Note: Descriptions are shown in the official language in which they were submitted.
il9B3~Q~
This invention relates to the production of an anti-
bacterial and anticoccidial agent, designated antibiotic
LL-C23024~ and iota, by fermentation of the microorganism
Ac-tinomadura yumaense sp. nov. or mutants thereof, to methods
for its recovery and centration from crude solutions and to
processes for its purfication. The invention also relates to
a biologically pure culture of Actinomadura yumaense sp. nov.
Antibiotic LL-C23024~ has the following postulated
s-tructure:
OCH3
H3CO"~ ~ 3
3 ~ ~2 ~ H
~ o o ~o~ ~\ o o
COOH 3
-- 1 --
3~6
wherein Rl and R2 are different and are each selected from
the group consisting of H and CH3~
The above postulated structure is based on the
elemental analysis, optical rotation, field desorption
mass spectroscopy moleculate weight, melting point, infra-
red (IR) spectrum, 13C nuclear magnetic resonance (13C~NMR)
spectrum, and proton magnetic resonance (lH-NMR~spectrum,
as detailed in the Examples and as shown in the accompanying
figures:
Fig. I: IR Spectrum of LL-C2302~ in KBR
Fig. II: 13C-NMR Spectrum of LL-C23024~. 20
MHz in CDC13, internal TMS reference
equivalent.
Fig. III: lH-NMR Spectrum of LL-C23024~. 80 MHz
in CDCl3, internal TMS reference equi-
valent.
TABLE I
3C-NMR SPECTRUM QF LL C23024~ (,c;pm relative to TMS)
I
,hemical Shift, ~ppm) Number of Carbons
10.5
11.0
12.2
17.0 1 D
17.7
17.g
22.3
26.1
26.8
27.~ 1
30.2
32.0
33.3
33.7 2
33.8 2
36.5
36.8
39.0
39.9
45.5 2
57.1
.J'`..l
60.7
66.9
67.6
70.2
71.3
'/~.9
74.~ 1
75.1
79.9
80.8 2
82.1
~; ~
TABLE I (eontinued~
Chemical Shift, (ppm) Number of Carbons
84.5
8~.7
85.6
86.9
95.8
96.9
97.7
107.5
179.2
Total 46
LL-C23024 iota is a polyether antibiotie with very
potent anticoecidial activity. It is at least ten times more
potent than most other known polyethers. Field desorption mass
spectral data indicates it has a molecular weight of 930, whieh
together with its earbon-13 nuclear magnetic resonanee (13C-NM~)
spectrum allows the proposal of a tentative moleeular formula of
C48H82O17. LL-C23024 iota appears to have four methoxy groups,
-the same sugar observed in polyether antibiotic X~14868A [United
States Patent 4,278,663], and one carboxyl group. The only other
reported polye-ther antibiotic with a moleeular weight of 930 is
an-tibiotie A28695B (hydroxyseptamycin) [J. Antibioties 33(2): 252
(1980)] whieh differs signifieantly in structure and biologieal
proper-ties. The above observations are based on the elemental
analysis, optieal rotation, ealeulated moleeular weight by field
desorption mass spectroseopy, infrared (IR) speetrum, C-NMR
spectrum, and proton nuclear magnetic resonanee ( H-NM~) speetrum,
- 4a -
as detailed in the Examples and as shown in the accompanying
figure 5:
~. ~ r
3~6
Fig. IV: IR spectrum of LL-C23024 iota in KBR.
Fig. V: lH-NMR spectrum of LL-C23024 iota.
80 MHz in CDC13, internal TMS re-
ference equivalent.
5 Fig. VI: 13C-NMR spectrum of LL-C23024 iota.
20 MHz in CDCl~, internal TMS re-
ference equiva~ent.
Fig. VII A 13C-NMR spectrum of LL~C23024 iota.
(expanded scale: 0-50 ppm).
20 MHz iN CDC13, internal TMS
Fig. VIII B: 13C-NMR spectrum of LL-C23024 iota
(expanded scale: 50 110 ppm).
20 MHz in CDC13, internal TMS re-
feren_e equivalent.
The 13C-NMR spectra of LL-C23024 iota were ob-
tained in deuterated chloroform (CDC13) at a field strength
of 20 MHz. The peaks with their respPctive chemical shifts
in parts per million relative to tetramethyl silane (TMS)
and the estimated number of carbons per peak are listed in
Table II. Peaks were ohserved for chloroform at 75.4,
77.0, and 78.6.
.~
3~
-- 6 --
TABLE I~
Chemical Shi~t No. of Carbons ~hemical Shift No. of Carbons
10.~ 1 59.9
S 10.9 1 60.8 2
11.7 1 68.5
~ 1 6~.8
17.6 1 71.3
1~ . o 1 72.2
21.7 1 76.1
22.g 1 76.9
24.1 1 ~8.5
28.2 1 81.0
31.2 2 81.3
32.2 1 81.8
33.4 1 84.8
33.8 2 85.3
34.2 1 85.6
36.7 1 86.8
37.2 1 88.5
39.4 1 95.9
- 40.3 1 97.9
44.5 1 99.6
45.5 1 107.2
47.6 1 173.0 _1
56.8 1 ~'otal 48
Antibiotic LL-C23024~, ~, and iota are organic
carboxylic acids and thus are capable of forming salts with
non-toxic pharmaceutically acceptable cations. Thus, salts
formed by admixture of ~he antibiotic free acid with stoi-
chiometric amounts of cations, suitably in a neutral sol-
vent, may be formed with cations such as sodium, potassium,
calcium, magnesium and ammonium, as well as organic amine
cations such as tri(lower alkyl)amine te.g., triethylamine,
triethanolamine), procaine and the like. The cationic salts
of antibiotic LL-C23024~ are, in general, crystalline solids,
..
<~
relatively insoluble in water and soluble in most common organic
solvents such as methanol, ethyl acetate, acetone, chloroform,
heptane, ether and benzene.
The antibiotic LL-C23024~ and iota are active in vitro
against gram-positive bacteria when tested by the standard agar
clilution procedure. The results are reported as minimal
inhlbitory concentrations (MIC) in mcg./ml. in Tables III and IV.
~ntibiotic LI.-C23024~ and iota are not active against gram-
neyative organisms at concentrations of 256 mcg./ml. or less.
TABLE III
IN VITRO ANTIBACTERIAL ACTIVITY OF LL-C23024~
MINIMAL INHIBITORY
ORGANISM CONCENTRATION (mcg./ml.)
Staphylococcus aureus Smith 64
SSC 80-11 64
" " SSC 80-32 64
" " SSC 80-38 64
" " LL-14 64
" " LL-45 64
" " LL-27 128
" " ATCC 25923 64
Streptococcus pyogenes C203
" ~-hemolytic
Keller T623 8
" pneumoniae 78-1 8
Enterococcus SSC-80-62 64
" SSC-80-63 128
331~
TABLE IY
In Vitro An~ibacterial Activity of LL-C23024 iota
Minimal Inhibitory
Concentration (mcg/ml)
LL-C23024 iota
Organism
Enteroeoccus OSU 75-1
Enteroeoccus SM 77-15
Staphylococcus aureus SSC 79-18
Staphylococcus aureus ~U 79-19-2 2
Micrococcus lutea PCI 1001
Staphylococcus aureus Smith
Staphylococcus aureus ATCC 25923
As demonstrated by the above data, antibiotic
LL-C23024~ ~, and iota inhibit the growth of certain
gram-posi-Eive-bacteria and are t-hus useful as a topical
or surface antiseptic in wash solutions for skin, equip-
ment, walls, floors, etc.
It has also been found that antibacterial agent
LL-C23024~ and iota are active in vivo as an anticoc-
cidial agent for poultry as demonstrated by the followin~
tests.
Two days before inoculation, medicated feed with
~0 several levels of drug was presented to the various groups
of one-day-old test chicks. The poultry feed used through-
out the test is prepared as follows:
..
, --
-
out the test is p,epared-as follows:
Vitamin-amino acid premix 0.5
Trace minerals O.l~
Sodium chloride 0.3
Dicalcium phosphate l.2
Cround limestone 0.5
Stabilized fat 4.0
Dehydrated a~fa~fa, 17g protein 2.0
Corn gluten meal, 41% protein 5.0
Menhaden fish meal, 60~ protein 5.0~
Soybean oil meal, 44% protein 30.0%
Ground yellow corn, fine to 100%
The vitamin-amino acid premix in the above feed
composition is prepared from the following formulation.
The expre~sions of quantity relate to units per kilogram
of the finished feed composition.
Butylated hydroxy toluene 125.0 mg.
dl-Methionine 500.0 mg.
Vitamin A 3300.0 I.U.
Vitamin D3 llO0.0 I.C.U.
.. . . . . . _ . .
Riboflavin 4.4 mg.
Vitamin E 2.2 I.U.
25 Niacin 27.5 mg.
Panthothenic acid 8.8 mg.
Choline chloride 500.0 mg.
Folic acid l.43 mg.
Menadione sodium bisulfats l.l mg.
Vitamin Bl2 ll.0 mcg.
Ground yellow corn, fine to 5.0 gm.
The test chicks were then inoculated by direct
inoculation into the crop of each chick with a mixed inocu-
lum of 5000 sporulated oocysts of Eimeria acervulina and a
sufficient number of oocysts of Eimeria tenella to produce
85-100% mortality in untreated controls. The chicks were
:
- 10 -
given free access to feed and water during the entire test
period. Seven days after inoculation the tests were ter-
minated and the birds were weighed, necropsied and their
intestinal tracts examined for lesions. The results appear
in Tables V and VI below and show that improved survival of
infected chicks is obtained when 10 ppm or less of antibio-
tic LL-C23024 or iota is administered in the diet. The
results also show a significant suppression of intestinal
lesions due to Eimeria tenella and Eimeria acervulina.
TABLE V
EVALUATION OF ANTIBIOl`IC LL-C23024~ AS AN ANTICO~CI~IAL AGENT IN CHICKS
Percent Birds with
Keduced Lesions
Concentration No. Birds Percent E. E.
Compoundin Diet, pp.~ Started Survival tenella acervulina
LL-C23024 ~ 15 3 100 100 100
LL-C23024 ~ 10 5 lOO 6Q 100
LL-C23024 B 5 5 80 -O 100
NNC~ 0 15 46.6 O O
b~
LL-C23024 ~ 120 15 100 100 100
100 100 100
100 100 100
0 15 20 0 0
NNC 15 100
LL-C23024 ~ 20 15 100 87 100
100 60 100
- 10 ~15 60 O O
47 7 0
0 15 2~ ~ 0
NNC 15 100
;NNC = Nontreated, noninfected control.
TABLE VI
EVALUATION OF ANTIBIOTIC LL-C23024 IOTA AS AN ANTICOCCIDIAL AGENT IN CHICKS
No. Birds Percent Percent Birds with Reduced Lesions
Concentration in diet (ppm) Started Survival Eimeria tenella Eimeria acervulina
100 100 100
100 100 lCO
~ ~
o 15 20 ' æ
100 100 100
7.5 10 100 70 100
33~6
13 -
The present invention provides a biologically pure
culture of Actinomadura yumaense sp. nov., said culture being
capable of producing the antibiotic LL-C23024~ and iota in
recoverable quantlties upon fermentation in a liquid nutrient
medium containing assimilable sources of carbon, nitrogen and
inorganic salts.
In another aspect r the invention provides a process for
the preparation of antibiotic LL-C23024~ and iota and antibiotic
X-14868A which comprises aerobically fermenting Actinomadura
yumaense sp. nov. or a mutant thereof in a liquid medium contain-
ing assimilable sources of carbon, nitrogen, and inorganic salts,
until substantial antibiotic activity is imparted to the
fermentation broth and then recovering the an-tibiotic therefrom.
A representative strain of Antinomadura yumaense sp. nov.
was isolated from a soil sample collected in Yuma County, Arizona
and is maintained in the culture collection of the Lederle
Laboratories Division, American Cyanamid Company, Pearl River,
New York as culture number LL-C23024. A viable culture of this
representative strain has been deposited with the Cu'ture
Collection Laboratory, Northern Regional Center, United States
Depar-tment of Agriculture, Peoria, Illinois 61604, and has been
added to its permanent collection under the accession number
NRRL 12515.
Taxonomic Charac-terization of NRRL 12515
The strain NRRL 12515 has been taxonomically character-
ized ancl identified as the type strain of a new species of the
cJenus Actinomadura to be known as Actinomadura yumaense sp~ nov.
Observations were made of the cultural, physiological
33~
- 13a -
and morphological features of representative strain NRRL 12515
using methods detailed by E. B. Shirling and D. Gottlieb,
"Methods for characterization of Streptomyces species", Internat.
J. Syst. Bacteriol. 16:313-340 (1966), and R. E. Gordon et al.,
"Nocardia coeliaca, Nocardia autotrophica, and the nocardin
strain," Internat. J. Syst. Bacteriol. 24 54-63 (1974). Media
used in this study were selected from those recommended by T. G.
Pric~am _ al., "A selection of media for maintenance and
ta~onomic study of Streptomycetes," Antibiotics Ann., pp. 947-953
(1956/1957); G. F. Gauze et al., "Problems in the classification
of antagonistic actinomycetes," State Publishing House for Medical
Literature, Medgiz, Moscow (1957); and R. ~. Gordon _t al.~ supra,
for the taxonomic study of actinomycetes and soil bacteria.
Chemical composition of the cell walls of the microorganism was
determined using the method of H. A. Lechevalier et al., "Chemical
composition as a criterion in the classification of
actinomycetes," Adv. Appl. Micro-
- 14 -
biol. 14:47~72 ~1971)~ Phospholipid patterns were determined
using the method o~ M P. Lechevalier et al., "Chemotaxonomy
of aerobic actinomycetes: phopholipid composition," Bio-
chem. Syst. Ecol. 5:249-260 (1977). Details are recorded
in Tables IV-IX, and a general descripti~n of the culture
is given below. Underscored deseriptive colors are taken
~rom K. L. Kelly and D. B. Judd, "Color~ Universal Language
and Dictionary of Names," U.S. Nat. Bur. Stand., Spec.
Publ. 440, Washington, D.C. (1976) and the accompanyin~
Inter-Society Color Council, Natl. Bur. Stand. Centroid
Color Charts.
The data observed for this novel species as re-
presented by strain NRRL 12515 were compared with the data
published for the known species of the genus Actinomadura
[M. Goodfellow et al., "Numerical Taxonomy of Actinomadura
and related actinomycetes," J. &en. Microbiol. 122:95-111
(1979); L. ~. Huang, "Actinomadura macra sp. nov., the
producer of antibiobic CP-47,433 and CP-47,434," Internat.
J. Syst. Bacteriol. 30:565-568 (1980); J. Meyer, "New
species of the genus Actinomadura," Z. Allgem. Mikrobiol.
19:37-44 (1979); H. Nomura and Y. Ohara, "Distribution of
_ a ~inomycetes in soil.-XI. Some new species of the genus
Actinomadura, Lechevalier, et al.," J. Ferment. Technol.
49:904-912 (1971); and T. P. Preobrazhenskaya et al., "Key
for identification o~ the species of the genus Actinomadura,"
The Biology o~ Actinomycetes and Related Organisms 12:30-38
(1977)~. Culture NRRL 12515 bears a slight resemblance to
Actinomadura pelletieri, but resembles no other described
species and d;f~ers from A. pelletieri in a number o~
characteristics. Therefore, strain NRRL 12515 has been
designated the type strain of a new species to be known as
Actinomadura yumaense, sp. nov., named for the site of
collection of the soil sample from which the type strain
was isolated.
Micromorphology
Spores are formed in short spiral chains (maxi-
mum length appr3ximately ~0 spores per chain) on branched,
almost verticillate aerial sporophores. The spores are
ovoid, 0.6 to 0.8 micron by l.O to 1.4 micron, with a smooth
surface.
Cell Wall ~omposition
Whole cell hy~rolysates o~ this culture contain
madurose (3-0-methyl-D-galactose) and the meso isomer of
diaminopimelic acid (DAP). The culture also has a Type P-l
phospholipid pattern and no other diagnostic phospholipid
other than some phosphatidyl glycerol. ~hese characteris-
tics are all very typical of members o~ the genus Actino-
madura.
Amount of Growtb
Good growth is observed on Bennett's agar, Gauze
No. 2 agar, NZ-amine-starch-glucose agar (ATCC Medium 172),
tomato:paste-oatmeal agar, and yeast extrac~-malt extract
agar; moderate growth is observed on Benedict's agar~ Cza-
pek's sucrose agar, glycerol-asparagine agar,;Hickey-Tresner
agar, and oatmeal agar; poor growth is observed on calcium
malate agar, Gauze No. l agar, and inorganic salts-starch
agar.
Vegetative Mycelium
On media where good growth occurred, the vegeta-
tive mycelium was observed to be raised and convoluted and
WflS generally yellowish-gray shades in color.
Aerial Mycelium and Spore Color
Aerial mycelia and/or spore masses were white to
264. light gray in color. Aerial mycelia production is
light on most media.
~ i
- 16 -
Soluble Pigments
Absent on many media; yel]ow pigment on Benedict's
and g]ycerol-asparagine agars; yellow-green pigment on
calcium malate agar; greenish brown pigment on NZ-amine-
starch-glucose agar; orange pigment on Bennett's and yeast
extract-malt extract agars.
Physiologica] Reactions
No melanin pigments on peptone-ir~n agar and
]o tyrosine agar (IS0-7j; strong peptonization of litmus
milk; str`ong pro~eolysis o~ nu~rient gelatin; moderate
reduction o~ nitrate; no hydro]ysis of adenine or guanine;
strong hydrolysis of hypoxanthine, tyrosine, and xanthine;
weak hydrolysis of starch; hydrolysis o~ e~culin; variable
hydrolysis o~ urea. No growth at 4C., 10C., or 55C.;
moderate growth at 25C. and 45C; good growth at 32C and
37C. Carbohydrate utilization as per the method of T. G.
Pridham and D. Gottlieb, "The utilization o~ carbon com-
pounds by some actinomycetales as an aid for species deter-
mination," J. Bacteriol. 56:107-114 (1948): good utilization
o~ g]ucose, glycerol and trehalose; moderate utilization
of maltose and sucrose; poor utilization of ~ructose,
galactose, inositol, mannose, and melezitose; no u~ilizat;on
o~ adonitol, arabinose, dulcitol, lactose, mannitol, melibi-
ose, raffinose, rhamnose, salicin, sorbitol and xy]ose.
Acid production ~rom carbohydrates by the method o~ Gordon,
et al., supra: Good ac;d production ~rom glucose, glycero],
maltose, sucrose and trehalose; weak acid production from
galactose, inos;tol and mannose. Utilization of organic
' acids by the method of Gordon et al., ~pra: utilization
o~ acetate, malate, propionate, pyruvate, succinate and
tartrate; no utilization of benzoate, citrate, lactate,
mucate and oxalate.
r
TA~LE VII
j~ ~t Cultural Characteristics of ~ctinomadura yumaense NRRL 12515
.,
Incubation: l4 days Temperature: 28C.
A~ount of Soluble
Medium Gcowth Aerial Mycelium and/or SporesPigment Reverse Color
Benedict's Moderate Flat, powdery colonies; aerial Yellowish 89. pale yellow
Agar to poor mycelia white to 264~ ht qray
Bennett's Good No aerial mycelia; convoluted Orange Bl. dark grayish
Agar ~egetative growth 93. yellowish yellowish ' ~
2~ to 80. grayish yellowish brown brown _ ~9
' g~
Calcium Poor Flat growth; sparse white aerial Yellow- 90. greenish
Malate mycelia green yellow
Agar
Czapek's Poor to Flat growth; moderate aerial mycelia None 89. ~ yellow
Sucrose Moderate vegetative growth 90. grayish ~ellow
Agar
Gauze No. 1 Poor Colorless flat growth; sparse white None Colorless
Agar aerial mycelia
TABL~ VII (continued)
Amount of Solu~le
Medium Growth Aerial Mycelium and/or Spores Pigment ~evecse Color
Gauze No. 2 Good Raised convoluted colonies; vegeta- None 72. dark orange
Agar tive mycelia 93. yellowish ~ ; yellow
moderate aerial mycelia, 92. y~el-
lowish white
Glycerol- Poor to Flat, powdery colonies; whiteYellow8-9. pale yellow
Asparagine Moderate aerial rnycelia
Agar
Hickey- Modera~e Flat waxy colonies, 90. grayish None 90.~ grayish
Tresner yellow sparse aerial mycelia, white yellow
Agar to 264. light qray
Inorganic Poor Fla~, colorless, powdery colonies; None Colorless
Salts- white aerial mycelia
Starch Agar
NZ-amine- Good Heavy convoluted growth, 61. grayish Greenish 78. dark y~
Starch- yellowish brown to 65. brownish brown lowish brown
Glucose black; moderate aerial mycelia,
Agar white to 26~ ght gray
`-:
TAB~E VII (contlnued)
~mount of Soluble
Medium Growth Aerial Mycelium and/or Spores Pi9ment ~everse Color
Oatmeal Moderate Flat waxy growth, 90. grayish None 90. grayish
Agar yellow; moderate aerial mycelia, yellow
white
Tomato Good Flat waxy growth, 91. dark grayish None ' ~
Paste yellow; trace of white aerial ~ ~0
Oatmeal mycel i2
Agar
Yeast Good Raised, waxy, convoluted colonies, Orange 78. dark yel-
Ex~ract 93. yellowish ~ to ~0. grayish lowlsh bro'wn
Malt yellowish brown; no aerial mycelia
Extract
Agar
TABLE VIII
Micromorphology of Actinomadura yumaense NRRL 12515
Aerial Mycelium and/or Spore
Medium Sporiferous Structures Spore Shape Spore SizeSur~ace
Czapek's Aerial sporophores; branched, almost 0.6 - 0.8 micron
Sucrose verticilate; carrying relatively ovoid X Smooth
Agar short spiral chains of mature spores 1.0 - 1.4 .i~icron
TABLE IX
Physiological Reactions o~ Actinomadura
yumaense ~RRL 12515
Incubation Amount o~ Physiological
MediumPeriod GrowthReactinn
Peptone- 7 Days GoodSlight browning
Iron Agar14 Days GoodSlight browning
Tyrosine 7 Days Moderate -.No pigment
Agar 14 Days GoodYellowish pigment
Litmus 14 Days GoodGood peptonization ~ 6
Milk 2~ Days GoodStrong peptonization '
Nutrient14 i)ays Goodslight proteolysis
Gelatin 28 Days GoodTotal proteolysis
Nitrate 14 Days GoodVery weak reduction
Broth 28 Days GoodModerate reduction
Adenine 14 Days GoodNo hydrolysis
Agar 21 Days GoodNo hydrolysis
TABLE IX (continued~
7~ Incubation Amount of Physiological
~` Mediun)PeriodGrowth~eaction
Guanine 14 Days GoodNo hydrolysis
Agar 21 Days GoodNO hydrolysis
Hypoxanthine 14 Days Good Total hydrolysis
Agar 21 Days GoodTotal hydrolysis
.
Tyrosine 14 Days GoodS~rong hydrolysis
Agar Zl Days GoodStrong hydrolysis
.
Xanthine 14 Days GoodModerate hydrolysis
Agar 21 Days GoodStrong hydrolysis
N~-amine with 5 Days Poor or no growth at 4C.,
Soluble 10C. and 55C.; moderate
Starch and growth at 25C., 2~C. and
Glucose Agar 45C.; good gro~th at 32C. and
~ATCC Med. 37C.
No. 1723
Urea Broth2~ Days Good Decomposition
variable
f.
. `
TABLE IX (continued)
Incubation Amount ofPhysiologic~al
~iediumPeriod Growth Reaction
Esculin 14 Days Good Hydcolysis ' 0
8roth 28 Days ~ood Hydrolysis
Starch 5 Days Good No Hydrolysis
Agar 10 Days Good No Hydrolysis
- 24 -
TABLE X
Carbon Source Ucilization of Actinomadura
yumaense NRRL 12515 on ISP-9 Carbohydrate
Utilization Medium
Incubation: 28 days Temperature: 28C.
Carbon Source Utiliza~ion
AdoniCol
l-Arabinose
Dulcitol
Fruccose Poor
~-Galactose Poor
~-Glucose Good
Glycerol Good
i-lnosicol Poor
Laccose
Maltose Fair
~-Manni~ol
d-Mannose Poor
d-Melezitose Poor
d-Melibiose
~-Ra~;nose
l-Rhamnose
Salicin
Sorbi tol
Sucrose Fa i r
~.~
- 25 -
TABLE X ~continued)
Carbon Source ULilization
d-Trehalose Good
d-Xylose
Nega~ive Control
,,
- 26 -
TABLE XI
Acid Production from Various Carbohydrates
by Ac-inomadura yumaense NRRL.12515
on Go~don' s 8asal Inorganic Nltrogen Medium
Incubation: 28 days Tempera~ure: 28C.
Acid Produc ion'
Carbon Source 7 Davs 2 Days
Adonitol - -
l-Arabi no se - -
Dulcitol - -
Fruc~ose
d-Galactose
d-Glucose +~+ ~++
Glycerol ++ +++
l_ I nos i tol - +
Lac tose
Maltose . - ~++
d-Ma nni tol
d-Ma nnose
d-Melezi tose - -
d-Melibiose
d -Ra f f i nose - -
l-Rhamnose - _
Salicin - -
Socbitol
Sucrose - ++~
';;~
- 27 -
TABLE XI (continued)
Acid Production
Carbon Source7 Days 28 Days
~-Trehalose - +++
d-Xylose - -
Negative Control ~ - -
+ = Strong positive response
++ = Moderate positive res~onse
+ = Slight positive response
- = Negative response
r.-"
~3~
- 28 -
TABLE XII
Ucilizacion o~ Organic Acids by Arrinnm~ ra
yllm~n~e NRRL 12515 on Gordon's Modi~ication
of Koser's Basai Agar (Koser's Citrace A~ar)
Incubacion: 28 days Temperature: 28C.
Carbon Source U~iliæa~ion*
Acetate +
Benæoa~e
Cirrate
Laccate
Malate +
Mucic Acid
Oxalace
PropionaCe
Pyruvace
Succinace
Tartrate +
~: + ~ Positive response - - NegaLive response
- 29 -
It is to be understood that the term Actinoma-
dura yumaense is not limited to strain Actinomadura yumaen-
se NRRL 12515 or to strains fully answering the above growth
and microscopic characteristics, which are given for illus-
trative purposes only. Actinomadura yumaense described
herein includes all strains of Actinomadura yumaense which
produce the antibiotic LL-C23024~ and iota and which cannot
be definitely differentiated from Actinomadura yumaense
NRRL 12515 and its subcultures, including mutants and
variants thereof. The term "mutants" includes the natural
(spontaneous) mutants of this organism as well as induced
mutants produced from this organism by various mutagenic
means known to those skilled in the art, such as exposure
to x-ray radiation, ultraviolet radia~ion, nitrogen mustard,
actinophages, nitrosamines, and the like. It is also de-
sired and intended to include inter- and intraspecif;c
genetic recombinants produced by genetic techniques known
to those skilled in the art, such as for example conjuga-
tion, transduction and genetic engineering techniques.
Cultivation of Actinomadura yumaense may be car-
ried out with a wide variety of solid or liquid culture
media. Media which are useful for the production of anti-
biotic LL-C23024~ and iota include an assimilable sour e
of nitrogen such as protein, protein hydrolysate, polypep
tides, amino acids, corn steep liquor, etc., and inorganic
anions and ca~ions, such as potassium, sodium, ammonium,
calcium, sulfate, carbonate, phosphate, chloride, etc.
Trace elements such as boron, molybdenum, copper, etc., are
supplied as impurities of other consti~uents of the media.
Aeration in tanks and bottles is provided by forcing ster-
ile air through or onto the surfact of the fermenting
medium. Further agitation in tanks is provided by a mechan-
ical impeller. An antifoaming agent such as lard oil or
silicone defoamer may be added as needed.
Actinomadura yumaense is grown and maintained on
agar slants, for example Bennett's agar, Yeast Malt Agar,
or ATCC ~edium #172. ATCC Med;um ~172 is preferred. The
slan~ is inocula~ed wiLh a culture of AcLinomadura yumaense
and incubaLed at 28-37C, preferably aL abouL 32C, for
approx;ma~ely 7 days. These stock cul~ures may be mainLained
S by serial Lransfers Lo Fresh agar slan~s, or a plug of
~he agar con~aining mycelia ~rom ~he well-grown agar
slanL may be used Lo inocula~e liquid media.
Shake flask inoculaLions of AcLinomadura yumaense
are prepared by inoculaCing 100 ml. of sLerile liquid
medium in 500 ml. flasks wi~h scrapings or washings of
spores from an agar slan~ of Lhe culLure. Examples of
su;~able seed media are:
Medium A
BeeF exLrac~............. 0.3%
BacLo~ tryptonel......... 0.5%
Glucose... ~..................... 1.0%
Yeas- extracL................... 0.5~/O
WaLer.......... qs............... 100%
[lA pep~one, registered ~rademark of Di~co LaboraLories,
DeLroiL, Michigan]
The pH is adjus-ed Lo 6.8-7.2 wi~h diluLe base,
e ~. sodium hydroxide.
Medium B
Glucose........................ 1%
SLarch......................... 2%
YeasL exLracL.................. 0.5%
N-Z Amine A~2~ 0~5%
Calcium carbona~e.............. 0.1%
Wa~er.......... qs...................... 100%
[2Casein diges-, regis-ered ~rademark oF Sheffield
Chemical Co., Div. NaL'l. Dairy Produc~s Corp., Norwich,
N.Y.~
Medium B is preferred.
The flasks are incubaLed a~ a LemperaLure of 25-
35C., preferably aL 32C., and agiLaLed vigorously on a
;,.-,
rotary shaker for 1-4 days. This seed inoculum is then
used to inoculate fermentation culture, or this culture
~ay be frozen and stored to provide inoculum for subsequent
seed cultures.
The following media are examples of those suitable
for the fermentation of Actinomadura yumaense to produce
antibiotic LL-C230~4~ and iota.
Medium C
Glucose.~..................................... .1.5%
~ Glycerol;..................................... .1.5%
Soy flour3.................................... .1.5~/O
Calcium carbonate............................. Ø l~/o
Sodium chloride............................... Ø3%
Water........... qs ............ .100%
~3May be substituted ~y cottonseed ~lour or mea~ solu-
bles witb equal ef~ect.]
Medium D
Glucose....................... .3%
Soy ~lour..................... .1.5%
Calcium carbonate............................. Ø1%
Sodium chloride............................... Ø3%
Water........... .qs............ .100%
Starch........................ .1%
Molasses....................... .2%
Soy ~lour...... ...~........... .1.5%
Calcium carbonate............................. Ø1%
Water.......... qs............. .100%
Medium F
Glucose....................... .3%
Meat solubles................. .2.5%
Sodium chlor;de............................... OØ2%
Calcium carbonate............................. Ø1%
Wa Ler .......... qs ............ .100%
33~
- 32 -
Med;um G
Glucose.... ~.......................... ~3%
Soy flour.................. ........... Ø5%
Ammonium sul~ate............. Ø3%
Sodium chloride.............. Ø2%
Calcium carbona~e............ Ø1%
Water...... qs.... :.................... .100%
Medium H
Glucose .... .................... 3/O
Ammonium sulfate...... ~................. 0.3%
Dibasic potassium phosphate...Ø1%
Calcium carbonate.............. ......... 0.2%
Sodjium chloride............... ......... 0.1%
Water....... ..qs................ 103%
Medium D is preferred.
The ~ermentation may be carried out in 100 mls. of
media in a 500-ml. flask inoculated with 3-10% (v/v~ o~ a
seed culture prepared as described above and incuba~ed at
25-35C, pre~erably at about 32C, ~or 24-72 hours with
aerat;on. Samples of ~he ~ermentation cul~ure may be frozen
and stored for later use as inoculum for seed cultures.
AlLernatively, the ~ermentation may be carr;ed ou~
in larger ~ermentation ~anks equipped with aerat;on and
agitation means. Each tank i5 inoculated w;th 3-10% (vlv)
oF inoculum prepared as described above. Aeration is sup-
plied aL the rate of 0.5 Co 2.0 liter o~ sterile air per
l;ter o~ broth per minute and the fermenting medjum is
agi~a~ed by an impeller driven at 200-400 rpm. The tempera
ture is maintained at 25-35C, pre~erably aL 32C. The
~ermentation is continued until antibiotic accumulate~ in
Lhe ~ermen~a~ion medium, usually af~er 100~150 hours, a~
which time the antibiotic is harvested.
The antibiotic may be harvested and puriFied accord-
ing Lo the me~hods described ;n U.S. Patent 4,278,663,
supra, or according ~o the following procedure:
The cr~de fermen~a~ion broth conLa;ning the whole
,~,,
- 33 -
cells, prepared as describ~d above9 is m;xed with an equal
volume o~ any non-hydrocarbon water-immiscible organic sol-
vent. Methylene chloride or ethyl acetate is pre~erred.
The organic phase is separated and concentrated in vacuo to
an oily syrup.
The oily syrup ;s dissolved ;n methylene chloride
and added to a column o~ silica gel, alumina, Sephadex LH-
20~ (Pharmacia Fine Chemicals Div. of Pharmacia, Inc.,
Piscataway, N.J.), or magnesium aluminum silicate. Examples
o~ suitable solvents ~or developing the column are die~hyl
ether, ethyl acetate, a 1:1 to 1:7 (v/v) m;xture o~ methyl-
ene chloride:e~hyl ether, 10 40% acetone in methylene chlor-
ide, 2-10% lower alcohol (methanol is pre~erred) in methyl-
ene chloride, 2-15% acetoni~rile in methylene chloride, or
2-15% dioxane Sn methylene chlor;de. Methylene chlor;de:
ethyl acetate l:l (v/v) ;s preferred. Fractions are collec-
ted and checked for the presence o~ antîbacterial activity
by bioassay against a susceptible organism, e.g. Bacillus
subtilis. Active ~ractions are combined and concentrated
in vacuo to a residue. This residue is dissolved in an or-
gan;c solvent, e.g. t-butanol ~pre~erred~, ben2ene, or
p-dioxane, and ~reeze-dried.
The ~reeze-dried solid is dissolved in an appropria~e
organic solvent, e.~. methylene chloride, hexane, methylene
chlor;de:ethyl acetate, diethyl ether9 hexane:ethyl aceta~e,
hexane:chloroform, or hexane:ether. Diethyl ether i5 pre-
~erred~ Th;s solution is shaken w;th water and the pH ;s
adjusted to pH 1.5-4.0, pre~erably about 2.5, w;th any
dilute mineral acid. The organic phase is separated, washed
with water to remove any excess ac;d, dr;ed over an appropriate
drying agent, ~iltered, and concentrated to a residue in
vacuo.
Th;s residue ;s d;ssolved ;n an appropriate solvent
and the solution is allowed to evaporate slowly, preferably
at about 4C. Examples o~ suitable solvents are methylene
chloride, hexane, methylene chloride:ethyl acetate, die-hyl
ether, hexane:ethyl acetate, hexane:chloro~orm, or hexane:
~,,.
3~
ether. Hexane:ether 5:2 (v/v) is preferred. The result-
ing crystals are collected and washed, preferably at about
40C, with any moderately boiling hydrocarbon such as for
example hexane or heptane, and air dried to yield as the
Einal product the antibiotic X-14868A in the free acid
form.
If the product is desired in the form of a salt,
the free acid may be converted by treatment with the appro-
priate cation, preferably in the form of a dilute mineral
base, according to procedures well--known to those skilled
in the art.
The following Examples illustrate the invention.
The Media A, B, C, etc. are those defined above. Unless
otherwise specified, all procedures were performed at room
temperature (approximately 22C) and at 1 atm pressure.
Example 1
Washed spores from an agar slant of Actinomadura
yumaense NRRL 12515 were used to inoculate a 500 ml. flask
containing 100 ml. of sterile Medium B. The flask was
incubated on a rotary shaker at 28C for 2 days.
A 5% inoculum of this culture was then transfer-
red to 100 mls. sterile Medium C in a 500 ml. flask and
incubated at 28C for 5 days on a rotary shaker.
The presence of antibiotic activity was monitored
daily by bioassay against Staphylococcus aureus ATCC 6S38
P, Bacillus subtilis, and Streptococcus faecalis, and an-
thelmintic activity was monitored by bioassay against the
free-living nematode Caenorhabditis elegans.
Example 2
Seven 500 ml. flasks were prepared, each con-
taining 100 mls. of one of the following sterile media:
Flask 1: 100 ml. Medium C
Flask 2: 100 ml. Medium C with 1.5~/~ cottonseed
flour substituted for the soy flour
Flask 3: 100 ml. Medium C with 1.5% meat solu-
bles substituted for the soy flour
Flask 4: 100 ml. Medium D
r'
Flask 5: 100 ml. Medium E
Flask 6: 100 ml. Medium F
Flask 7: 100 ml. Medium G
These flasks were each inoculated with a 5% inoculum of
Actinomadura yumaense NRRL 12515 grown in Medium B. T~e
flasks were then incubated on a rotary shaker at 28C for
four to six days.
Each of the above cultures was found to be active
when assayed for antibiotic activity as in Example 1 and
when assayed for anticoccidial activity against Eimeria
tenella in chick kidney tissue cultures.
Example 3
Frozen fermentation culture cells of Actinomadura
yumaense NRRL 12515 were used to inoculate a 500 ml. flask
containing 100 ml. sterile Medium B. The flask was then
incubated at 32C. for 4 days on a rotary shaker.
A 5% inoculum of this culture was then transer-
red to 100 ml. sterile Medium H in a 500 ml. flask and in-
cubated at 28C for 5 days on a rotary shaker.
Antibacterial activity was confirmed as in Exam-
ple 1 and anticoccidial ac~ivity as in Example 2.
The presence of antibiotic activity was also as-
sayed by thin layer chromatography on silica gel plates
developed in etbyl acetate:chloroform 70:30 (v/v).
_ Example 4
One hundred milliliters sterile Medium A in a
500 ml. flask was inoculated with washed spores from an
agar slant of Actinomadura yumaense NRRL 12515. The flask
was incubated at 32C for 2 days on a rotary shaker.
A 5lO inoculum of this culture was then transfer-
red to 100 ml. sterile Medium H in a 500 ml. flask and in-
cubated on a rotary shaker at 32C for 2 days.
A 5% inoculum of this culture was then transfer
red to a 500 ml. flask containing 100 ml. fresh sterile
Medium H and incubated at 28G for 6 days on a rotary sha-
ker.
J
3~1~
- 36 -
Antibacterlal activity was confirmed as in Exam-
ple l.
Example 5
Two 500 ml. flasks each containing lO0 ml. ster-
ile Medium A were inoculated with a frozen seed culture of
Actinomadura yumaense NRRL 12515 and incubated at 32C for
2 days on a rotary shaker.
The contents of the two flasks were then combin-
ed and added to 12 liters of fresh sterile Medium A in a
20 liter bottle. This culture was then incubated for 2
days at 28C with aeration.
The contents of this bottle were then transfer-
red to a 300 liter seed tank containing 288 liters sterile
Medium A and this culture was aerated and agitated for 25
hours at 32C.
At the end of 25 hours' incubation, the 300 li-
ters of seed culture were transferred to a 1500 li~er fer-
mentor containing 1200 liters of sterile Medium C. This
culture was incubated with aeration and agitation for 115
hours at 28C.
The fermentation broth was assayed for antibiotic
activity by thin-layer chromatography on silica gel plates
developed in ethyl acetate:cbloroform 70:30 (v/v). Alter-
natively, several of the developed plates were subjected
to bioassay against Bacillus subtilis which showed the pre-
sence of antibiotic activity.
Example 6
A typical medium used to grow the primary inocu-
lum was prepared according to the following formula:
Beef extract..................... 0.3%
Bacto~-tryptone.................. 0.5%
Glucose.......................... 1.0%
Yeast extract.................... 0.5%
Bacto~ agar...................... 0.15%
Water............ qs.............. 100%
,.
, ;,
Washed or scraped spores and mycelia from an agar
slant of Actinomadura yumaense NRRL 12515 were used to in-
oculate a 500 ml. flask containing 100 ml. of the above
sterilized medium. The flask is placed on a ro~ary shaker
and agitated vigorously for 48 hours at 32C. The result-
ing flask inoculum (100 ml.) is used to inoculate one
liter of the same sterile medium in a 2 li~er bottle. This
inoculum is aerated with sterile air while growth is con-
tinued Eor 48 hours at 28C. This one-liter culture was
then used to inoculate a 30-liter fermentor tank contain-
ing the same sterile medium, and this tank was aerated with
sterile air and incubated at 28C for 42 hours.
Example 7
A fermentation medium was prepared according to
the following formula:
Dextrose............ O.............. ...1.5%
Glycerol................... ........ ...1.5%
Soy flour................. ........ ...1.5%
Calcium carbonate.......... ........ ..Ø1%
Sodium chloride........... ........ ..Ø3%
Water............... qs............. ...100%
The pH was adjusted to 7.0 with 6N sodi~m hydrox-
ide and the medium was sterilized. A 30 liter portion of
inocullum prepared as described in Example 1 was used ~o
inoculate 250 liters of ~he above medium in a 300 liter
fermentor. Sterile aeration was supplied to the mash and
the mash was agitated by an impeller driven at 220 rpm.
Fermentation was carried out at 32C. for 97 hours at which
time the mash was harvested.
Example 8
A fermentation medium was prepared according to
the Eollowing formula:
Dextrose........... O.............. 3.0%
Soy flour........................ . 1.5%
Calcium carbonate................ . 0.1%
Sodium chloride.................. . 0.3%
Water.......... qs.................................. 100%
t
The pH was adjusted to 7.0 with 6N sodium hy-
droxide and the medium was sterilized. A 300 liter por-
tion of inoculum prepared as described in Example 1 was
used to inoculate 3000 liters of the above medium in a
fermentor. Sterile aeration was supplied to the mash.
The mash was agitated by an impeller driven at 100 rpm.
Fermentation was carried out at 32C for 115 hours at
which time the mash was harvested.
Example 9
1. The fermentation mash (100 1.) was adjusted to
pH 4.0 using 6N HCl. The acidic mash was then stirred for
1 to 2 hr; with an equal volume of methylene chlorideO The
mixture of mash and methylene chloride was then filtered
using diatomaceous earth as a filter aid. The methylene
chloride extract was drawn off and concentrated in vacuo
to approximately 2 1. of partially purified antibiotics.
2. Chromatography of the Partially Purified Anti-
biotics on Silica &el
A glass column with a diameter of 7 cm. was
2~ packed to a height of 91 cm. with Woelm Silica Gel TSC.
The crude concentrate tSee 1 above) with a volume of ap-
proximately 2 1. was allowed to seep into the column of
silica gel. The column was then first developed with 3
1. of methylene chloride, followed by methylene chloride:
ethyl acetate (1:1 by volume) to give a total of 126 frac-
tions, each with a volume of 80 ml. This was then develop~
ed further using e~hyl acetate-ethanol (7:33 to give an
additional 87 fractions.
Fractions, 177 to ~03, rich in C23024~ were com-
bined and concentrated in vacuo to a residue weighing
90.4 g. This residue was dissolved in a mixture of 600
ml. of diethyl ether and 600 ml. of hexane. Th~ resulting
suspension was filtered to give a clear filtrate. The
clear filtrate was concentrated in vacuo until crystals
started to form. This suspension was allowed to age over-
night. The crystalline C23024~ was collected on a funnel,
washed with cold ether and air dried to give 6.7 ~- of
~,
i!33~ii
- 39 -
crystalline C23024~.
Fractions 204 to 213 from elution with ethyl
acetate-ethanol and enriched in LL-C23024 iota were com-
bined and concentrated in vacuo to a paste and repeatedly
extracted with methanol. The methanol extract was extract-
ed with methylene chloride which was charged on a column
containing Woelm silica gel. The column was washed with
me~hylene chloride and then eluted with methylene chloride:
ethyl acetate (2:3). The cuts were monitored by thin layer
chromatography and the active fractions were pooled, treat-
ed with charcoal, concentrated and repeatedly extracted
with heptane. The final heptane extract was chilled at
0C. for 48 hours and the crystals removed by filtration
from the mother liquor.
This mother liquor was dissolved in methylene
chloride and allowed to seep into a glass column packed
with Woelm silica gel. The column was then eluted in
succession wi h 2 liters of methylene, 8 liters of methyl-
ene chloride:ethyl acetate ~1.1) and 4 liters of ethyl
acetate:methanol (9:1). The eluate was collected in frac-
tions and monitored for their antibiotic composition.
The active fractions were combined and concentrated in
vacuo to give a residue containing LL-C23024 iota.
Example 10
3. Further Purification of C23024~ from Chromato-
graphy on Silica Gel
Sephadex~ LH20 was allowed to swell in a mix-
ture of hexane-methylene chloride-methanol (10:5:1). A
glass column with a diameter of 5 cm. was packed to a height
of 86 cm. with the gel. The charge, 3 g. of purified
LL-C23024~ was dissolved in 10 ml. of methylene chloride,
3~
- 40 -
1 ml. of methanol and 10 ml. of hexane and allowed to seep
into the column of gel. The column 3 was then developed
with solvent with the same composition as that used to de-
liver the antibiotic to the column. Fractions were collec-
ted using a collector. The first 23 fractions had a volume
of 17 ml. each. Fractions 17 to 26 contained C23024~ by
TLC. These fractions were combined and concentrated in
vacuo to give pure amorphous LL-C23024~, weighing 1269 mg.
Example 11
Crystallization of LL-C23024~ as Sodium Salt
Purified LL-C23024 (2.4 g.) prepared, as des-
cribed in sections B and C was, dissolved in a mixture of
diethyl ether (500 ml.) hexane (160 ml.) and methylene
chloride (25 ml.). The resulting solution was transfer-
red to a separating funnel containing 300 ml. of distilled
water. Dilute HCl (IN) was added dropwise until the pH
reached 2.0 to 2.5 after shaking and settling. The acidic
aqueous phase was discarded and the acid treated organic
layer was washed three successive times with 400 ml. vol-
umes of water. The washed organic layer was stirred with
a teaspoon to Darco G-69 powder for 15 min. and filtered
through celite. The decolorized organic layer was placed
over 500 ml. of distilled water and 5N NaOH was added drop-
wise until the pH reached 11.0 to 11.5 after shaking and
settling. The alkaline water phase was discarded and the
remaining organic layer was washed twice with portions of
water having a volume of 400 ml. The washed organic layer
was dried over sodium sulfate and then concentrated in
vacuo to a volume of 150 ml. This concentrate was allowed
to stand in a hood for several hours. The crystalline
LL-C23024~ was collected on a funnel, washed with cold
hexane and air dried to give 402 mg. of the crystalline
salt of LL-C23024~. This sample was sent for microanaly-
ses and selected spectral data.
, ~
3~i
- 41 -
Anal. Calculated for C46H77017Na C~59 7;
H,8.33; 0,29.46; Ash, 2.49. C, 61.55,H,8.79;
Ash, 3.31; N,0. MP.(Fisher-Johns Apparatus= 174
(FDMass Spec) = 924 [~26 = +3.2
in methanol)
Example 12
Purification of LL-C23024 iota
A Waters Prep. 500A high performance liquid
chromatography instrument was fitted with a silica gel
cartridge under a pressure of 550 psi. The charge, which
was S.0 g. of the crude material from Example 3, was dis-
solved in 50 ml. of heptane:ethyl acetate (45:55) and
injected onto the colu~N of silica gPl. T~e column was
then developed with the same solvent mixture at a flow
rate of 200 ml/minute with collection of forty 200 ml.
fractions. Frartions 21-32 were combined, concentrated to
a residue, triturated with hexane and evaporated, giving
pure LL-C23024 iota. The above procedure was repeated
four times giving a combined total of 280 mg. of amorphous
LL-C23024 iota.
A 90 mg. portion of amorphous LL-C23024 iota was
dissolved in 10 ml. of diethyl ether, mixed with 10 ml. of
water, and adjusted to pH 2.5 with O.lN hydrochloric acid.
The ether layer was washed with water, adjusted to about pH
11 with O.lN sodium hydroxide, separated, and again washed
with water. The ether phase was dried over sodium sulfa~e,
filtered, and conce~trated to a residue. A solution of
this residue in ether-hexane was allowed to slowly evapor-
ate to give an amorphous white solid.
This amorphous white solid has the following
properties:
Elemen~al analysis: C, 61.79; H, 8.84; ash, 0.3~;
[~26 = +27 (C 0.724, methanol);
Field desorption mass spectrometry: (M~Na)~~ = m/e 953 9
therefor the calculated molecular weight is 930;
;.
