Note: Descriptions are shown in the official language in which they were submitted.
876
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
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.
- 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
,
--5--
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~
--6--
... , _ . .. .. . ... .
~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.
:~: :
, '
376
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
--10--
. . . _ . _ ~ _ _ . . .
~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
ATCC 11163 9 6
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
ATCC 13048 49 6
.
-12-
. _ .. . .
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.
`
-15-
.
1120876
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-
76
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
~ - .'
--17--
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.
--19--
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
-20-
. .
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
-21-
_ _ .
876
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.
:
: :
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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
-- 25 --
_.... . .
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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
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
- 28 -
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.
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