Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
lQ48430
The subject of the invention is a new process for
the manufacture of desacetoxycephalosporin C (3-methyl-7-
(o-aminoadipoyl?-ceph-3-em-carboxylic acid) of the formula
I
.
> Ci~ 2)3 ~ CO - ~ C.13
COOH
and of its salts. mis compound is known, compare
Stedman et al. J. Med. Chem., 7 (1964), 117-119 and US
Patent 3,124,576. It has been obtained by catalytic
hydrogenation of cephalosporin C and was converted directly
into derivatives, without being characterised. Deriva-
tives of compound I, for example 3-methyl-7-(phenoxy-
acetyl)-ceph-3-em-4-carboxylic acid, have also been pre-
pared by catalytic high pressure hydrogenation of the
corresponding 3-acetoxymethyl compound, compare Morin et
al., J. Am. Chem. Soc. 85 (1963), 1896-97. Another
method of manufacture of such derivatives is the sulphoxide
rearràngement of corresponding penicillinsj compare Morin
et al., loc. cit.
The compound of the formula I is of considerable
practical interest, less so because of its relatively weak
antibiotic action than because of its utilisability as a
starting material for the manufacture of valuable
- 2 - ~ .
1~48430
derivatives of the formula II
- ~H
N ~ 3
-COOH
wherein R is an acyl radical other than the aminoadipoyl
radical, especially an acyl radical such as occurs in thera-
peutically utilisable acyl derivatives of 6-aminopenicil-
lanic acid and 7-aminocephalosporanic acid, compare, for
example, Sassiver et al., Adv. Appl. Microbiol. 3 (1970),
163-235. To manufacture such acyl derivatives, the ~-
aminoadipoyl radical is first split off from compound I, for
example by the process of French Patent No. 1,394,~20 or
Belgian Patent No. 720,185, giving the compound of the
above formula II, wherein R represents hydrogen. m is
compound can then be acylated in the desired manner.
The present invention makes compound I accessible
by fermentation. It has been found that a micro-organism
which produces cephalosporin C can be subjected to mutation
in such a way that it produces desacetoxycephalosporin C.
me biosynthesis of cephalosporin C in the micro-
organism has not hitherto been clarified. Desace-tyl-
cephalosporin C, but not desacetoxycephalosporin C, was
known as a by-product from the production of cephalosporin
C. It was therefore very surprising that it proved pos-
sible to obtain mutants which produce desacetoxycephalo-
~48430
sporin C. m e discovery of the mutants permits the con-
clusion that desacetoxycephalosporin C is an intermediate
product in the biosynthesis of cephalosporin C by micro-
organisms which form cèphalosporin C and that in the mutant
the reaction steps which convert desacetoxycephalosporin C
into cephalosporin C or desacetylcephalosporin C are blocked.
Since biosynthetic reactions occur with the co-operation of
enzymes, it can be assumed that in the new mutant one or
more enzymes which participate in the biosynthesis of cepha-
losporin C from desacetoxycephalosporin C are no longer pre-
sent or are wholly or partially blocked. mis means that
one or more enzymes cannot fulfil, or cannot completely ful-
fil, their function of catalysing the conversion of desacet-
oxycephalosporin C into cephalosporin C (as ~he end product).
In the first case, no cephalosporin C at all is produced
whilst in the second case a lesser or greater amount of
cephalosporin C or desacetylcephalosporin C can be formed
alongside desacetoxycephalosporin C. The view that t~e
new mutants possess a defect with regard to one or more
enzymes which participate in the conversion of desacetoxy-
cephalosporin C into cephalosporin C in the micro-organism
is supported by the fact that it was possible to detect,
in cephalosporin C-producing micro-organisms or in their
cell-free extracts, an enzyme which in the presence of o~y-
gen and a co-factor (H-donor: NADH or NADPH) converts des-
acetoxycephalosporin C into desacetylcephalosporin C. The
enzyme (desacetoxycephalosporin C-hydroxylase) which very
probably belongs to the group of the oxido-reductases, is
1q)48430
a soluble protein which has a pH optimum of 6.5 and which is
denatured by heating (5 minutes at 100C). This hydroxy-
lase was not detectable in the new mutants or their cell-
free extracts. From this it can be concluded that due to
the lack of hydroxylase in the new mutants the biosynthesis
of the cephalosporin C is broken off at the stage of the
desacetoxycephalosporin C. The new mutants which can be
described as desacetoxycephalosporin C-hydroxylase-defect~ve
mutants or leaky mutants, have not hitherto been found in
nature.
Hence the subject of the invention are mutants of
cephalosporin C-producing micro-organisms in which the con-
version of desacetoxycephalosporin C into cephalosporin C
or desacetylcephalosporin C is entirely or partially pre-
vented. The invention also relates to the use of the said
desacetoxycephalosporin C-hydroxylase-defective mutants for
obtaining desacetoxycephalosporinC by fermentation using the
said mutants. A further subject of the invention is a pro-
cess for obtaining the new mutants in accordance with methods
which are in themselves known, namely by treatment with
mutagenic agents. A further subject of the invention is
the process for the manufacture of desacetoxycephalosporin
C which is characterised in that a mutant of the cephalc-
sporin C-producing micro-organism, in which the conversion
of desacetoxycephalosporln C into cephalosporin C is
entirely or partially prevented, is cultured in a manner
which is in itself known, and desacetoxycephalosporin C is
isolated.
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As cephalosporin-producing micro-organisms which
are to be subjected to mutagenesis, the known fungi of the
genus Emericellopsis or Cephalosporium, are used, such as
Cephalosporium acremonium, for example the Brotzu strain
I.M.I. 49~137 (ATCC 11,550) or mutants thereof, for example
the Clevedon mutant 8,650 (ATCC 14,533), compare U.S. Patents
-~,831,797 and 3,396,083, and also their mutants which produce
more cephalosporin C, for example the methionine-auxotrophic
mutants described in German O~fenlegungsschrift 2,101,345
or the mutants accordlng to German Offenlegungsschrift
2,239,321.
The new mutants are obtained according to the methods
known for obtaining mutants,-by the action of mutation-
causing rays or chemical agents on gro~th forms OL the fun~al
strain, such as above all, conidia and also arthrospores,
vegetative mycelia or, possibly, ascospores.
m us, radioactive rays which cause mutation, such as
a~ or ~-rays, or neutrons, o~ ultraviolet rays, can be
used. Kno~ chemical mutagens are, for example, alkylating,
especially lower-alkylating agents, such as die~hyl-sulphate,
ethyl metha~esulphonate, ethyl ethanesulphonate or analogues
of nucleotide bases such as 5-bromouracil or 2-aminopurine,
or compounds which chemically modify the ~ucleotide bases,
such as hydroxylamine, nitrous acid or l-methyl-3-nilro-
nitrosoguanidine, or compounds which cause an omission or an
additional insertion of one or more nucleo,tide bases, for
example acridines, such as pro~lavin. The mutation-causing
rays or chemical agents are caused to act for example on
conidia of the
-- 6 --
, .
.
1048430
fUngal strain. me time of action is so chosen that the
number of mutants is as great as possible; using such a
period of action, most of the.conidia (approx. 90 - 99%)
are destroyed, compare, for example, German Offenlegungs-
schrift 2,101,345 (C~se 4-G~40,L), Figure 1. Preferably,
the mutagenic treatment is carried out in such a way that
the survival rate is app.rox. 1%.
It has been found experimentally that the ratio of
the number of conidia surviving the mutagenic treatment to
the number of desacetoxycephalosporin C-producing mutants
obtained is constant and reproducible.within statistical
limits, regardless of the method of treatment. On average,
one desacetoxycephalosporin C-producing mutant is found
amongst 5,000 to 10,000 conidia which sur~ive the mutagenic.
treatment.
m e mutagenic treatment can be carried out, for
example, as follows:
The conidia (2.5 x 104/ml) suspended in M/15 phos-
phate buffer of pH 7 are uniformly distributed over the
- surface of a solid agar nutrient medium which contains all
the requisite nutrient materials, for example on a "complex
complete medium 1 H" (10 g of glucose, 4 g of Difco-Bacto~
yeast extract, 4 gof D,L-methionine, ~5 g of Difco agar,
tap water to make up to 1,000 ml, sterilisation for 20 min-
utes at 120C and 1.2 atmospheres gauge 7 pH after sterili-
sation 7.0); 0.1 ml are smeared per agar plate. In this
way, 2.5 x 103 live conidia are brought onto the agar sur-
face. The latter is exposed for 14 seconds to the W
-- 7 --
~/e ~ Rrk
~¢~48430
light of a Philips T W sterilisation lamp of maximum radia-
tion intensity at a wavelength of 2,537 ~. mis gives
lO,OOO erg/sec/cm incident on the surface. me amount of
radiation can be measured by means of a precision W
measuring instrument and can be so chosen that the desired
number of surviving conidia, for example 1% (30 + 5) is
obtained.
The isolation of the desired mutants from the muta-
genically treated conidia is carried out by incubating the
agar plates, for example for 7 days at 25, and testing
the separate colonies, in the above case 25 - 35 colonies
(diameter approx. ~ mm) to see whether they form desacetoxy-
cephalosporin C or whether they contain the hydroxylase
enzyme which is responsible for converting desacetoxycepha-
losporin C into desacetylcephalosporin C.
mese tests are, as usual, not carried out on the
individual colonies themselves but on the daughter genera-
tion which is obtained by culturing the individual colonies
in a liquid nutrient medium, for example in shaking flasks,
for 3 to 7 days at 23C-. It is possib'e either to examine
the culture filtrate to ascertain whether it contains des-
acetoxycephalosporin C, or little or no cephalosporin C,
or to examine the mycelium to ascertain whether the hydroxy-
lase mentioned is present therein.
Since, as already mentioned, only one desacetoxy-
cephalosporin C-producing colony is found on average amongst
5,000 to 10,000 colonies which have resuited from the conidia
which have survived the mutagenic treatment, it is
11~)48430
technologically important to have available a preselection
process by means of which the cephalosporin C-forming colo-
nies can be separated out. A suitable method for this is,
above all, the microbiological plate diffusion test with
micro-organisms which react to cephalosporin C but are not
equally sensitive to desacetoxycephalosporin C, for example
with Alcaligenes faecalis, Sarcina lutea or Neisseria catar-
rhalis. Desacetoxycephalosporin C only shows a very weak
activity against these strains, that is to say a small
inhibition zone, whilst cephalosporin C is strongly active
and accordingly forms a large inhibition zone. Hence,
those which produce a large inhibition zone against the
said micro-organisms can be left out of the further test
for culture filtrates containing desacetoxycephalosporin C.
On average, 95% of the cultures can be exc]uded in this way
from further testing. The fermentation liquors which are
less active against the said micro-organisms can be tested
for the presence of desacetoxycephalosporin C, for example
biologically, biochemically and by thin layer chromato-
graphy.
m e biological test can be carried out, for example-,
by preparing a bioautogram with the said micro-organisms,
for example Alcaligenes faecalis, for the case of several,
for example 5 - 10, culture filtrates and a standard amount
of authentic desacetoxycephalosporin C. For this purepose,
the culture filtrates and the standard desacetoxycephalo-
sporin C are chromatographed on paper (for example Whatman
No. 1) in one of the solvent systems mentioned in Table 1
_ 9 _
~048430
(migration time approx. 8 - 10 hours) and the paper is then
placed on an agar plate of the particular micro-organism.
After 30 minutes at 4C, the paper is again removed and the
agar plate is incubated (approx. 12 - 20 hours at 37C).
Desacetoxycephalosporin C shows an inhibition zone in the
position of the Rf value (see Table 1). It is therefore
possible to read off the result as to which culture fil-
trates contain desacetoxycephalosporin C, and draw conclu-
sions regarding the amount of desacetoxycephalosporin C from
the size of the inhibition zone. In this way it is also
possible to detect whether the mutant tested produces
exclusively desacetoxycephalosporin C or whether it produces,
in addition, also cephalosporin C, desacetylcephalosporin C
or some other microbiologically active substance.
The biochemical test for the presence of desacetoxy-
cephalosporin C-hydroxylase can be carried out, for example,
by testing for the presence or absence of the said hydroxy-
lase in the mycelium of the mutated strain or in cell-free
extracts obtained therefrom. The mycelium can be obtained,
for example, by incubating the strain which is to be inves-
tigated (from a colony of the abovementioned agar plates) in
a synthetic nutrient solution ("synthetic base medium C3",
see below) to which 4 g of D,L-methionine has been added,
for example for 96 hours at 23C, and separating off the
mycelium, for example by centrifuging. (In that case a
synthetic nutrient medium is used preferably, to avoid the
presence of other unknown or similar enzymes from ihe con-
stituents of the nutrient solution, for example from
-- 10 --
lQ48430
groundnut flour). To produce the cell-free extract, the
mycelium is washed twice with M/15 phosphate buffer of pH
7.0 and the cells are then broken up in a X-press (of
Messrs. AB Biox, NACKA 2, Sweden) analogously to the method
described, for example, for D-aminoacid oxidase by Benz et
al., Eur. J. Biochem. 20 (1971) 82. The cell-free crude
extract thus obtained, in M/~5 phosphate buffer of pH 7, is
tested for its capacity to convert desacetoxycephalosporin
C into desacetylcephalosporin C in the presence of oxygen,
for example by allowing it to react with desacetoxycephalo-
sporin C for 4 hours at 23C whilst shaking. If the
hydroxylase is present, desacetylcephalosporin C is formed.
This is converted into cephalosporin C by means of a further
enzyme (desacetylcephalosporin C-0-acetyl-transferase) and
acetyl-l-C14-coenzyme A. If no hydroxylase is present, no
desacetylcephalosporin C, and hence also no (radioactive)
cephalosporin C is formed. me desacetoxycephalosporin C
accumulates during the culture of the fungal strain ~ld can
then be detected in the culture filtrate.
Synthetic base medium C 3
(NH4)2S04 2.5 g
KN03 5.0 g
MgS04-7H20 0.2 g
KH2P04 ; 0.2 g
CaC03 5 g
Trace element solution 1), see above 10 ml
Maltose 40.0 g
Methyl oleate 7.0 e
-- 11 --
1048430
~eso-inositol 2.0 g
Distilled H20 to 1,000 ml
pH before sterilisation 7.3
pH after sterilisation 7.0 + 0.2
Sterilisation: In an autoclave, 20 minutes, 120C, 1.2
atmospheres gauge.
The test, by thin layer chromatography, for the
presence of desacetoxycephalosporin C can be carried out in
the pre-purified fermentation solution of the strain to be
investigated (from a colony of the abovementioned agar
plates). m e fermentation of the strain and the pre-
purification of the fermentation liquor are preferably
carried out in the manner l~nown for the isolation of cepha-
losporin C. Thus, for example, the strain is fermented
for 144 hours in a complex complete medium and the fermen-
tation liquor is pre-purified as described in Belgian Patent
750,292 (Case 4-6770), for example by acidification, filtra-
tion, extraction, adsorption on non-ionic resins of large
surface area, such as Amberlite ~ XAD-2, if necessary
absorption on weakly basic ion exchangers such as Amberlite
IR-68, elution and lyophilisation. The lyophilisate thus
obtained is examined by thin layer chromatography. Table 1
gives the Rf values on cellulose in 9 differe~t solvent sys-
tems. In addition, the table contains the corresponding
Rf values for cephalosporin C, desacetylcephalosporin C
and desacetylcephalosporin C-lactone. The solvents are:
A: Isopropanol-formic acid-water (77:4:19)
B: n-Butanol-acetone-diethylamine-water (37:37:8:18)
~48430
C: n-Butanol-acetic acid-pyridine-water (37.5:7.5:25:30)
D: n-Butanol-ethanol-water-glacial acetic acid (50:15:20:15)
E: 66% strength aqueous acetonitrile
F: 80% strength aqueous phenol
G: n-Butanol-formic acid-water (4:1:~)
H: n-Butanol-glacial acetic acid-water (upper phase) [11:3:7]
I: 70% strength aqueous n-propanol.
1(3148430
. ~ ~ ,
H I
O O O
~ ~D ~ ~
~ O O O O.
. ~ U~ ~
. .
O O O
~3
C~i
L~
~q ~, . .
O O O O
. ~ ~ ~ ~ 0
a) ~3
O O O O
~Q~ ~ ~ C~l
C~l
,~ ~O O O O
O C-
V . .
tQO O O O
"1 ~ mO O O o
~3 ~D O U~
E~ , 'C ~ ~ C~
o o o o .
. ~d
. V
V C~
.
O h h
~, O O
' O ~ ~
O O
1~ l ~I V
,1
O o~ h
O
O O O
o ~ a) a) a
V~ ~ .~ ~)
-- 14 --
.. . . .. ... .. . . .. . ..... ... .. . .. .. .. . .. . ... .. . . . ...
~048430
Desacetoxycephalosporin C obtained by fermentation
has the same Rf values as the synthetically obtained compound.
As Table 1 shows, desacetoxycephalosporin C can, if desired,
be separated by thin layer chromatography from the remaining
compounds.
me aminoacid analyser [AA] (Technicon TSM 1 of
Messrs. Technicon Corp., Tarryton, N.Y., USA) is also suit-
able for carrying out the separation. Table 2 shows the
retention times of the abovemèntioned compounds (20 ~1
samples of a 5-millimolar solution of the compound in 0.2 M
sodium citrate buffer of pH 2). Furthermore, desacetoxy-
cephalosporin C can be detected and can be separated from
the remaining compounds mentioned by high pressure liquid
chromatography [HPLC] (see J. Konecny et al., J. Antibiotics
26, 135 [1973]). m e extreme right-hand column in Table 2
gives the corresponding retention times in minutes.
Table 2
.
Compound Retention time in
minutes
- - AA HPLC
.
Desacetoxycephalosporin C 59 11.0
Desacetylcephalosporin C 93 10.0
Desacetylcephalosporin C-lactone 93 5.5
Cephalosporin C 54 12.4
me fermentation of the mutants according to the
invention and the isolation of desacetoxycephalosporin C
from the fermentation liquor are carried out in accordance
1(~48430
with the methods known for the isolation of antibiotics. Since desacetoxy-
cephalosporin C shows a very similar physico-chemical behaviour to cephalo-
sporin C, all known processes for isolating cephalosporin C from fermenta-
tion ~quids are applicable.
The isolation of desacetoxycephalosporin C from the fermentation
liquor can therefore be carried out, for example, in accordance with the
processes of British Patents 810,196; 938,758; 968,324; 1,036,125; 1,109,362
and 1,205,226, French Patents 1,429,873 or 2,011,520, Belgian Patent 750,292
or German Offenlegungsschriften 2,101,345, 1,933,187, 2,031,754 or 1,939,341.
The fungus is cultured aerobically, preferably in submerged cul-
ture, whilst shaking or stirring with air or oxygen in shaking flasks or in
the known fermenters. The fermentation solution contains a source of car-
bon and a source of nitrogen and, optionally, growth-promoting substances
as well as inorganic salts. Possible sources of carbon are, for example,
assimilable carbohydrates, such as glucose, sucrose, lactose, maltose and
starch and also manitol, inositol and glycerol. As nitrogen-containing
nutrients and optionally growth-promoting substances there may be mentioned:
aminoacids, especially methionine, peptides and proteins and their degrada-
tion products, for example groundnut flour, soya bean flour, peptone or tryp-
tone, and also meat extracts, water-soluble constituents of cereal grains
or distillation residues from the manufacture
- 16 -
1048430
of alcohol, for example dry corn steep, ammonium salts and
nitrates. Amongst other inorganic salts, the nutrient solu-
tion can contain, for example, chlorides~ carbonates and
sulphates of alkali metals or alkaline earth metals, iron,
copper, zinc and manganese.
Culturing takes place at a temperature between 18
and 37C, preferably at approx, 23C, for 4-7 days.
Various methods can be used to isolate the desace-
toxycephalosporin C. Preferably, desacetoxycephalosporin
C is isolated from the fermentation liquor in accordance
with the methods known for the isolation of cephalosporin C.
The isolation can either be carried out in accord-
ance with the whole broth process direct from the fermenta-
tion liquor or, preferably, from the culture filtrate after
separating off the mycelium, for example by filtration, if
appropriate in the presence of filtration auxiliaries, such
as diatomaceous earth. A treatment with acids, for example
mineral acids such as sulphuric acid or organic acids such
as oxalic acid, to precipitate sparingly soluble salts, for
example calcium salts, from the nutrient solution and to
destroy penicillin N which may be present, can be carried
out before or after filtration. me culture filtrate can
further be pre-purified by removing lipophilic impurities
from it. Suitable methods for this are, for example,
extraction with a water-immiscible solvent or solvent mixture
and/or adsorption on ion exchangers.
Examples of possible water-immiscible solvents are
aliphatic, cycloaliphatic, araliphatic and aromatic
1(} 48430
hydrocarbons with at most 12 carbon atoms, which are option-
ally substituted by halogen atoms, such as bromine, fluorine
and especially chlorine, for example hexane, heptane, cyclo-
hexane, benzine, petroleum ether (boiling point 110-140C),
kerosene, (boiling point 210-240C), carbon tetrachloride,
chloroform, methylene chloride, methylchloroform, ethylene
chloride, perchloroethylene, perfluoroethylene, isopropyl
bromide, benzene, toluene and xylenes, and also esters,
especially lower alkyl esters of lower fatty acids such as
ethyl acetate, butyl acetate and amyl acetate, ketones such
as methyl isobutyl ketone and methyl isoamyl ketone, ethers
such as diisopropyl ether, and water-immiscible or sparingly
water-miscible alcohols such as butanol, 2-ethylbutanol,
ethylhexanol, cyclopentanol and cyclohexanol.
The extraction with a water-immiscible solvent is
preferably carried out in the presence of a liquid ion
exchanger, for example "Amberlite" LA-2 (of Meesrs. Rohm
and Haas). Suitably, an acid pH range is used, for example
pH 1-6.
Instead of pre-purifying the culture filtrate by
treatment with acid and/or solvent extraction, it can also
be subjected to a successive treatment with a strong cation
exchange resin, for example "Amberlite" IR-120 or "Dowex"
50-8 (of Messrs. Dow Chemical Co.,) and a strong anion
exchange resin, for example "Amberlite" IRA-400 or "Dowex"
1, as described in British Patent 968,324.
Various methods are suitable for extracting desacetoxy-
cephalosporin C from the culture filtrate which has option~ly
- 18 -
:~048430
been pre-purifed as mentioned; these methods can be employed
once or repeatedly, individually or in any desired combina-
tion. In particular, absorption or adsorption, solvent
distribution and precipitation should be mentioned.
Examples of absorbents or adsorbents which can be
used are active charcoal, aluminium oxide, cellulose, ion
exchange resins and non-ionic adsorption resins whilst
aqueous solutions, above all, can be used for the elution.
The use of active charcoal and aluminium oxide is described,
for example, in British Patent 810,196. Examples of ion
exchangers which should be mentioned are weakly basic anion
A exchangers such as "Amberlite'' IR-4B or "Deacidite" E, from
which the desired product can be eluted with, for example,
aqueous pyridine acetate at a pH of approx. 6. It is also
possible to use strongly bas c anion exchange resins, such as
"Amberlite" IRA-400, "Dowex" 1, "Dowex" 2 or "Deacidite" FF,
from which the products can be eluted with approx. 1 N acetic
acid, compare British Patents 810,196 and 968,324. Non-
ionic adsorption resins which should be singled out are
- styrene polymers, crosslinked with divinylbenzene, of large
surface area, such as "Amberlite" XAD-2, from which the
desacetoxycephalosporin C can be eluted with, for example,
aqueous alcohols, for example lower alkanols such as methanol,
ethanol and especially isopropano~ compare Belgian Patent
750,292.
The solvent distribution method can be employed, ~or
example, in the form of counter-current distribution. Suit-
able solvent systems are mixtures of water and phenol or
-- 19 --
1~48430 -
lower alkyl-substituted phenols, compare British Patent
810,196.
Suitable materials for precipitating desacetoxy-
cephalosporin C from aqueous solutions are, for example,
water-miscible organic solvents such as ketones, for example
acetone, or lower alkanols, for example isopropanol. m e
compound can also be precipitated from concentrated aqueous
solutions in the form of their metal salts, such as alkali
metal salts, for example sodium salts, or alkaline earth metal
salts, for example calcium salts or barium salts. Preci-
pitation as sparingly soluble heavy metal complexes, for
example as complex with copper, mercury, cadmium, lead,
manganese, iron, cobalt or nickel or especially with zinc,
should be singled out particularly, compare French Paten~
2,011,520. ^
Chromatography on cel]ulose is particularly suitable
for-separating desacetoxycephalosporin C and cephalosporin
C which may also have been formed by fermentation. If the
content of cephalosporin C is high it can be necessary to
carry out the chromatography in more than one step. It can
also be advan-tageous to convert the cephalosporin C, before
one of the chromatography stages, into desacetylcephalo-
sporin C, since the latter can more readily be separated
from desacetoxycephalosporin C. The conversion of cepha-
losporin C into desacetylcephalosporin C is carried out in
a known manner by desacetylation, for example by means of
desacetylases such as citrus desacetylase or desacetylase
from micro-organisms, such as are described in British
- 20 -
.
` ~)48430
Patent 1,080,904 for the desacetylation of 7-amino-cephalo-
sporanic acid, especially using the desacetylase from Bac.
subtilis ATCC 6,633. m e desacetylation can be carried
out with the cell lyophilisate of the micro-organism or
with enzyme obtained from the micro-organism.
The elution is carried out, for example,-with
aqueous-alcoholic or aqueous-phenolic solutions such as
aqueous lower alkanols, for example propanol or butanol or
aqueous phenol.
-~ Desacetoxycephalosporin C of the formula I has the
following properties: In the UV spectrum in water, ~max =
260 nm. In ~he NMR spectrum (in D20) a sharp signal is
present at 1.92 ~, which can be attributed to 3 proteins
of the CH3 group in the 3-position. In the IR spectrum
(in potassi~m bromide), the bands at 8.15 ~ and 9.7 ~ which
are typical of cephalosporin are absent and instead a band
is present at 3.36 ~. The optical rotation of desacetoxy-
cephalosporin C is [a]D = + 125 + 1 (c = 1, in water).
The hydrolysis of desacetoxycephalosporin C with 6 N HCl ln
a bomb tube (24 hours, 110C) gives a-aminoadipic acid in
addition to other decomposition products. Further physico-
chemical characteristics are given above in Tables 1 and 2.
In all the characteristics mentioned, the fermen-
tatively prepared desacetoxycephalosporin C completely
matches chemically prepared desacetoxycephalosporin C.
The invention is described in the examples which
follow.
- 21 -
1~48430
Example 1
The contents of an ampoule containing lyophilised
mycelium spore suspension of desacetoxycephalosporin C-
hydroxylase-defective mutant 127a/2, which have been
obtained by irradiation of Cephalosporium acremonium ATCC
14,533 with W light, as indicated in the general part of
the description, are dissolved in 5 ml of M/15 phosphate
buffer of pH 7. A 500 ml Erlenmeyer flask with 4 flow
breakers, containing 100 ml of the nutrient solution a),
is inoculated with the 5 ml of the Cephalosporium suspension
and incubated for 72 hours on a rotary shaking machine at
250 revolutions per minute (rpm), amplitude 50 mm, at 25C.
The main culture solution b), which is also contained in a
500 ml Erlenmeyer flasks, with l flow breaker and 100 ml of
nutrient solution, is inoculated with 5 ml, per flask, of
the pre-culture thus obtained. The flasks are incubated
for 168 hours on a rotary shaking machine at 250 rpm and
25C. From the 4th day onwards, samples are taken daily
for biological (plate diffusion test) and chemical (thin
layer chromatogram) determination of the activity. After
168 hours, the maximum amount of desacetoxycephalosporin C
has formed. The culture solution then has a pH of 7.5
and a concentration of approx. 750 mg/l of desacetoxy-
cephalosporin C.
Nutrient solution a)
Corn steep powder 12.0 g/l
Ammonium acetate 4.5 g/l
Sucrose 20.0 g/l
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pH ~efore sterilisation 7.5
pH after sterilisation 7.0
Nutrient solution b)`
Groundnut flour 30.0 g/l
Beet molasses 20.0 g/l
Maize flour 20.0 g/l
Methyl oleate . 6.7 g/l
DL-methionine 10.0 g/l
Borax ~ 0.5 g/l
CaC03 5.0 g/l
Adjust pH to 7.0 with KOH before sterilisation.
3 litres of culture soiution containing 0.75 g of
desacetoxycephalosporin C per litre are acidified to p~ 3.0
with 50~0 strength aqueous sulphuric acid (volume/volume),
and centrifuged. The cul-ture filtrate, containing 6&0 mg
of desacetoxycephalosporin C per litre, is extracted ~ith
1.5 litres of a solution of 10% of Amberllte LA-2 (acetate
form) in 2-ethylhexanol in 3 portions of 0.5 litre, in the
course of which the desace-toxycephalosporin C remains in
the aqueous phase. The extracted culture filtrate is
acidified to pH 2 . 6 with 50/0 strength sulphuric acid and is
percolated through 1~ litres of Amberlite XAD-2. me resin
is washed ~ith 750 ml of deionised water and is then eluted
with 3 litres of 12% strength aqueous isopropanol. The
eluate is collec-ted in 4 fractions of- 600 ml. me frac-
tion with the highest concentration of desacetoxycephalo-
sporin C is lyophilised. m e crude lyophilisate contains
24% of desacetoxycephalosporin C. It can be converted into
the zinc complex and then into the free desacetoxycep'nalo-
sporin C as described in example 2.
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Example 2
27 litres of culture liquor obtained as described in
Example 1 are adjusted to pH 2.8 with 50~0 strength sulphuric
acid and filtered, after addition of 810 g of filtration aid,
on a filter press. me acid filtrate is adsorbed on 18
litres of "Amberlite" XAD-2 (polystyrene-divinylbenzene
resin). me speed of percolation is 18 l/hour. me
charged XAD-2 column is eluted with 20% strength, volume/
volume, aqueous isopropanol. me rate of elution is
36 l/hour. me eluate is collected in fractions of 2
litres. More than 70~ of the adsorbed activity are con-
tained in fractions 7 - 18, that is to say in 24 litres of
eluate. mese fractions are adsorbed on 500 ml of Amber-
lite IRA-68 (acetate form). m e rate of percolation is
5 l/hour. me column is then eluted with pyridine acetate
buffer of pH 5.5 which is 0.44 molar in pyridine and 0.2 molar
in acetic acid. me rate of elution is 1 l/hour. 200 ml
portions of eluate are collected. m e fractions 2-13,
that is to say 2.4 litres, contain the entire activity.
mey are concentrated to 100 ml in vacuo at 30 - 35C. m e
concentrate is introduced into 1,200 ml of isopropanol whilst
stirring. me precipitate thereby produced is filtered
off, washed with isopropanol, suction-dried and dissolved,
as such, in 200 ml of water. 20 g of zinc acetate are
added to the clear solution and 250 ml of isopropanol are
then added to the homogeneous solution over the course of
15 minutes. Hereupon desacetoxycephalosporin C precipl-
tates as the zinc complex. After cooling for 4 hours to
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1~48430
0 C, the mixture is filtered. The filter residue is washed
with water and isopropanol and dried in vacuo at 50C. The
product is obtained in the form oP light beige crystals -
which according to HPLC and thin layer chromatogram are
identical with desacetoxycephalosporin C (since in both
these analytical test methods the zinc complex behaves in
the same way as the free desacetoxycephalosporin C).
- From the zinc complex the free desacetoxycephalo-
sporin C can be prepared in the same way as cephalosporin
C from its zinc complex, cf. U.S.patent 3,661,901. For
example strong acid cation exchanger in the H-form e.g.
- Dowex WX-12R (sulfonated polystyrene cross-linked with divinyl
benzene) is added to an aqueous suspension of 1~% strength
of the zinc complex, until a pH of 2,5 is obtained. Then
the solution is filtered and evaporated. If desired, the
sodium salt can be prepared by adding sodium hydroxide
up to pH 6,5, concentrating and crystallizing the sodium
salt.
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