174 29.3 ~
ATCC 11163 4.0 -IF0 3048 24.0 NRR L B- 11342 15.2 NRR L B--11341 17.6 ATCC 14537 22.3 NRR L B - 11345 15.6 NRRL B-11344 3.2 NRRL B--11348 10~6 IFO 3731 18.3 NR R L B- 113468.2 ATCC 13048 48.5 _.... . .
~IZQ~76 Example 19 In the method shown in Example 1, D-arabinofuranose-l -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 result-ant reaction mixture was separated by preparqtive high speed 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 crystalline products, the products were ascertained as the arabinosides of the respective purine sources used as the starting materials.
Conversion ratio of the purine sources used to the purine arabinosides was determlned by measuring molecular extention coefficient and shown in Table 10.
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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~ P04~ and the reaction mixture was held at 60C for 15 hours.
Cells were removed from the reaction mixture by centrifugation. Adenine arabinoside accumulated was iden-tified by high speed liquid chromatography.
Table 11 arabinose donor
Suitable bacteria include, by way of example:
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 ~RRL B-11340 (CCM 69), Salmonella typhimurum ~RRL B-11347 (FERM-P3735), Citrobacter freundii ATCC 8090, Citrobacter freundii ATCC 6750, (Citrobacter intermedium) Escherichia coli ATCC 9637, Escherichia aurescens ATCC 12814, ~}ebsiella pneumoniae ATCC 9621, (Enterobacter aerogenes) Serratia liquefaciens ATCC 14460, (Enterobacter liquefaciens) Enterobacter aerogenes ATCC 13048, Aeromonas punctata ATCC 11163, ! ' 8~76 Aeromonas salmonicida ATCC 14174, Serratia marcescens IFO 3048, Erwinia carotovora NRRL B-11342~CCM 872), Erwinia amylovara NRRL g-11341(CCM 1017), Erwinia herbicola ATCC 14537, Proteus vulgaris NRRL B-11345(FERM-P3394), Proteus rettgeri NRRL g-11344(FERM-P3395), Bacterium cadaveris IFO 3731, and Xanthomonas 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 conven-tional culture media. The culture media contain conven-tional carbon sources, nitrogen sources, inorganic ions, and when required minor organic nutrients such as vitamines and amino acids. 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 40C.
As the enzyme source, intact cells, culture liquids containing the cells are used preferably. Additionally, cells dried with acetone, frçeze-dried cells, homogenated cells, cells treated with super sonic waves, cells treated with toluene, surfactants or lysozyme are employed giving deslrable results. Moreover protein fractions having the enzyme activity capable of transarabinosylation from the arabinose donor to unsubstituted or substitut-ed purine of the purine source can be used preferably as , 112~876 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 have grown sufficiently, and thereafter the temprerature is maintained at 40C to 70C.
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, "aqueous mediuml' means culture medium or reaction medium (reaction mixture). The reaction media are maintained preferably at a temperature from 40C to 70C, and at a pH of 4 to 10 for 5 to 100 hours.
The reaction temperature (40C to 70C) of this invention is specific in the point that the temperature is higher than the ordinarly enzyme reaction temperature, and 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~
... , _ . .. .. . ... .
~Z~)876 The invention i9 illustrated by reference to the drawings in which:
Figure 1 is an ~MR spectrum of the crystalline product produced by the process of the invention in Example 5;
Figure 2 is an W spectrum of the product of Example 5, Figure 3 is an IR spectrum of the product of Example 5;
Figure 4 is an NMR spectrum of the crystalline product produced by the process of the invention in Example 6, Figure 5 is an W spectrum of the product:of Example 6,.and : Figure 6 is an IR spectrum of the product of Example 6.
:~
: ~ , ' - 6a -Example 1 l~Z'J876 An aqueous culture medium of pH 7.2 was prepared which contained, per deciliter, 0.5g yeast extract, l.Og peptone, 0.5g bouillon, and 0.5g NaCl. Five ml batches of the aqueous culture medium were placed in test tubes, and heated to sterilize. Each one loopful inoculum of the bacteria listed in Table 1 was transferred into each batch of the aqueous culture medium. Cultivation was carried out at 30C for 36 hours with shaking. The cells produced in the culture liquid were collected by centri-fugation and washed with physiological saline. The cells thus obtained (50mg(wet)/ml) were suspended in samples of 0.05M phosphate buffer of pH7.0, and 0.5ml of the suspen-sion of the cells was mixed with 0.5ml of reaction mixture of pH7.5 containing 0.5g/dl uracil arabinoside, 0.2g/dl hypoxanthine and 50mg/dl KHlPO4. Each mixture was held at 60C for 15 hours, and thereafter heated to 100~ for 5 minutes.
Each product in the reaction mixture was identified as 9-~-D-arabinofuranosylhypoxanthine(hypoxanthine arabino-side) by high speed liquid chromatography, and the amounts of the hypoxanthine arabinoside in the reaction mixture were determined by the high speed liquid chromatography, and are shown in Table 1.
)8~76 Table 1 microorganismhypoxanthine arabinoside used accumulated mg/dl NRRL B-11343 3.7 ATCC 8366 6.6 ATCC 12841 6.7 NRRL B-11340 5.7 NRRL B-11347 7.5 ATCC 8090 11.3 A TCC 6750 13.2 ATCC 9637 10.5 ATCC 12814 17.0 ATCC 9621 126.0 ATCC 14460 17.0 A TCC 14174 36.0 ATCC 11163 4 .1 IF 0 3048 23.0 NRR L B- 11342 14.0 NRRL B-11341 18.0 ATCC 14537 21.0 NRRL B-11345 9.6 NRRL B-11344 2.4 NRRL B-ll348 11.0 IF0 3731 12.0 NRRL B-11346 7.5 ATCC 13048 55.7 llZ~876 Example 2 In the method shown in Example 1, adenine was sub-stituted 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 l,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 : -S'-monophosphate was substituted for hypoxanthine, and the amounts of adenine arabinoside shown in Table 4 were accumulated in the reaction mixture.
:~: :
, ' Table 2 microorganismadenine arabinoside usedaccumulated mg/dl NRRL B-113434.5 ATCC 8366 8.2 ATCC 12841 8.0 NRRL B-113406.5 NRRL B-113478.6 ATCC 8090 13.3 ATCC 6750 15.0 ATCC 9637 10.6 ATCC 12814 18.8 A TCC 9621L32.0 ATCC 14460 26.0 A TCC 1417441.0 ATCC 1116318.5 IFO 3048 32.6 NRRL B-1134220.5 NRRL B-1134122.5 ATCC 1453731.5 NRRL B-1134526.3 NRRL B-1134428.6 NRRL B-1134813.5 IF0 3731 21.2 NRRL B-113468.6 A TCC 1304871.8 . . . _ . _ ~ _ _ . . .
~lZr~876 Table 3 .
micr~organismhypoxanthine arab~noside used accumulated mg/dl NRRL B-11343 4.2 ATCC 8366 5.5 ATCC 12841 8.2 NRRL B-11340 2.6 NRRL B-11347 4.8 ATCC 8090 6.5 ATCC 6750 10.3 ATCC 9637 6.3 ATCC 12814 3.6 ATCC 9621 82.1 ATCC 14460 15.0 ATCC 14174 20.5 ATCC 11163 0.8 IF0 3048 13.6 NRRLB~11342 2.6 NRRL B- 11341 8.7 A TCC 14537 15.0 NRRLB--11345 8.1 NRRLB-11344 0.5 NRRLB--11348 0.8 IF0 3731 10.6 NRRLB~11346 3.2 ATCC 13048 40.2 _ .
-11~
:112~8~76 Table 4 microorganismadenine arabinoside usedaccumulated mg/dl _ NRRL B-11343 3.8 ATCC 8366 5.6 ATCC 12841 7.2 NRRL B-11340 3.5 . NRRL B-11347 8.3 ATCC 8090 10.2 ATCC 6750 8.6 ATCC 9637 5.5 ATCC 12814 6.9 ATCC 9621 82.3 ATCC 14460 13.5 ATCC 14174 25.5 IF0 3048 21.5 :: NRRL B-11342 15.5 ` NRRL B-11341 11.5 ATCC 14537 18.3 NRRL B-11345 12.6 NRRL B-11344 15.8 NPRL B-11348 8.3 IFO 3731 14.5 NRRL B-11346 8.5 .
. _ .. . .
Example 5 llZ~876 A hundred ml batches of the aqueous culture medium shown in Example 1 were placed in 500ml shaking flask and heated to sterilize. Klebsiella pneumoniae ATCC 9621 was inoculated in the aqueous culture medium and cultured at 30C for 36 hours with shaking. Cells produced in the resultant culture liquid were collected by centrifugation, and 30g twet) of the cells was put into 1~ of the reaction mixture of pH 7.0 containing 1.5g 2-methylhypoxanthine, 7.3g uracil arabinoside and 3.4g KH2PO~. The reaction mixture was held at 60C for 36 hours.
Cells were removed from the reaction mixture by centrifugation, the supernatant was passed through cation exchange resin("Amberlite CG-120"), and the resin was washed with O.lN ammonium acetate(pH 6.8). After eluting with O.lN ammonium hydroxide, the eluate was evaporated and cooled, and 710mg crystals were obtained.
The crystalline product was dertermined as 9~ D-arabinofuranosyl)-2-methylhypoxanthine(2-methylhypoxanthine arabinoside) by NMR spectrum, W specturm, 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 Figure 1.
UV spectrum : shown in Figure 2.
IR spectrum : shown in Figure 3.
* trade mark -13-Example 6 11~876 Thirty grams o~ the cells obtained in Example 4 was put into lQ of reaction mixture containing 1.7g 2-chloro -hypoxanthine, 7.3g uracil arabinoside, and 3.4g KH7PO4, and the reaction mixture was held at 60C for 36 hours.
After removing the cells from the reaction mixture, the supernatant was passed through anion exchange resin ("Dowex IX4"), and the resin was washed with O.lN ammonium acetate of pH6.8. After eluting with O.lN ammonium acetate of pH4.0, the eluate was evaporated, and charged on *
"Sephadex G-10", and developed with water. The eluate portions showing first peak of UV absorption of the two were collected, evaporated and cooled. Then, 326mg crystals were obtained.
The crystalline product was determined as 9-(~-D-arabinofuranosyl~-2-chlorohypoxanthine(2-chlorohypoxanthine arabinoside) by 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 Figure 4 UV spectrum : shown in Figure 5 IR spectrum : shown in Figure 6 Beilstein test : positive (green) * trade mark -14-..... . _ 1~2~876 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, 0.2g/dl hypoxanthine was replaced with 0.4g/dl inosine, and the amounts of hypo-xanthine 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 500ml shaking flask,heated to sterilize, and inoculated with Aeromonas salmonicida ATCC
14174. cultivation was carried out at 30C for 36 hours with shaking.
Ce~ls produced in the resultant culture liquid were collected by centrifiguation, and 2.0g (wet weight) of the cells were put into IOOml reaction mixture of pH 7.5 containing lOOmg hypoxanthine, 300mg uracil arabinoside and SOmg KH~P04. The reaction mixture was then held at 60OC for 15 hours.
Twenty five m~j crystals of hypoxanthine arabinoside were obtained from the reaction mixture.
`
.
Table 5 m;.cro~rganism 2-methylhypoxan- 2-chlorohypo-thine arabinoside xanthine accumulated arabinoside mg/dl accumulated _ . m~ l NRRL B-11343 2.1 0.5 ATCC 8366 3.4 0.8 ATCC 12841 -4.0 2.1 NRRL B-11340 5.5 2.5 NRRL B-11347 4.8 2.8 ATCC 8090 8.7 3.6 ATCC 6750 9.5 8.2 ATCC 9637 4.7 5.1 ATCC 12814 12.0 10.5 ATCC 9621 80.5 51.6 ATCC 14460 18.5 11.3 A TCC 14174 21.6 10.0 ATCC 11163 0.8 0.05 IF0 3048 15.4 10.8 NRRL B-1134 22. 5 0.1 NRRL B-11341 12.0 10.5 ATCC 14537 15.5 12.1 NRRL B-11345 0.6 0.05 NRRL B-11344 8. 2 O. 5 NRR L B- 1134 8 12. 5 0.8 IFO 3731 21.6 2.1 NRRL B- 11346 15.3 10.3 ATCC 13048 40.2 28.7 . -16-Table 6 microorganism used hypoxanthine arabinoside .accumulated mg/dl NRRL B-11343 2.8 ATCC 8366 3.6 ATCC 12841 5.5 NRRL B-11340 4.3 NRRL B-11347 6.2 ATCC 8090 8.8 ATCC 6750 7.4 ATCC 9637 1.6 ATCC 12814 13.6 ATCC 9621 83.3 ATCC 14460 6.2 ATCC 14174 16.8 ATCC 11163 0.9 IF0 3048 15.3 NRR L B-11342 6.B
NRR L B-11341 10.2 ATCC 14537 8.9 WRR L B- 11345 8.5 NRRL B-11344 0.8 NRRL B-11348 7.2 IF O 3731 5.8 NRRL B-11346 3.3 ATCC 13048 40.4 ~ - .' Example 10 ~V876 Klebsiella pneumoniae ATCC 9621 was cultured by the manner shown in Example 9. Cells in the resultant culture liquid were collected by centrifugation, and 2g (wet weight) of the cells were suspended in 100ml reaction mixture of pH7.5 containing 100mg hypoxanthine, 300mg cytosine arabino-side, 50mg XH~PO4, and the reaction mixture was held at 60C
for 15 hours.
The cells in the reaction mexture were removed by cen-trifugation, and a concentrate of the supernatant was passed through anion exchange resin ("Dowex-~" OH form,pH6.8).
After eluting with 0.lN formic acid of pH4.0, the eluate was passed through "Sephadex G-l~". Eluate (250ml) 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, 35mg purified crystals were ob~ained.
The crystalline product was identified with authentic hypoxanthine arabinoside by NMR spectrum, IR spectrum and UV spectrum.
* trade mark -18-Example 11 112~876 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 60C 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 with 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 collect-ed by centrifugation.
The cells thus obtained (2g (wet weight)/dl)were suspended in lOOml of a reaction mixture of pH7.5 containing lOOmg/dl adenine, 300mg/dl cytosine arabinoside and 50mg ::
KH~PO4, and held at 60C for 15 hours.
After removing the cells from the reaction mixture, the reaction mixture was concentrated to 20ml, and cooled.
The crystals thus obtained were recrystallized with water and 55mg purified crystals were obtained. The crystalline product was identified with adenine arabinoside by NMR
spectrum, IR spectrum and W spectrum.
Exa~ple 13 i~2~876 Cells (5g (wet)~dl) of Aeromonas salmonicida ATCC
14174 were suspended in lOOml batches of a reaction mixture containing 30mM cytosine arabinoside, 25mM KHlP04, and lOmM
of one of the purines shown in Table 7. The reaction mixtures were placed in test tubes and held at 60C for 15 hours.
Newly formed product having UV absorption in the resal-tant reaction mixture was separated by liquid chromatography The eluate of the chromatography was concentrated and added with ethano], whereby crystals were formed in the eluate.
From NMR spectra and UV spectra of the purified cry-stalline products, the products were ascertained as the arabinosides of the respective purines used as the starting materials.
Conversionratio of the purine arabinosides from purine source were determined by measuring molecular extinction co-efficient, and are shown in Table 7.
Table 7 Starting material Product Conversion ratio (~) xanthine xanthine arabinoside 15 guanine guanine arabinoside 8 purine purine arabinoside 23 6-mercaptopurine 6-mercaptopurine arabinoside 8 2~6-diaminopurine 2~6-diaminopurine arabinoside38 6-mercaptoguanine 6-mereaptoguanine arabinoside 7 2-methylhypoxanthine 2-metlylhypoxanthine arabinoside 35 -chlorohypoxanthine 2-chlorohypoxanthi~,e arabinoside,,, 18 . .
Example 14 llZ~876 Cells t5g twet)/dl) of Klebsiella pneumoniae ATCC 9621were suspended in lOOml batches of a reaction mixture placed in test tubes, containing 30mM uracil arabinoside, 25mM
K~I~Po4, and one of the purine sources (19 mM) listed inTable 8, and the reaction mixture was held at 60OC for 15 hours.
Newly formed product having UV-absorption in the resul-tant reaction mixture was separated by liquid chromato-graphy. The eluate of the chromatography was concentrated and added with 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 molecular extinction.
coefficient, and are shown in Table 8.
Table 8 5tarting material Product Conversion ratio(%) xanthine xanthine arabinoside 65 guanine guanine arabinoside 20 purine purine arabinoside 36 6-mercaptopurine 6-mercaptopurine arabinoside 8 2~6-diaminopurine 276-diaminopurine arabinoside 52 6-mercaptoguanine 6-mercaptoguanine arabinoside 5 _ _ .
Example 15 In the method shown in Example 5, 2-methylhypoxanthine was replaced with 2-ethylhypoxanthine. The resultant reaction mixture was charged on silica-gel thin-layer, 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 O.lNHCl, and sillca-gel was removed from the suspension.
When the supernatant of the suspension was made 6N 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-ethyl-hypoxanthine 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.~5M
phosphate buffer of pH 7.5 to obtain 100 g (wet)/l, and treated with super sonic waves, A hundred ml of a reaction mixture, of pH 7.5 contain-ing 50ml/dl the supernatant, 500m~/dl uracil arabinoside -5-monophosphate, 100 mg/dl hypoxanthine and 30 mg/dl KHjPO~, was held at 60C for 15 hours. Then the reaction mixture was centrifiuged to remove precipitates, and the supernatant was passed through cation exchange resin ("Chromobead C-2'~).
* trade mark -22-'376 Elution was made with 0.3N formic acid, and the eiuate was charged on anion exchange resin ("Dowex lX4~
Hypox~nthine ~rabinoside was eluted by gr~dient elution with ammonium formate of pH 9 to ~ and 8 m~ of crystals were obtained from the eluate.
Example 17 One ml of a reaction mixture containing, per millili-ter, 0.2 ml of the supernatant shown in Example 16, 10 mg uracil arabinoside, 2 mg KH2PO~, ~ mg of one of the purine sources shown below was held at 600C in a test tube for L5 hours, and heated at 100C 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 cutted, and put into O.lN HCl.
/Then the O lN HCl was made 6N by adding concentrated HCl after removing filter paper, and boiled for 10 minutes, arabinose was found by orcinol-ferric chloride reaction in the boiled 6NHCl.
Thus, it is expected that arabinosides of the purine sources used as the starting materials were produced in the reaction mixtures 6-chloropurine 6-mercaptopurine ~-~hlorohypoxanthine 6-methylthiopurine 2-aminopurine 2-amino-6-mercaptopurin~
~-methylthiohypoxanthine 6-carboxypurine 8-chloroadenine 8-bromoadenine * trade mark -23-- ~z~76 Example 18 In the method shown in Example 16, uracil arabino-side-5'-monophosphate was replaced with cytocine arabino-side-5'-monophosphate. In the resultant reaction mixture, hypoxanthine arabinoside was found.
:
: :
:~ :
;~ -24-llZ~)876 Table 9 microorganism hypoxanthine arabinoside used accumulated mg/dl NRRL B-11343 2.8 ATCC 8366 5.5 ATCC 12841 6.3 NRR L B- 11340 6.0 NRRL B-11347 5.2 ATCC 8090 8.8 ATCC 6750 10.6 ATCC 9637 8.5 ATCC 12814 12.3 ATCC 9621 103.6 ATCC 14460 12.5 ATCC 14174 29.3 ~
ATCC 11163 4.0 -IF0 3048 24.0 NRR L B- 11342 15.2 NRR L B--11341 17.6 ATCC 14537 22.3 NRR L B - 11345 15.6 NRRL B-11344 3.2 NRRL B--11348 10~6 IFO 3731 18.3 NR R L B- 113468.2 ATCC 13048 48.5 _.... . .
~IZQ~76 Example 19 In the method shown in Example 1, D-arabinofuranose-l -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 result-ant reaction mixture was separated by preparqtive high speed 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 crystalline products, the products were ascertained as the arabinosides of the respective purine sources used as the starting materials.
Conversion ratio of the purine sources used to the purine arabinosides was determlned by measuring molecular extention coefficient and shown in Table 10.
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. ,q ~ ~a ~J . . a~ h h O
V ~ O h ~
U ~t ~ o n~ -r~ rl D~ O S S
O U~ V V
O ~ O U~
h ~1 ~ O ~rl h ~ 1 nl R. R ,1 ~: h~:1lU X X
lo Q ~ s o o h ,a ,CI ~ O tJ~ ~ ~
1~ a o~: o :~ :,.
la h V~rl V S .C
a) ~ ~ ~ R, ~ O
s~ la ~ h .,1 ~ O U ~rl U S
,r: ~1 S:: h ~ h V --1 v ~: ~,1 IL\ I a) a) ~:
1 h ~~) E ~ u X
~ Q~
.~ .
Q~ a) ~ ~
~ ~ ~ ~ V V
.,~
)~ ~1 h ~: 10 1~
: ~ ~ V h 11~ oX o E~ oQ~ o~
: t~ V~ ~ V~ ~ ~::
F. ~ ~ ~ ~
~rl ~ U -1 U S
O V S ~ h~:1 h V -1 c-l ~ v ~ aJ S
~ F. ~ D E3 E u a) v cn X ~ ~ ~ ~ ~D
E~ _ . ...
.. - . , , , " . . - - . .
., . . , . ,. .-,' .' .. , ' :. , .
. .
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~ P04~ and the reaction mixture was held at 60C for 15 hours.
Cells were removed from the reaction mixture by centrifugation. Adenine arabinoside accumulated was iden-tified by high speed liquid chromatography.
Table 11 arabinose donor
4-thiouracil arabinofuranoside 4-(S~methyl-)thiouracil arabinofuranoside 2-thiouracil arabinofuranoside
5-nitrouracil arabinofuranoside 5-hydroxymethyl uracil arabinofuranoside isocytosine àrabinofuranoside 5-fluorouracil arabinofuranoside 5-bromouracil arabinofuranoside 5-Iodouracil arabinofuranoside ~; thymine arabinofuranoside llZ~8"76 Example 22 In the method shown in Example ll, adenine in the reaction mixture was replaced with 200 mg adenylic acid, and the reaction mixture was held at 60C 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 with NMR spectrum IR spectrum, and W 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 60C for 15 hours.
The crystals of 2~ D-arabinofuranosyl)guanine (guanine arabinoside) (28mg) were obtained from the supernatant of the resulted reaction mixture.
~.
Example 24 ~: .
In the method shown in Example 13, adenosine, deoxy-adenosine, deoxyadenylic acid, guanylic acid, deoxyguanylic acid, xanthosine, deoxyxanthosine, deoxyinosine or deoxy-inosinic acid were used in place of hypoxanthine as the purine source. From the above adenine source, adenine ar-abinoside 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.
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 with NMR spectrum IR spectrum, and W 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 60C for 15 hours.
The crystals of 2~ D-arabinofuranosyl)guanine (guanine arabinoside) (28mg) were obtained from the supernatant of the resulted reaction mixture.
~.
Example 24 ~: .
In the method shown in Example 13, adenosine, deoxy-adenosine, deoxyadenylic acid, guanylic acid, deoxyguanylic acid, xanthosine, deoxyxanthosine, deoxyinosine or deoxy-inosinic acid were used in place of hypoxanthine as the purine source. From the above adenine source, adenine ar-abinoside 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.
Claims (19)
1. A method for producing a purine arabinoside, which comprises:
(a) holding at a temperature of 40°C to 70°C, in an aqueous medium, an arabinose donor selected from the group consisting of (i) D-arabinofuranose-1-phosphate (ii) a compound of Formula I
Formula I
wherein X represents 0, S or NH; Y represents OH, NH2, SH
or SR (R is lower alkyl group); and Z represents H, halogen, NO2, CH3 or CH2OH and (iii) a phosphate of said compound of Formula I, and a purine source selected from the group consisting of purine, purine substituted at one or more of the 2-, 6- and 8- positions and ribofuranosides, ribofuranotides, deoxyribofuranosides and deoxyribofuranotides thereof, in the presence of an effective amount of an enzyme produced by bacteria, and capable of transarabinosylation from the arabinose donor to the unsubstituted or substituted purine of the purine source, whereby a .beta.-D-arabinofuranosyl radical is attached to the 9-posit-ion of the unsubstituted or substituted purine of the purine source and (b) recovering the resulting purine arabinoside,
(a) holding at a temperature of 40°C to 70°C, in an aqueous medium, an arabinose donor selected from the group consisting of (i) D-arabinofuranose-1-phosphate (ii) a compound of Formula I
Formula I
wherein X represents 0, S or NH; Y represents OH, NH2, SH
or SR (R is lower alkyl group); and Z represents H, halogen, NO2, CH3 or CH2OH and (iii) a phosphate of said compound of Formula I, and a purine source selected from the group consisting of purine, purine substituted at one or more of the 2-, 6- and 8- positions and ribofuranosides, ribofuranotides, deoxyribofuranosides and deoxyribofuranotides thereof, in the presence of an effective amount of an enzyme produced by bacteria, and capable of transarabinosylation from the arabinose donor to the unsubstituted or substituted purine of the purine source, whereby a .beta.-D-arabinofuranosyl radical is attached to the 9-posit-ion of the unsubstituted or substituted purine of the purine source and (b) recovering the resulting purine arabinoside,
2. The method of claim 1, wherein the purine source comprises a substituted purine substituted at one or more of the 2-, 6- and 8- position by one or more substituents selected from the group consisting of halogen, hydroxyl, amino, lower alkyl, alXoxyl, aryl, aralkyl, mercapto, alkyl-amino alkylmercapto, alkylsulfonyl, alkylsulfenyl, carboxyl, alkoxycarbonyl, nitro and cyano radicals.
3. The method of claim 1, wherein the purine source comprises a substituted purine substituted at one or more of the 2-, 6- and 8- positions with one or more substituents selected from the group consisting of amino, hydroxyl, and mercapto radicals.
4. The method of claim 1, wherein the purine source is selected from the group consisting of adenine, adenosine, deoxyadenosine, deoxyadenylic acid, adenylic acid, hypoxanthine, inosine, deoxyinosine, deoxyinosinic acid, inosinic acid, guanine, guanosine, deoxyguanosine, deoxy-guanylic acid, guanylic acid, xanthine, xanthosine and the deoxyxanthosine of xanthylic acid.
5. The method of claim 1, wherein the arabinose donor is 1-.beta.-D-arabinofuranosyl-cytosine or 1-.beta.-D-arabinofuranosyl-uracil.
6. The method of claim 4, wherein the arabinose donor is 1-.beta.-D-arabinofuranosyl-cytosine or 1-.beta.-D-arabinofuranosyl-uracil.
7. The method of claim 1, 4 or 6 wherein the bacterium is one from a genus selected from the group consisting of Pseudomonas, Flavobacterium, Achromobacter, Salmonella, Serratis, aeromonas, Erwinia, Proteus, Bacterium, Xanthomonas, Klebsiella and Citrobacter.
8. The method of claim 1, 4 or 6, wherein the bacterium is of the genus Escherichia.
9. The method of claim 1, 4 or 6, wherein the bacterium i9 of the genus Enterobacter.
10. The method of claim 1, 4 or 6, wherein the bacterium is selected from the group consisting of Pseudomonas stutzeri NRRL B-11346, Flavobacterium rhenanum NRRL B-11343, Flavobacterium acidoficum ATCC 8366, Flavobacterium proteus ATCC 12841, Achromobacter lacticum NRRL B-11340, Salmonella typhimunum NRRL B-11347, Citrobacter freundii ATCC 98090, Citrobacter freundii ATCC 6750, Klebsiella pneumoniae ATCC 9621, Serratia liquefaciens ATCC 14460, Aeromonas punctata ATCC 11163, Aeromonas salmonicida ATCC 14174, Serratia marcescens IF0 3048, Erwinia carotovora NRRL B-11342, Erwinia amylovara NRRL B-11341, Erwinia herbicola ATCC 14537, Proteus vulgaris NRRL B-11345 Proteus rettgeri NRRL B-11344 Bacterium cadaveris IFO 3731, and Xanthomonas citri NRRL B-11348.
11. The method of claim 1, 4 or 6, wherein the bacterium is selected from the group consisting of Escherichia coli ATCC 9637, Escherichia aurescens ATCC 12814 and Enterobacter aerogenes ATCC 13048.
12. The method of claim 1, 4 or 6, wherein the pH of the aqueous medium is from 4 to 10.
13. The method of claim 1, wherein cells of the bacterium are employed as a source of the enzyme.
14. The method of claim 1, wherein said purine source is 2-chlorohypoxanthine or 2-methylhypoxanthine.
15. The method of claim 14, wherein said purine source is 2-chlorohypoxanthine.
16. The method of claim 14, wherein said purine source is -2-methylhypoxanthine.
17. A 9-(.beta.-D-arabinofuranosyl 2-substituted hypoxanthine, wherein the 2-substituent is selected from the group consisting of the 2-chloro-and the 2-methyl whenever prepared by the method of claim 14 or by an obvious chemical equivalent.
18. 9-(.beta.-D-Arabinofuranosyl)-2-chlorohypoxanthine, when-ever prepared by the process of claim 15, or by an obvious chemical equivalent.
19. 9-(.beta.-D-Arabinofuranosyl)-2-methylhypoxanthine, when-ever prepared by the process of claim 16, or by an obvious chemical equivalent.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9576677A JPS5432695A (en) | 1977-08-10 | 1977-08-10 | Preparation of purine arabinosides |
| JP95766/1977 | 1977-08-10 | ||
| JP15873877A JPS5492695A (en) | 1977-12-28 | 1977-12-28 | 2-substituted hypoxanthinearabinoside and its preparation |
| JP158738/1977 | 1977-12-28 | ||
| JP180278A JPS5495793A (en) | 1978-01-11 | 1978-01-11 | Preparation of purine arabinoside |
| JP1802/1978 | 1978-01-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1120876A true CA1120876A (en) | 1982-03-30 |
Family
ID=27275082
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000309031A Expired CA1120876A (en) | 1977-08-10 | 1978-08-09 | Method for producing purine arabinosides |
Country Status (5)
| Country | Link |
|---|---|
| CA (1) | CA1120876A (en) |
| DE (1) | DE2835151A1 (en) |
| FR (1) | FR2400033A1 (en) |
| GB (1) | GB2006185B (en) |
| NL (1) | NL7808321A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3379274D1 (en) * | 1982-04-01 | 1989-04-06 | Yamasa Shoyu Kk | Process for producing 3'-deoxyguanosine |
| EP1464708A1 (en) * | 2003-04-03 | 2004-10-06 | Pro. Bio. Sint. S.p.A. | A process for the preparation of fludarabine phosphate from 2-fluoroadenine |
| RU2563257C1 (en) * | 2014-11-07 | 2015-09-20 | Федеральное государственное бюджетное учреждение науки Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова РАН (ИБХ РАН) | METHOD OF PRODUCING 9-(BETA-D-ARABINOFURANOSYL)-6-(Nα-L-SERYLAMIDO)-2-CHLOROPURINE |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3269917A (en) * | 1963-03-18 | 1966-08-30 | Takeda Chemical Industries Ltd | Process for producing purine-nucleosides |
| AT281301B (en) * | 1966-12-30 | 1970-05-11 | Parke Davis & Co | Process for the preparation of 9- (β-arabinofuranosyl) adenine |
-
1978
- 1978-08-08 GB GB7832560A patent/GB2006185B/en not_active Expired
- 1978-08-09 NL NL7808321A patent/NL7808321A/en not_active Application Discontinuation
- 1978-08-09 CA CA000309031A patent/CA1120876A/en not_active Expired
- 1978-08-10 DE DE19782835151 patent/DE2835151A1/en active Granted
- 1978-08-10 FR FR7823633A patent/FR2400033A1/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| GB2006185A (en) | 1979-05-02 |
| FR2400033B1 (en) | 1982-07-30 |
| DE2835151A1 (en) | 1979-02-22 |
| GB2006185B (en) | 1982-04-28 |
| DE2835151C2 (en) | 1988-07-14 |
| NL7808321A (en) | 1979-02-13 |
| FR2400033A1 (en) | 1979-03-09 |
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