close

Вход

Забыли?

вход по аккаунту

?

Патент USA US3053750

код для вставки
3,053,740
United States Patent O?ice
Patented Sept. 11, 1962
2
1
in the aforesaid U.S. patents to Duggar and to Minieri
3,053,740
et al. and which, in turn, are generally the same as for
BIOLOGICAL CONVERSION OF ANHYDROTETRA
CYCLINES TO TETRACYCLINES
Philip Andrew Miller, Valley Cottage, and Jerry Robert
Daniel McCormick, New City, N.Y., assignors to Amer
ican Cyanamid Company, New York, N.Y., a corpora
the presently known methods for producing various tetra
cyclines by fermentation. That is, the fermentation me
dium contains the usual nutrients and mineral substances.
Suitable nutrients include starch, dextrose, cane sugar,
glucose, molasses, soybean meal, peanut meal, yeast,
tion of Maine
No Drawing. Filed Aug. 25, 1960, Ser. No. 51,775
9 Claims. (Cl. 195-80)
meat extracts, peptone, urea, corn steep liquor, distillers
solubles,‘ ?sh meal and other conventional substances.
This invention relates to a new process for producing
bonate, ammonium sulfate, ammonium chloride, and salts
tetracyclines and, more particularly, is concerned with
a novel process for biologically transforming anhydro
of the various trace elements such as manganese, cobalt,
Zinc, copper, iron and the like.
The inorganic salts include such things as calcium car
tetracyclines to the corresponding tetracyclines.
The other general conditions of the fermentation, such
The tetracyclines are disclosed in U.S. Patent 2,482,055 15 as hydrogen ion concentration, temperature, time rate of
to Duggar, U.S. Patent 2,734,018 to Minieri et al.,
aeration, preparation of the inoculum, sterilization, in
oculation and the like are conventional and are similar
U.S. Patent 2,878,289 to McCormick et al., and U.S.
to those for the production of chlortetracycline shown
Patent 2,516,080 to Sobin et al. They have been pro
duced by aerobically fermenting an ‘aqueous nutrient
in U.S. Patent No. 2,482,055, to Duggar.
medium with various strains of the genus Streptomyces, 20
After the fermentation has been continued for the
the morphological and cultural characteristics of which
desired period of time and the transformation of the
anhydrotetracycline compound to the corresponding
are adequately described in the aforesaid ‘U.S. patents.
The anhydrotetracyclines, the starting materials for
tetracycline is substantially complete, the tetracycline com
pound may be isolated from the fermentation mash in
the novel process of the present invention, are well known
compounds and may be readily prepared by the dehy 25 any convenient manner such as are set forth in the afore
dration of the corresponding tetracycline with a strong
mineral acid. Anhydrochlortetracycline is described in
U.S. Patent 2,744,931 to Broschard et al., and anhydro
tetracycline is described in U.S. Patent 2,744,932 to
the sterile inoculum medium has the following com
Waller et al.
position:
mentioned U.S. patents to Duggar and to Minieri et al.
The invention will be described in greater detail in
conjunction with the following speci?c examples wherein
Anhydro~4-epi-tetracycline, anhydro—5 30
,
hydroxytetracycline, anhydro-6-demethylchlortetracycline,
Grams
and 10-benzenesulfonyl-anhydro~5 -hydroxytetracyclinoni
Corn steep liquor
Dextrin No. 162
trile have been disclosed in the Journal of the American
Chemical Society 79, 2849 (1957); 74, 4976 (1952); 79,
4563 (1957); and 74, 4976 (1952), respectively. In ad
dition, the synthesis of dedimethylamino-l2a-deoxy-6
demethylanhydrochlortetracycline has ben described by
____ __
_
(181102304 -
CaCOa _
20
30
-
2
_---
7
Water to 1000 milliliters.
and the sterile fermentation medium has the following
J. H. Boothe et al., J.A.C.S. 81, 1006 (1959). The an
composition:
hydrotetracyclines exhibit antimicrobial activity particu
larly against certain strains of bacteria which are re 40 Corn steep liquor____________________ __grams__ 27.5
sistant to the tetracyclines.
Starch
___
____do__.__ 55.0
Our invention is based upon the discovery that it is
(NH4)2SO4 _________________________ __do____ 5.0
possible to effect the biological transformation of an
CaCO3
____
do____. 9.0
hydrotetracyclines to the corresponding tetracyclines; that
MgClz
.__
is, the biological hydration at the 5a-6 bond of the an 45 NH, Cl.
hydrotetracyclines. This transformation is accomplished
Water to 1000 milliliters.
mentation medium inoculated with a strain of a species
1Trace elements solution:
of the genus Streptomyces such as, for example S.
aureofaciens ATCC No. 10,762, S. aureofaciens ATCC
No. 12,551, S. aureofaciens ATCC No. 13,192, S. rimosus
ATCC No. 10,970, and S. viridifacz'ens ATCC No. 11,989;
viable cultures of which have been deposited with the
American Type Culture Collection (ATCC) in Washing
version of the anhydrotetracycline to the corresponding
tetracycline is from about 25% to about 75% complete.
It is most surprising that the anhydrotetracyclines can
serve as substrates which can be acted upon by the micro
organism so as to transform the anhydrotetracycline
2.0
do_.___
1.5
Trace elements solution 1 __________ __milliliters__ 10.0
by adding an anhydrotetracycline compound to a fer
ton, D.C. After fermenting for a suitable time, for ex
ample, from 12 to 96 hours, it is found that the con
_do____
FeSOr- 7H2O
ZnSOa
MHSO4-1'I2O
zramsdo
___________________________ __d0____
12
20
1
Water to 1000 ml.
AdJust pH:1.3 with concentrated H2504.
‘EXAMPLE 1
55
Conversion of Anhydrotetracycline to Tetracycline
Spores of Streptomyces aureofaciens ATCC No. 10,762
were Washed from a streaked agar slant with sterile dis
tilled Water and yielded a suspension containing 73x10“
spores per milliliter. A 1.0 ml. aliquot of this suspen
sion was added to 100 ml. of the sterile inoculum medium
in a 500-ml. Erlenmeyer ?ask. The mixture was incu
bated at 265° C. for 24 hours on a reciprocating shaker
operating at 116 oscillations per minute. A 1.0 ml.
medium serve as the substrate from which the antibiotic
is synthesized. It is unexpected to discover that a known 65 portion of this 24-hour inoculum was added to each of
compounds to the corresponding tetracyclines. In the
normal fermentation, the ingredients of the nutrient
stable chemical compound, especially one resulting from
vigorous chemical degradation, may serve as the sub
strate for producing an entirely different tetracycline com
pound.
two 250-ml. Erlenmeyer ?asks containing 25 ml. vol
umes of the sterile fermentation medium. The i?asks
and contents were incubated at 25° C. for 48 hours on a
rotary shaker operating at 180 revolutions per minute.
.The conditions of the fermentation for the biological 70 Upon completion of this 48-hour incubation period, 10
conversion of the anhydrotetracyclines to the corre
sponding tetracyclines are generally the same as set forth
mg. of C14-labeled anhydrotetracycline (300 disintegra
tions per minute per microgram of anhydrotetracycline)
3,053,740
3
4.
was added as a ?ne powder to one of the two 250-ml.
reciprocating shaker, then a 1.0 ml. portion of this 24
Erlenmeyer ?asks containing the 48-hour fermentation
hour vegetative inoculum was removed and added to a
25-ml. portion of the sterile fermentation medium in a
mashes; the other ?ask and its contents being retained as
a control. The two ?asks were incubated for an addi
250-ml. Erlenmeyer ?ask. The ?ask contents were in
tional 72 hours at 25° C. on a rotary shaker; making 5 cubated at 25° C. for 48 hours on a rotary shaker, then
this a total incubation time of 120 hours. At this time,
3.0 ml. samples of the growing mash were transferred
3.0 ml. portions were removed from each of the two
to two 20><150 mm. sterile shaker tubes. A 200 mcg.
?asks before the mash assay was performed.
quantity of C14-labe1ed anhydrotetracycline (1000 disin
tegrations per minute per microgram) in powder form
Mash assay for
was added to one tube; the other being retained as a con
trol. The tubes were mounted on a rotary shaker and
incubated at 25 ° C. for an additional 72 hours; making
tetracycline, mcg/ ml.
Control (no anhydrotetracycline added) ________ __ 50
Test (10 mg. anhydrotetracycline added) _______ __ 270
a total incubation period of 120 hours. Upon comple
tion of this incubation period, the tubes were removed,
the mash in each diluted with 10 ml. of 0.2 N hydro
The 220 meg/ml. difference between the control value
of 50 mcg./ml. and the test value of 270 meg/ml. is
the amount of tetracycline derived from the conversion
chloric acid and the mycelium removed by ?ltration.
of the anhydrotetracycline which was added. This repre
sents an overall conversion from anhydrotetracycline to
Each ?ltrate was extracted by shaking with 0.5 ml. of
parachlorophenol.
tetracycline of 55 percent.
Ten-microliter portions of each phenol phase were
chromatographed according to systems A and B described
in Example 1, The developed, air-dried chromatograms
were examined visually, then scanned for radioactivity
The 3.0 ml. portions of control and test 120-hour
fermentation mashes, removed and saved before the mash
assay was performed, were each diluted with 10 ml.
of 0.2 N hydrochloric acid and the mycelium removed by
by means of a Geiger-Miiller counter.
?ltration. Each ?ltrate was extracted by shaking with
1.5 ml. of parachlorophenol.
Ten microliters of each phenol phase were spotted on
The appearance of zones of radioactivity having R;
values of 0.31 (system A) and of 0.41 (system B); cor
responding to the R; values for tetracycline on the con
trol strips; con?rmed the conversion of anhydrotetra
cycline to tetracycline. By measurement of radioactive
peak areas, the extent of conversion of anhydrotetra
cycline to tetracycline was estimated at about 10 percent.
EXAMPLE 3
individual 1-inch wide by 22-inch long strips of What
man No. 1 chromatograph paper which previously had
been buffered by immersion in a solution of 0.3
M Nari-IP04 adjusted to pH 3.0 with phosphoric acid, then
air dried.
Subsequently the strips were subjected to
descending development; using the organic phase of a
Conversion of Anlzydrotetracycline to Tetracycline
A 1.0 ml. spore suspension of S. viridifaciens ATCC
No. 11,989 containing 63><106 spores per milliliter, was
n—butanol:O.3 M NaH2PO4::l:1 solvent system wherein
the butter was adjusted to pH 3.0 with phosphoric acid
(system A).
Two l-inch wide by 22-inch long strips of Whatman
No. 1 chromatograph paper which previously had been
buffered by immersion in a solution composed of equal
volumes of 0.4 M Nazi-IP04 and 4.5 percent citric acid,
added to 100 ml. of the sterile inoculum medium con
tained in a 500-ml. Erlenmeyer ?ask. This mixture was
incubated at 26.5° C. ‘for 24 hours on a reciprocating
shaker operating at 116 oscillations per minute. At the
then air dried, were hung overnight in a chromatograph 40 termination of this incubation period, a 25 ml. portion
tank containing Water on the floor of the tank and ethyl
of the sterile fermentation medium in a 250-ml. Erlen
acetate in an open container on the bottom of the tank.
meyer ?ask was seeded with a 1.0 ml. aliquot of this
About an hour before developing, a 10-microliter vol
24-hour vegetative inoculum. This seeded ?ask was
ume of the phenol phase of the extract of control ?l
incubated at 25° C. for 48 hours on a rotary shaker oper
trate was spotted on one strip and an equal volume of 415 ating at 180 revolutions per minute. Upon completion
the phenol phase of the extract of test ?ltrate was spotted
of this 48-hour incubation period, 3.0 ml. aliquots of the
on the other strip. Subsequently, the strips were sub
growing mash were transferred to two 20><150 mm.
jected to descending development; using the organic phase
sterile shaker tubes. A 200 mcg. quantity of Chi-labeled
of an ethyl acetate:phosphate-citrate buffer::1:1 sol
vent system at pH 4.5 (system B). The phosphate
citrate buffer is composed of equal volumes of 0.4
M Na2HPO4 and 4.5 percent citric acid. Developed spots
were detected by fuming the strips with ammonia and
anhydrotetracycline (11000 disintegrations per minute per
microgram) in powder form was added to one tube; the
other being retained as a control. The tubes were mount
ed on a rotary shaker and incubated at 25° C. for an
additional 72 hours; making a total incubation period of
120 hours. Upon completion of this incubation period,
viewing under ultraviolet light.
The developed, air-dried strips were scanned for radio- ‘
the tubes were removed, the mash in each diluted with
10 m1. of 0.2 N hydrochloric acid and the mycelium
removed by ?ltration. Each ?ltrate was extracted by
activity by means of a Geiger-Muller counter. The ap
pearance of zones of radioactivity on the test strips at
R; 0.31 (system A) and at R, 0.41 (system B); corre
shaking with 0.5 ml. of parachlorophenol.
sponding to the R; values for tetracycline on the control
Ten-microliter portions of each phenol phase were
strips, con?rmed the conversion of anhydrotetracycline 60 chromatographed according to systems A and B described
to tetracycline as previously demonstrated by the mash
in Example 1. The developed, air-dried chromatograms
assay shown above. By measurement of radioactive peak
were examined visually, then scanned for radioactivity
areas, the extent of conversion of C14-1abeled anhydro
tetracycline to C14-labeled tetracycline was estimated at
50 percent.
EXAMPLE 2
by means of a Geiger-Muller counter.
The appearance of zones of radioactivity having R;
' values of 0.31 (system A) and of 0.41 (system B); cor
Conversion of Anlzydrotetracycline to Tetracycline
An aqueous suspension of S. rimosus ATCC No. 10,
970 spores containing approximately 68x106 spores per
milliliter, was prepared by washing a streaked agar slant
with sterile distilled water. A 1.0 ml. of volume of this
suspension was added to 100 ml. of the sterile inoculum
medium in a 500-ml. Erlenmeyer ?ask. The inoculated
material was incubated at 26.5° C. ‘for 24 hours on a 75
responding to the R; values for tetracycline on the control
strips; confirmed the conversion of anhydrotetracycline to
tetracycline. By measurement of radioactive peak areas,
the extent of conversion of anhydrotetracycline to tetra
cycline was estimated at about 30 percent.
EXAMPLE 4
Conversion of 7-Chloroanhydrotetracycline to
7-Chlorotetracycline
A 1.0 ml. spore suspension of S. aurcofaciens ATCC
3,053,740
5
6
added to 100 ml. of the sterile inoculum medium con
tained in a 500-ml. Erlenmeyer ?ask. This mixture was
the strips were subjected to descending development; using
the organic phase of an ethyl acetate:phosphate-citrate
bu?er::1:1 solvent system at pH 4.5 (system B). The
phosphate-citrate buifer is composed of equal volumes
incubated at 26.5 ° C. for 24 hours on a reciprocating
shaker operating at 116 oscillations per minute.
Next, a 25 ml. portion of the sterile fermentation
medium was placed in each of four 250-ml. Erlen
meyer ?asks. An inhibitor of fermentative chlorination,
2(2-furyl)-5-mercapto-l,3,4-oxadiazole, was added; in the
of 0.4 M Na2HPO4 and 4.5 percent citric acid. Developed
proportion of 7 meg/ml. of fermentation medium, that 10
of the 24-hour inoculum described above, the four ?asks
were incubated at 25° C. for 48 hours on a rotary shaker
operating at 180 revolutions per minute.
At the termination of this incubation period, 8.75 mg.
of anhydrochlortetracycline was added to the contents
of one ?ask; 12.5 mg. of Chi-labeled anhydrochlortetra
shown above. .By measurement of radioactive peak
areas, the extent of conversion of Chi-labeled anhydro
chlortetracycline to C14-labeled chlortetracycline was
estimated at 50 percent; while the conversion of C136
labeled anhydrochlortetracycline to Chis-labeled chlor
tetracycline was estimated at 40 percent.
EXAMPLE 5
cycline (320 disintegrations per minute per microgram of
anhydrochlortetracycline) was added to the contents of
the second ?ask; 12.5 mg. of Cl36-labeled anhydrochlor
tetracycline (50 disintegrations per minute per micro
gram of anhydrochlortetracycline) was added to the con
tents of the third ?ask; the fourth ?ask being retained
25
All four ?asks were again incubated at 25° C. on a
rotary shaker for an additional 72 hours; making a total
incubation time of 120 hours. At this time, 3.0 ml.
portions were removed from each of the two 25 ml., 120
hour fermentation mashes containing C14-labeled anhy
droohlortetracycline and Clse-labeled anhydrochlortetra
cycline, respectively, and also from the control fermen
tation mash, and saved for further testing. The remain
ing portion of the control fermentation mash and that to
spots were detected by fuming the strips with ammonia
and viewing under ultraviolet light.
The developed, air-dried strips were scanned for radio
activity by means of a Geiger-Muller counter. The
appearance of zones of radioactivity on the test strips
at Rf 0.54 (system A) and at Rf 0.76 (system B); corre
sponding to the Rf values for chlortetracycline con?rmed
the conversion of anhydrochlortetracycline to chlortetra
cycline as previously demonstrated by the mash assay
is, a total of 175 mcg. to each of the four ?asks. After
being sterilized, cooled, and seeded with 1.0 ml. aliquots
as a control.
.
volume of the phenol phase of the extract of Chis-labeled
test ?ltrate was spotted on a third strip. Subsequently,
No. 10,762 containing 65><106 spores per milliliter, was
Conversion of 7-Br0mzonnhydrotetracycline to
7-Br0m0tetracyc‘line
Spores of S. aureofaciens ATCC No. 12,551 were
washed from a streaked agar slant with sterile distilled
water to form a suspension containing approximately
70><l06 spores per milliliter. A 100 ml. portion of the
sterile inoculum medium in a 500-ml. Erlenmeyer ?ask
was inoculated with a 1.0 ml. aliquot of this suspension
and incubated at 26.5° C. on a reciprocating shaker
operating at 116 oscillations per minute for 24 hours.
which nonradioactive anhydrochlortetracycline had been 35 At the termination of this incubation period, a 1.0 ml.
added were assayed ?uorometrically for chlortetracycline
portion of this inoculum was added to each of two
content, with the following results:
Mash assay for
chlortetracycline,
meg/ml.
250-ml. Erlenmeyer ?asks containing 25 ml. of the sterile
fermentation medium plus 175 mcg. of 2-(2-furyl)-5
mercapto-1,3,4-oxadiazole, an inhibitor of fermentative
chlorination. For a period of 48 hours these two inocu
Control (no anhydrochlortetracycline added) _____ __ 310
lated ?asks were incubated at 25° C. on a rotary shaker
Test (8.75 mg. anhydrochlortetracycline added)____ 460
The 150 mcg./ml. of chlortetracycline diiference be
of anhydrobromtetracycline Was added, in powder form,
operating at 180 revolutions per minute; then 8.0 mg.
tween the control value of 310 mcg./m‘l. and the test value 45 to one of the two ?asks. Both ?asks were again incu
bated under the above conditions for 72 hours; making
of 460 mcg./ml. is the amount of chlortetracycline de
a total of 120 hours in all. The ?asks were then removed
rived from the conversion of the anhydrochilortetracycline
from the incubator and their contents assayed for
to chlortetracycline, a yield of 43 percent.
The 3.0 ml. portions of control, Chi-labeled anhydro~
ohlortetracycline and Cl36-lalbeled anhydrochlortetracy
cline 120-hour fermentation mashes were each diluted
bromtetracycline, with the following results:
Mash assay for
bromtetracycline,
meg/ml.
with 10 ml. of 0.2 N hydrochloric acid and the mycelium
removed by ?ltration. The ?ltrate, in each instance, was
Control (no anhydrobromtetracycline added)____
0
extracted by shaking with 0.5 ml. parachlorophenol.
Test (8.0 mg. of anhydrobromtetracycline added__ 180
Ten microliters of each phenol phase were spotted on 55
The overall conversion of anhydrobromtetracycline to
individual l-inch wide by 22-inch long strips of Whatman
bromtetracycline was 56 percent.
No. 1 chromatograph paper which previously had been
EXAMPLE 6
buffered by immersion in a solution of 0.3 M NaH2PO4
adjusted to pH 3.0 with phosphoric acid, then air dried.
Conversion of 6-Demethylanhydrotetracycline to
Subsequently the strips were subjected to descending de 60
6-Demethyltetracycline
velopment; using the organic phase of a n-butanol:0.3
A water suspension, containing approximately 70x106
M NaH2PO4::1:1 solvent system wherein the buffer is
spores per milliliter of S. aureofaciens ATCC No. 13,192
adjusted to pH 3.0 with phosphoric acid (system A).
was produced by washing a streaked agar slant with sterile
Three l-inch wide by 22-inch long strips of Whatman
No. 1 chromatograph paper, which previously had been 65 distilled water. One milliliter of this suspension was
used to inoculate 100 ml. of the sterile inoculum medium
buffered by immersion in a solution composed of equal
in a 500-ml. Erlenmeyer ?ask. The seeded medium was
volumes of 0.4 M Na2HPO4 and 4.5 percent citric acid,
incubated at 26.5 ° C. for 24 hours on a reciprocating
were air dried. The buifered strips were hung overnight
shaker operating at 116 oscillations per minute. At the
in a chromatograph tank containing water on the ?oor of
the tank and ethyl acetate in an open container on the 70 termination of this incubation period, two 25 ml. portions
of the sterile fermentation medium in two 250-ml. Erlen
bottom of the tank. About an hour before developing,
meyer ?asks were seeded with 1.0 ml. aliquots of this
a 10-microliter volume of the phenol phase of the extract
24-hour inoculum. These two seeded ?asks were incu
of control ?ltrate was spotted on one strip, an equal
bated at 25° C. for 48 hours on a rotary shaker operat
volume of the phenol phase of the extract of C14-labeled
test ?ltrate was spotted on a second strip, and an equal 75 ing at 180 revolutions per minute. Upon completion of
3,053,740
7
this 48-hour incubation period, 3.0‘ ml. aliquots of both
contents were incubated at 25 ° C. for 48 hours on a
growing mashes were transferred to 20X 150 mm. sterile
shaker tubes; A 200 mcg. quantity of Cl‘i-labeled
rotary shaker, then 8.0 mg. of 7-bromo-6-d‘emethylan
6-demethylanhydrotetracycline (1,000 disintegrations per
hydrotetracycline was added, in powder form, to one
of the two ?asks. Both ?asks were again incubated at
25° C. on a rotary shaker for an additional 72 hours;
minute per microgram) in powder form was added to one
tube; the other being retained as a control. The tubes
thus achieving a total incubation time at 25° C. of 120
were mounted on a rotary shaker and incubated at 25° C.
hours. Assay of the 120-hour fermentation mashes from
for an additional 72 hours; making a total incubation
these two ?asks yielded the following results:
period of 120 hours. Upon completion of this incuba
tion period, the tubes were removed, the mash in each
diluted with 10 ml. of 0.2 N hydrochloric acid and the
mycelium removed by ?ltration. Each ?ltrate was ex
tracted by shaking with 0.5 ml. of parachlorophenol.
Two strips of l-inch wide by 22-inch long Whatman
No. 1 chromatograph paper were buffered by immersion
in a pH 3.4 buffer solution composed of 30 volumes of
0.2 M Na2HPO4 and 70 volumes of 2.24 percent citric
acid, and air dried. The two buttered strips were sus
pended in a chromatograph tank containing water on the
?oor of the tank and a mixture of organic solvents (nitro
methaneibenzenezpyridine::20:10:3) in an open con
tainer on the bottom of the tank. About an hour before
Mash assay for
7-bromo-6-demethyl
Control
cline
tetracycline, mcg./ ml.
7-brom0-6-demethylanhydrotetracy
added) _____________________________ __
0
Test (8 mg. of 7-bromo-6-demethylanhydrotetracy
cline
added) _____________________________ __ 215
The overall conversion of 7-bromo-6-demethylanhydro
tetracycline to 7-bromo-6-demethyltetracycline was 61
percent.
EXAMPLE 8
Conversion of Anhydrotetracyclinonitrile to Tetracyclino
nitrile
developing, a 10 microliter quantity of the phenol phase
of the extract of control ?ltrate was spotted on one strip
(no
A SOO-ml. Erlenmeyer ?ask containing 100 ml. of the
and an equal volume of the phenol phase of the extract
sterile inoculum medium was seeded with 1.0 ml. of an
of Chi-labeled test ?ltrate was spotted on the other.
aqueous suspension of spores of S. aureofaciens ATCC
No. 13,192. The concentration of spores in this sus
pension was approximately 65 X106. The seeded inocu
Subsequently, the strips were subjected to descending
development; utilizing the organic phase of a nitro
methane:benzene:pyridine:pH 3.4 butter: :20: 10:3 :3
solvent system wherein the pH 3.4 buffer was composed
of 30 volumes of 0.2 M Na2HPO4 and 70 volumes of
2.24 percent citric acid (system C).
Two strips of l-inch wide by 22-inch long Whatman
No. 1 chromatograph paper were buttered by immersion
in a solution of 0.3 M NaH2PO4 adjusted to pH 2.0 with
phosphoric acid, then air-dried. The two buttered strips
were suspended in a chromatograph tank containing
water on the ?oor of the tank and an organic solvent
(butyl acetate) in an open container on the bottom of
the tank. About an hour before developing, a 10 micro
liter quantity of the phenol phase of the extract of control
?ltrate was spotted on one strip, and an equal volume
of the phenol phase of the extract of Cl‘i-labeled test
?ltrate was spotted on the other. Subsequently, the strips
were subjected to descending development; using the
organic phase of a butyl acetate:5% trichloracetic acid:
pH 2.0 butfer::5:1:4 solvent system wherein the but?“
solution was 0.3 M NaH2PO4 (system D).
The developed‘, air-dried strips were examined visually
and scanned for radioactivity by means of a Geiger- '
Miiller counter. The appearance of zones of radio
lum was incubated at 26.5 ° C. for 24 hours on a recip
rocating shaker. At the end of this period, a 1.0 ml.
aliquot of this inoculum was added to each of two 25
ml. portions of the sterile fermentation medium in sepa
rate 250~ml. Erlenmeyer ?asks. These two seeded ?asks
were incubated at ‘25° C. on a rotary shaker for 48
hours.
At this point, 7.0 mg. of powdered anhydro
tetracyclinonitrile was added to one of the ?asks; the
other ‘being retained as a control. Both ?asks were again
incubated at 25° C. and with agitation for 72 hours
more; thus resulting in a total incubation time at 25° C.
of 120 hours. Three milliliter aliquots of both test and
control 120-hour fermentation mashes were each diluted
with 10 ml. of 0.2 N hydrochloric acid and the mycelium
removed by ?ltration. The ?ltrate, in each instance, was
extracted by shaking with 0.5 ml. of parachlorophenol.
Four l-inch wide by 22-inch long strips of Whatman
No. 1 chromatograph paper were buffered by immersion
in a solution of 0.3 M NaH2PO4 adjusted to pH 2.0 with
phosphoric acid, then air dried.
Two buffered strips
were suspended overnight in a chromatograph tank con
taining water on the ?oor of the tank and a mixture of
organic solvents (chloroformzamyl alcohol::4:1) in an
activity having R! values of 0.33 (system C) and 0.24
(system D), corresponding to those of 6-demethyltetra
open container on the bottom of the tank.
methylanhydrotetracycline to C14-labeled ‘6-demethyl
ing development; utilizing the organic phase of a chloro
formcamyl alcohol:pH 2.0 bu?er::4:1:5 solvent system
About an
hour before developing, a 10 microliter quantity of the
cycline, testi?ed to the conversion of C14-labeled 6-de
phenol phase of the extract of control ?ltrate was spotted
methylanhydrotetracycline to C14-labeled 6-demethyl Cr Ur on one strip and an equal volume of the phenol phase
tetracycline. Measurement of radioactive peak areas
of the extract of test ?ltrate was spotted on the other.
indicated the extent of conversion of C14-labeled 6-de
Subsequently, the two strips were subjected to descend
tetracycline to be 50 percent.
EXAMPLE 7
Conversion of 6-Demethyl-7-Brom0anhydrotetracycline
t0 6-Demethyl-7-Br0m0tetracycline
An aqueous suspension of S. aureofaciens ATCC No.
10,762 spores, containing approximately 70x106 spores
per milliliter, was prepared by washing a streaked agar
slant with sterile distilled water. A 1.0 ml. quantity of
this suspension was added to I100 ml. of the sterile
inoculum medium in a 500-ml. Erlenmeyer ?ask. The
inoculated material was incubated at 26.5 ° C. for 24
hours on a reciprocating shaker, then 1.0 ml. portions
of this 24-hour inoculum were removed and added to
each of two 25 m1. quantities of the sterile fermentation
medium in two 250-rn1. Erlenmeyer ?asks. The ?ask
wherein the pH 2.0 buffer solution was 0.3 M NaH2PO4
(system B). The other two buffered strips were sus
pended overnight in a chromatograph tank containing
water on the ?oor of the tank and a mixture of organic
solvents (chloroform:dimethylforrnamide:24:1) in an
open container on the bottom of the tank.
About an
hour before development, a IO-microliter quantity of the
phenol phase of the extract of control ?ltrate was spot
ted on one buttered strip and an equal volume of the
phenol phase of the extract of test ?ltrate was spotted
on the other. Subsequently, the two strips were sub
jected to descending development using the organic phase
of a chloroform:dimethylformamidezpH 2.0 butter: :4: 1 :6
solvent system wherein the pH 2.0 buffer solution was
0.3 M NaH2PO4 (system F). The developed air-dried
strips were scanned ?uoroscopically. The appearance
3,053,740
1%
of ?uorescent zones having Rf values of 0.14 (system
B) and ‘0.12 (system F), corresponding to those of
tetracyclinonitrile, thus con?rmed the fact that anhydro
tetracyclinonitrile was converted to tetracyclinonitrile.
The extent of conversion of anhydrotetracyclinonitrile
sisting of S. aureofaciens, S. rimosus and S. viridifaciens,
and continuing the fermentation until the 6-demethyl
anhydrotetracycline is substantially converted to 6-de
methyltetracycline.
6. The process for the biological transformation of
to tetracyclinonitrile was estimated at 35 percent.
What is claimed is:
1. The process for the biological transformation of
6-demethyl-7-chloroanhydrotetracycline to 6-demethyl-7
chlorotetracycline which comprises adding 6-demethyl
cycline.
6-demethyl-7-bromoanhydrotetracycline to 6-demethyl-7
bromotetracycline which comprises adding 6-demethyl-7
7-chloroanhydrotetracycline to an aqueous nutrient me
dium, aerobically fermenting the aqueous nutrient me
an anhydrotetracycline to the corresponding tetracycline
which comprises adding an anhydrotetracycline to an 10 dium with a strain of a species of the genus Streptomyces
selected from the group consisting of S. aureofaciens, S.
aqueous nutrient medium, aerobically fermenting the
rimosus and S. viridifaciens, and continuing the fermenta
aqueous nutrient medium with a strain of a species of
tion until the 6-demethyl-7-chloroanhydrotetracycline is
the genus Streptomyces selected from the group consist
substantially converted to 6-demethyl-7-chlorotetra
ing of S. aureofaciens, S. rimosus and S. viridifaciens, and
continuing the fermentation until the anhydrotetracycline 15 cycline.
7. The process for the biological transformation of
is substantially converted to the corresponding tetra
'2. The process for the biological transformation of
anhydrotetracycline to tetracycline which comprises add
bromoanhydrotetracycline to an aqueous nutrient me
tetracycline.
cycline.
ing anhydrotetracycline to an aqueous nutrient medium, 20 dium, aerobically fermenting the aqueous nutrient me
dium with a strain of a species of the genus Streptomyces
aerobically fermenting the aqueous nutrient medium with
selected from the group consisting of S. aureofaciens, S.
a strain of a species of the genus Streptomyces selected
rimosus and S. viridifaciens, and continuing the fermenta
from the group consisting of S. aureofaciens, S. rimosus
t-ion until the 6-demethyl-7-bromoanhydrotetracycline is
and S. viridifaciens, and continuing the fermentation un
til the anhydrotetracycline is substantially converted to 25 substantially converted to 6-demethyl-7-bromotetra
3. The process for the biological transformation of
7-chloroanhydrotetracycline to 7-chlorotetracycline which
comprises adding 7-chloroanhydrotetracycline to an
aqueous nutrient medium, aerobically fermenting the
aqueous nutrient medium with a strain of a species of
8. The process for the biological transformation of
anhydrotetracyclinonitrile to tetracyclinonitrile which
comprises adding anhydrotetracyclinonitrile to an aque
ous nutrient medium, aerobically fermenting the aqueous
nutrient medium with a strain of a species of the genus
Streptomyces selected from the group consisting of S.
the genus Streptomyces selected from the group consist
aureofaciens, S. rimosus and S. viridifaciens, and continu
ing of S. aureofaciens, S. rimosu‘s and S. Viridifaciens,
ing the fermentation until the anhydrotetracyclinonitrile is
and continuing the fermentation until the 7-chloroanhy
drotetracycline is substantially converted to 7-chlorotetra 35 substantially converted to tetracyclinonitrile.
9. The process for the biological transformation of
cycline.
12a-deoxyanhydrotetracycline to 12a-deoxytetracycline
4. The process for the biological transformation of
7-bromoanhydrotetracycline to 7-bromotetracycline which
comprises adding 7~bromoanhydrotetracycline to an
aqueous nutrient medium, aerobically fermenting the
aqueous nutrient medium with a strain of a species of
which comprises adding 12a-deoxyanhydrotetracycline to
an aqueous nutrient medium, aerobically fermenting the
aqueous nutrient medium with a strain of the species of
the genus Streptomyces selected from the group consisting
of S. aureofaciens, S. rimosus and S. viridifaciens, and
continuing the fermentation until the 12a-deoxyanhydro
tetracycline is substantially converted to 12a-deoxytetra
the genus Streptomyces selected from the group consist
ing of S. aureofaciens, S. rim‘osus and S. viridz'faciens,
and continuing the fermentation until the 7-bromoanhy
drotetracycline is substantially converted to 7-‘bromo 45 cycline.
tetracycline.
5. The process for the biological transformation of
6-demethylanhydrotetracycline to 6-demethyltetracycline
which comprises adding 6-demethylanhydrotetracycline
to an aqueous nutrient medium, aerobically fermenting
the aqueous nutrient medium with a strain of a species
of the genus Streptomyces selected from the group con
References Cited in the ?le of this patent
UNITED STATES PATENTS
2,952,587
2,965,546
Miller et al. _________ __ Sept. 13, 1960
McCormick et al. ____ _.. Dec. 20, 1960
Документ
Категория
Без категории
Просмотров
22
Размер файла
851 Кб
Теги
1/--страниц
Пожаловаться на содержимое документа