862
THE
HERBICIDINS
JOURNAL
F AND
G,
Yo TAKIGUCHI,
OF
TWO
NEW
Research
(Received
A Mutant
of Streptomyces
Tokyo,
for publication
saganonensis
1979
ANTIBIOTICS
AKIRA TERAHARA,
MICHIYA TERAO
Laboratories,
1-12-11, Tanashi
SEPT.
NUCLEOSIDE
HIROJI YOSHIKAWA,
AKIO TORIKATA and Fermentation
ANTIBIOTICS
Sankyo
Co., Ltd.
188, Japan July 9, 1979)
No. 4075, obtained
with N-methyl-N'-nitro-N-
nitrosoguanidine treatment, produced herbicidins F and G without herbicidins A and B. Isolation of the antibiotics was performed by adsorption on resinous adsorbent followed by elution with aqueous MeOH. Herbicidin F was obtained as colorless needles after extraction of the eluate using methylene dichloride. Purification of herbicidin G was completed with silica-gel chromatography to give a crystalline powder. Physico-chemical characterization revealed that herbicidins F and G were new nucleoside antibiotics having an adenine moiety in their structures. There was no inhibition activity at 100,ug/ml of herbicidins F and G against all of bacteria and yeast tested. Herbicidin F, as well as herbicidin G, are inhibitory activity against some of fungi such as Tricophyton rubrum (MIC; 6.25 p.-/ml), T. asteroides (6.25 pg/ ml), T. mentagrophytes (6.25' 12.5 lug/nil), Botrytis cbrcrea (12.5 pg/ml), Blastomyces brasiliensis (12.5-25 pg/ml).
S/reptomyces
saganoncnsis
No.
4075
produced
new
antibiotics,
herbicidins
A,
B''-',
C and
E3
with 9-/-D-arabinofranosyl adenine and deoxycoformycin. We isolated some mutants of the organism by means of the treatment with N-methyl-N'-vitro-N-nitrosoguanidine (NTG). A strain of these mutants produced herbicidins F and G, new nucleoside antibiotics, without the production of herbicidins A and B. The biological
present
paper
properties
describes
the
of herbicidins
fermentation,
F and
G.
isolation,
Structural
physical
elucidation
and
chemical
of the antibiotics
properties
and
will be reported
elsewhere. Materials Mutagenesis A strain
and
selection
of S. saganonensis
and Methods
of the mutants was
cultivated
for
10 days
on
a slant
YM
agar
medium
containing
per liter; 4 g yeast extract (Difco Lab., Detroit, U.S.A.), 10 g malts extract (Difco Lab.), 4 g glucose and 20 g agar powder (Eikenkagaku, Tokyo, Japan) (pH 6.5 before sterilization). Spores of the organism grown on the slant were suspended in 1/15 M phosphate buffer (pH 7.0), and treated with N-methylN'-nitro-N-nitrosoguanidine (NTG) at 2,000 pg/ml at 27°C for 2 hours (killing rate 99.7%). After washing twice with saline, the mutagenized spores were spread on the YM agar plates and incubated at 27°C for 3 days. Colonies,
well separated,
on the plates
were
transferred
to the fresh
slants
and
incubated
at 27'C
for 10 days. Each strains on the slants were inoculated into 500-ml shake flasks containing 50 nil of HC-4 medium consisting per liter; 20g soybean meal, 10g glucose, 20g soluble starch, 20g fresh baker's yeast, 10 g KH2PO.,, 3 g CaCO,, 5 g NH4C1, 1 g ZnSO;.7H_O and 0.2 g MgSO,.7H20 (pH 6.5 before sterilization). Each of the culture broths was assayed by the method described in the previous paper'. Fermentation The fermentation
of a mutant
strain
of the organism
was performed
in 30-liter jar fermentors
VOL.
XXXII
NO.
9
THE
JOURNAL
OF
ANTIBIOTICS
863
containing 15 liters of the HC-4 medium. The preculture was conducted at 27°C for 2 days in 2-liter Erlenmeyer flasks containing 500 ml of the HC-4 medium. Each flask of a good preculture was then inoculated into each jar fermentor (inoculum size; 3 %). During 120-hour incubation period of the main culture at 24°C, dissolved oxygen concentration in the broth was held at more than 2 ppm by agitation and aeration. Isolation Fermentation broth (30 liters) containing herbicidins F and G was filtered with the aid of infusorial earth (Celite 545 from Johne-Manville Products Corp., Calif., U.S.A.) The filtrate (20 liters) was adsorbed on 2 liters of Diaion HP-20 (Adsorbent resin from Mitsubishi Co., Ltd., Japan) column. The spent broth and water washes (10 liters) were discarded. The adsorbate was washed with 10 liters of 40 % aqueous MeOH again, and eluted with 20 liters of 45 % aqueous MeOH to obtain herbicidin G. Herbicidin F was then eluted from the adsorbate (Diaion HP-20) with 25 liters of 70Y. aqueous McOH. Fifteen
liters
aqueous
solution,
dryness
to yield
of the eluate which
5.8 g of crude
700 ml of Sephadex tion
containing
was extracted
containing
LH-20
solid.
column
herbicidin
herbicidin twice
G was
with
The
3 liters
solid
was
to be developed
G was concentrated
aqueous
solution,
to dryness
in vacuo
decolorized obtained
which
extracted
at 45°C.
by means from
was
The
the decolorized
twice
crude
of 5 g of active McOH
with
solid
carbon. solution
Results Mutagenesis A mutant
of S. saganonensis
No.
4075,
dissolved
thus after
at 45°C
extract
was
hundred
at 45°C.
to 3 liters
and
placed
milliliters
One
of
concentrated
to on
of CH2C12.
obtained
was
grams
The
hundred
dissolved
of needle
concentration
extract
was
ninety
milliof
concentrated
in 500 ml of MeOH
crystals
a
of the frac-
and recrystallized from EtOAc. concentrated in vacuo at 45°C to 3 liters
3 liters
Twelve
Three
in vacuo
powder F was
in vacuo The
in 10 ml of MeOH,
by MeOH.
to dryness
grams of herbicidin G was obtained as a crystalline Twenty liters of the eluate containing herbicidin
concentrated of EtOAc.
of herbicidin
and
F were
in vacuo at 45°C.
and Discussion and
Fermentation
m 17-F strain,
which
mainly
produce
herbicidins
F and G
without herbicidins A, B, C and E, was obtained by mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) at 2,000 pg/ml for 2 hours. in 30-liter jar fermentor 15
liters
After
of
the
120-hour
mentation
dins per liter;
HC-4
medium.
incubation,
the fer-
contained
herbici-
broth
Large-scale fermentations of the mutant were conducted
containing Fig. 1.
Isolation
Culture
However,
C and
E was not
herbicidins
A, B,
detected
in the
filtrate
washed eluted
final broth. Fraction
Isolation The
isolation
herbicidins
F
Fig.
1.
tion,
the resinous
HP-20
For
the
a satisfactory
used
for
G is outlined
in
first
step
purifica-
adsorbent,
Diaion
was selected,
each
other
because
separation and
I (15 liters)
concentrated extracted with EtOAc
Sephadex
LH-20
developed
(0.7 liter)
with MeOH
it gave
(20 liters)
with 40%
aq. MeOH (10 liters) with 45 % aq. MeOH (20 liters) eluted with 70° , aq.
Fraction
II (20 liters)
concentrated extracted with CH-Cl~ CH2Cl2 solution
(6 liters)
concentrated
of herbiciHerbicidin
dins
F and G.
MeOH (25 liters)
method
and
of herbicidins
DiaionHP-20(2liters)
1,100 mg F, and 30
mg G.
and purification
satisfactory
re-
G (190 mg)
(crystalline powder)
Herbicidin F (12 g) (colorless needles)
864
THE
covery
of the antibiotics
ates.
Herbicidin
CH2CI2
from
in their
elu-
F was extracted
by
the
eluate,
and
JOURNAL Table
OF 1.
ANTIBIOTICS
Physico-chemical
as colorless
cation
of
pleted
needles.
herbicidin
G
Herbicidin
on Sephadex
after
extraction
LH-20
with
Solubility
comcolumn
EtOAc
from Melting
Physical
and
Chemical
1.
F and
in MeOH,
insoluble
in
in EtOH,
acetone
but insoluble
respectively.
142
and
154°C (dec.)
C, 51.58; H, 5.46; N,
acetone
146°C (dec.)
C, 50.67 ; H, 5.22: N, 13.43
13.08
Rf*
0.71
0.53
260 nm (245)
260 nm (200)
but UV
Herbicidin
E'~, Merck,
solu-
DC-Ferting
platten
(CHCI3 - McOH,
Kieselgel
60 F254
7 : 3)
EtOAc,
in n-hexane.
mass spectrum.
150-
in MeOH,
EtOH, EtOAc.
Calcd for C23H29N5010 (535.51) C, 51.59; H, 5.46, N, 13.08
solu-
in MeOH,
powder
Soluble
535
her-
and CH2C12
n-hexane.
G
White crystalline
needle
Elementary
soluble
and acetone,
soluble
point
in Table
is readily
EtOAc
G is readily ble
F
EtOH
ble in H20,
of
G are listed
Herbicidin
F and G.
Herbicidin
Soluble in H20, MeOH, EtOH, acetone, EtOAc, CH2CI2
analysis
data
F
M. W.
Properties Physico-chemical
of herbicidins
crystals
the eluate.
bicidins
Colorless
Purifiwas
properties
1979
obNature
tained
SEPT.
Molecular
weight
The IR and NMR spectra
of herbicidin
of herbicidins
F was derived
from analysis
F and G are shown
of its FD
in Figs. 2, 3, 4 and 5,
Signals at 8 2.05 ppm in Fig . 4 and at 8 3.18 in Fig. 5 were due to (CD3)2CO and CD3OD
used as a solvent, respectively. Their
physico-chemical
glets
due to heteroaromatic
ppm
corresponding
ion peak
tures
herbicidins
of both
described
protons
to the
and fragment
activities
properties,
chemical
at 135 in FD
below,
F and
G.
such
shift
of anomeric
mass
spectrum,
From
one can conclude
Fig. 2.
as UV maxima
at 8 7.8 - 8.3 ppm
Infrared
the results
doublet
proton suggested
elementary
due
of usual adenine
mentioned
that herbicidins
spectrum
at 260 nm,
and
to one nucleosides
nucleoside
above
and
analysis, proton
in NMR moiety
characteristic
F and G are two new nucleoside
of herbicidin
F (KBr).
two
sin-
at 8 5.9 - 6.2 spectra,
in the strucbiological antibiotics
VOL. XXXII
NO.
9
THE
Fig. 3.
Fig. 4.
with
very
closely
related
NMR
JOURNAL
OF
Antimicrobial
865
Infrared spectrum of herbicidin G (KBr).
spectrum
of herbicidin
F in (CD3).CO
at 60 MHz.
structures. Biological
laboratories').
ANTIBIOTICS
activities
of herbicidins
There were no activity
tested such as Staphylococcus
F and
Properties G were
tested
with
at 100 ltg/ml of herbicidins
aureus FDA 209P (JC-1), Escherichia
smegmatis ATCC 607, Pseudomonas
aeruginosa
the
method
reported
F and G against coli NIHJ
SANK 73575, Proteus
from
our
all of the bacteria
QC-2), Mycobacterium
rettgeri SANK 73775, Klebsiella
pneumoniae PCI 602, Serratia marcescens SANK 73060, Bacteroides fragilis SANK 71176, Bacillus subtilis PCI 219, and all of the yeast tested such as Candida albicans SANK 50157, Crvptococcus neoformans
SANK
mentagrophytes
57963, Saccharornvices
SANK
11868 was inhibited
cererisiae
SANK
by herbicidin
50170.
However,
F at 6.25-12.5lrg/ml
growth
of Tr.i•choplrnton
and herbicidin
G at
THE
866
Fig. 5.
NMR
spectrun,
Table
JOURNAL
of herbicidin
2.
OF
ANTIBIOTICS
G methyl
Antifungal
SEPT.
ester in CDC13 and CD3OD
spectrum
of herbicidin
1979
at 60 MHz.
F. MIC (pg/ml)
Test
organism 2 days
Trichophyton mentagrophytes SANK 11868 Trichophyton rubrcan SANK 11768 Trichophyton toavurans SANK 30970 Trichophyton Trichophyton Trichophyton Microsporum
volume of horse serum. rubrum
SANK
activities
6.25
30970, _Microsporum gypseum
SANK
50
6.25
50
12.5
12.5
12.5
12.5
50
50
100
> 100
> 100
> 100
> 100
> 100
100
100
100
> 100
> 100
25
50
100
12.5
25
25
100
100
50 12.5
12.5
12.5
> 100
> 100
> 100
> 100
> 100
> 100
100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
100
> 100
> 100
F and G were not affected F also inhibited
T. megninii
11668, Blastomyces
6.25
50
>100
10362,
6.25
50
50
of herbicidins
SANK
12.5
6.25
25
As shown in Table 2, herbicidin
11768, T. asteroides
12.5
6.25
asteroides SANK 10362 interdigitale SANK 10469 megninii SANK 30870 gypseum SANK 11668
The inhibitory
7 days
6.25
Sporothrix schenckii SANK 30166 Aspergillus oryzae SANK 11262 Hormodendrum pellrosoi SANK 11758 Mucor mncedo IFO 5776 Coccidioides immitis IFO 5960 B/astoinvices brasiliensis SANK 20567 Alternaria solani SANK 16676 Botrytis cinerea SANK 16976 Cocltllobolus miyabeanus SANK 16476 Colletotrichum lagenarium SANK 16576 Fusarium oxyspor un SANK 17076 Pellicularia filomentosa SANK 16376 Penicillium chrysogenuin SANK 12768 Pyricularia oryzae SANK 14758
100 /ig/ml.
4 days
SANK
brasiliensis
by addition
of 10
some of fungi, such as T. 30870,
SANK
T. tonsurans
20567,
Botrytis
SANK cinerea
VOL. XXXII
SANK The
NO. 9
16976, acute
600 mg/kg.
THE
Coccidioides toxicity
Herbicidal
immitis
JOURNAL
IFO
of herbicidin
OF
ANTIBIOTICS
867
5960.
F, LD5o,
administered
activities of the antibiotics
intraperitonealy
will be reported
to mice,
was
more
than
elsewhere.
References
1)
ARAI, M.; T. HANEISHI, N. KITAHARA, R. ENOKITA, K. KAWAKUBO & Y. KONDO: two new antibiotics biotics 29: 863-869,
2)
activity.
I.
Producing
organism
and
Herbicidins
biological
activities.
A and
B,
J. Anti-
HANEISHI, T.; A. TERAHARA, H. KAYAMORI, J. YABE & M. ARAI: Herbicidins A and B, two new antibiotics with herbicidal activity. 11. Fermentation, isolation and physico-chemical characterization. J. Antibiotics
3)
with herbicidal 1976
29: 870-875,
1976
TAKIGUCHI, Y.; H. YOSHIKAWA, A. TERAHARA, A. TORIKATA & M. TERAO: nucleoside antibiotics. J. Antibiotics 32: 857-861, 1979
Herbicidins
C and E, two new