Manufacturing Process
Two thousand parts by volume of an aqueous culture medium (pH 7.2)
comprising 0.5% of glycerol, 0.5% of polypeptone, 0.5% of yeast extract and
0.3% of meat extract is inoculated with Escherichia coli R11 (IFO-13560). The
medium is incubated at 37°C under aeration for 18 h. The culture broth is
subjected to centrifuge to recover 4.4 parts of wet cells. The cells are
suspended into 17.6 parts by volume of 0.05 M phosphate buffer (pH 7.0).
The suspension is subjected to ultrasonic oscillation (Kaijo Denki Co., Ltd.; TA-4201, 4280-type, 2A) to disintegrate the cells, followed by removing the
debris (insoluble materials) by centrifugation, whereby 17 parts by volume of
crude enzyme solution is obtained.
To 17 parts by volume of the crude enzyme solution are added 5 parts of
kanamycin B, 50 parts by volume of 0.5 M phosphate buffer (pH 7.0), 100
parts by volume of 1 M adenosine triphosphate solution, 50 parts by volume
of 0.1 M magnesium acetate solution and 50 parts by volume of 0.1 M 2-
mercaptoethanol, which is filled up to 500 parts by volume with distilled
water. The mixture is subjected to enzymic reaction at 37°C for 20 h.
The reaction mixture is heated at 80°C for 5 min to cease the reaction,
followed by centrifugation. The supernatant is run onto a column of 100 parts
by volume of cation-exchange resin [Amberlite IRC-50, NH4
+-form]. The
column is washed with water, and then eluted with 1 N-aqueous ammonia to
give fractions which contain kanamycin B-3'-phosphate. The fractions are
collected and concentrated under reduced pressure, and then the concentrate
is run onto a column of 100 parts by volume of cation-exchange resin
[carboxy-methyl Sephadex C-25, NH4
+-form]. The column is washed with
water, and eluted with 0.2 N-aqueous ammonia to give fractions which contain
kanamycin B-3'-phosphate. The fractions are collected, concentrated and
lyophilized, whereby 4.5 parts of kanamycin B-3'-phosphate.
A solution of one part of kanamycin B-3'-phosphate, 10 parts by volume of
bis(trimethylsilyl)acetamide, 2 parts by volume of trimethylchlorosilane and
0.4 part of triphenylphosphine is heated at 115°C for 30 h. After cooling, the
reaction mixture is concentrated under reduced pressure, and to the
concentrate is added 100 parts by volume of methanol and 50 parts by
volume of water, and then the mixture is stirred for 1 h. Methanol is removed
by distillation, and ethyl acetate-soluble portion is removed. The water layer is
run onto a column of 60 parts by volume of cation-exchange resin [Amberlite
CG-50, NH4
+-form]. The column is washed with 200 parts by volume of water,
and fractionated by linear gradient method with 600 parts by volume of water
and 600 parts by volume of 0.5 N-aqueous ammonia, each fraction being 10 parts by weight. Upon concentration of some fractions 0.61 part of 2',3'-
epimino-2'-deamino-3'-deoxykanamycin B is obtained.
In 40 parts by volume of water is dissolved 0.6 part of 2',3'-epimino-2'-
deamino-3'-deoxykanamycin B, and in the presence of 9 parts by volume of
Raney nickel the mixture is stirred while introducing hydrogen gas at a
pressure of 100 kg/cm2 at 60°C for 6 h. After the reaction Raney nickel is
separated by filtration. The Raney nickel is washed well with 300 parts by
volume of 1 N-aqueous ammonia and the washing is added to the filtrate. The
whole is concentrated to about 100 parts by volume. The precipitated
insolubles are removed by filtration, and the pH of the supernatant is adjusted
to about 5.0 with hydrochloric acid. The mixture is run onto a column of 50 ml
of cation-exchange resin [Amberlite CG-50, NH4
+-form].
The column is washed with 150 parts by volume of water, and fractionated by
linear gradient method with 1400 parts by volume of water and 1400 parts by
volume of 0.3 N-aqueous ammonia, each fraction being 14 parts by weight.
From No. 146 to 162 fractions 0.30 part of 3'-deoxykanamycin B (Tobramycin)
is obtained.
water. The mixture is subjected to enzymic reaction at 37°C for 20 h.
The reaction mixture is heated at 80C for 5 min to cease the reaction,
followed by centrifugation. The supernatant is run onto a column of 100 parts
by volume of cation-exchange resin [Amberlite IRC-50, NH4
+-form]. The
column is washed with water, and then eluted with 1 N-aqueous ammonia to
give fractions which contain kanamycin B-3'-phosphate. The fractions are
collected and concentrated under reduced pressure, and then the concentrate
is run onto a column of 100 parts by volume of cation-exchange resin
[carboxy-methyl Sephadex C-25, NH4
+-form]. The column is washed with
water, and eluted with 0.2 N-aqueous ammonia to give fractions which contain
kanamycin B-3'-phosphate. The fractions are collected, concentrated and
lyophilized, whereby 4.5 parts of kanamycin B-3'-phosphate.
A solution of one part of kanamycin B-3'-phosphate, 10 parts by volume of
bis(trimethylsilyl)acetamide, 2 parts by volume of trimethylchlorosilane and
0.4 part of triphenylphosphine is heated at 115°C for 30 h. After cooling, the
reaction mixture is concentrated under reduced pressure, and to the
concentrate is added 100 parts by volume of methanol and 50 parts by
volume of water, and then the mixture is stirred for 1 h. Methanol is removed
by distillation, and ethyl acetate-soluble portion is removed. The water layer is
run onto a column of 60 parts by volume of cation-exchange resin [Amberlite
CG-50, NH4
+-form]. The column is washed with 200 parts by volume of water,
and fractionated by linear gradient method with 600 parts by volume of water
and 600 parts by volume of 0.5 N-aqueous ammonia, each fraction being 10
parts by weight. Upon concentration of some fractions 0.61 part of 2',3'-
epimino-2'-deamino-3'-deoxykanamycin B is obtained.
In 40 parts by volume of water is dissolved 0.6 part of 2',3'-epimino-2'-
deamino-3'-deoxykanamycin B, and in the presence of 9 parts by volume of
Raney nickel the mixture is stirred while introducing hydrogen gas at a
pressure of 100 kg/cm2 at 60°C for 6 h. After the reaction Raney nickel is
separated by filtration. The Raney nickel is washed well with 300 parts by
volume of 1 N-aqueous ammonia and the washing is added to the filtrate. The
whole is concentrated to about 100 parts by volume. The precipitated
insolubles are removed by filtration, and the pH of the supernatant is adjusted
to about 5.0 with hydrochloric acid. The mixture is run onto a column of 50 ml
of cation-exchange resin [Amberlite CG-50, NH4
+-form].
The column is washed with 150 parts by volume of water, and fractionated by
linear gradient method with 1400 parts by volume of water and 1400 parts by
volume of 0.3 N-aqueous ammonia, each fraction being 14 parts by weight.
From No. 146 to 162 fractions 0.30 part of 3'-deoxykanamycin B is obtained.
The aqueous solution containing 3'-deoxykanamycin B in free base form by
addition of concentrated sulfuric acid give the 3'-deoxykanamycin B sulfate
(tobramycin sulfate). The solution of this compound is decolorized by stirring
of Darco G-60, filtered and purified by column chromatography.
Clinical Use
Introduced in 1976, tobramycin sulfate (Nebcin) is the mostactive of the chemically related aminoglycosides called nebramycinsobtained from a strain of Streptomyces tenebrarius Five members of the nebramycin complex have beenidentified chemically.
Factors 4 and 4' are 6＂-O-carbamoylkanamycin B andkanamycin B, respectively; factors 5' and 6 are 6＂-O-carbamoyltobramycinand tobramycin; and factor 2 isapramycin, a tetracyclic aminoglycoside with an unusual bicycliccentral ring structure. Kanamycin B and tobramycinprobably do not occur in fermentation broths per se but areformed by hydrolysis of the 6-O＂-carbamoyl derivatives inthe isolation procedure.
The most important property of tobramycin is its activityagainst most strains of P. aeruginosa, exceeding that of gentamicinby twofold to fourfold. Some gentamicin-resistantstrains of this troublesome organism are sensitive to tobramycin,but others are resistant to both antibiotics. OtherGram-negative bacilli and staphylococci are generally moresensitive to gentamicin. Tobramycin more closely resembleskanamycin B in structure (it is 3'-deoxykanamycin B).
