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

Herbicidins F and G, two new nucleoside antibiotics.

862 THE HERBICIDINS JOURNAL F AND G, Yo TAKIGUCHI, OF TWO NEW Research (Received A Mutant of Streptomyces Tokyo, for publication sagan...
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