19591

CHLORINATION INHIBITORS IN BIOSYNTHESIS

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Chlorination Inhibitors Affecting the Biosynthesis

of Tetracycline
J. LEIN, L. F. SAWMILLER, AND L. C. CHENEY
Research Division, Bristol Laboratories, Inc., Syracuse, New Yor1k

Received for publication September 29, 1958

In a previous publication (Gourevitch et al., 1955),

a scheme was proposed postulating that the difference
in the biosynthesis of chlortetracycline and tetracycline resided in the chlorination of a common precursor for the two antibiotics. The amount of synthesis
of either compound depends upon the rate at which
the precursor is chlorinated. Bromide was shown to
inhibit competitively the chlorination reaction but
led to the biosynthesis of some bromtetracycline.
Other workers reported similar phenomena (Sensi
et al., 1955; Arishima et al., 1956; Doerschuk et al.,

1956).
An alternative method of inhibiting chlorination
was reported by Sekizawa (1955). He found that certain organic compounds had the property of shifting
synthesis from chlortetracycline to tetracycline. The
most active compound reported was 2-mercaptobenzothiazole. It was most effective at levels which were
somewhat toxic since the highest percentages of tetracycline were obtained with amounts that inhibited
the over-all synthesis of the antibiotics. The problem
was further examined in this laboratory and a variety
of compounds tested to determine if any structure-

activity relationship could be determined.

MATERIALS AND METHODS

The organism used for the fermentation work was a
mutated strain derived from a culture of Streptomyces
autreofaciens isolated from soil.
Fermentations were carried out in the following
medium: sucrose, 7.0 per cent; (NH4) 2SO4, 0.45 per
cent; CaCO3, 0.6 per cent; Pharmamedia,1 2.0 per
cent; dechlorinated corn steep (Hatch et al., 1956),
1.5 per cent (50 per cent solids); ZnSO4, 0.003 per
cent; NaCl, 0.4 per cent.The amount of chloride added
was sufficient to enable the strain under control conditions to produce preferentially high percentages of
chlortetracycline.
Fermentations were done in 125-ml Erlenmeyer
flasks on a rotary shaker ascribing a 5 cm circle at
200 rpm. Temperature was maintained at 27 C. Flasks
were sampled after 7 days for assay. The quantitative
assays were carried out as follows.
Samples were brought to pH 1.8 by addition of concentrated phosphoric acid. After centrifuging, the
sample was placed in duplicate test tubes. To one was
added pH 2.0 buffer (Pharmacopeia of the United
States, 1955) and to the other, 2 N HCl. The acid'

Proflow Division, Traders

Texas.

Oil Company, Fort Worth,

J. LEIN, L. F. SAWMILLER, AND L. C. CHENEY

150

treated samples were placed in a 70 C water bath for

15 min, removed, and cooled in ice water immediately.
A spectrophotometer was used at the 355 m,u wave
lenigth to determine absorption differences in the
samples. The spectrophotometer was adjusted to zero
with the acid blank and a direct reading was obtained
from the buffer blank. Referral to a standard curve
obtained with different levels of tetracycline yielded
potency in tetracycline equivalents. One thousand jig
per ml of chlortetracycline under these conditions
assays as 532.2 ,ug per ml of tetracycline equivalents.
The percentage of tetracycline was determined by
acidifying broths to pH 2 with hydrochloric acid and
carrying out a quantitative paper chromatographic
assay on the supernatant. The solvent used for chromatography was a water-butanol system. Comparison
of zone sizes with those of tetracycline and chlortetracycline standards permitted estimates of percentage
of each antibiotic.
RESULTS
Fermentations were carried out using a variety of
compounds to determine which types were active in
inhibiting chlorination. These compounds are listed
in table 1 and are divided into four groups. One group
was deemed inactive, the second slightly active, a
third moderately active, and a fourth very active. The
results given were for the levels showing no or only
moderate decrease in over-all antibiotic production.
The control level with no added chlorination inhibitor
was 3500 ,ug per ml with the percenitage of tetracycline
being less than 40 per cent.
The compounds in group 1, all except no. 4, were
very toxic at levels above 0.002 per cent. The lack of
activity of these compounds might be attributable to
the fact that they could not be tested at higher concentrations due to their toxicity.
DISCUSSION
From this study, it appears that a wide variety of
materials have the property of inhibiting chlorination to varying degrees. There seem to be certain
relationships between activity and structure. The
moderately active and very active compounds in this
series all have in common the grouping (I) or tautomer (II) in a cyclic system wherein X may represent
a nitrogen, oxygen, or sulfur atom.
H
r

- - -

-N\\

_ _-

- -

-N\

C-SH

TABLE 1
Effect of variouts compounds on the relative production of
tetracycline and chlorination
Leviel

Compound

Tetracycline

lents)

Inactive
1.

Compouinds

HS-CH2-CO2H

lAg/ml

%70

0.002

1575

< 50

0.002

1805

< 50

0.002

2165

<50

0.004

1475

<50

0.002

1650

< 50

0.002

1450

<50

0.006

1415

55-60

0.005

2145

50

Thioglycollic acid
(mercaptoacetic acid)
2.

CH:,- (CH2)1o-CH2-SH
1 -Dodecanethiol

3.

H,N-C-NH-C-NH2
2, 4-Dithiobillret,

4.

H2N-C-NH-NH2
Thiosemicarhazide
NaSCN

5.

Thiocyanic acid

(sodiuim

salt)

6.

NH-C

CH3-C

NH

CH-C
(HC)
6-Methyl -2-thiomracil

Slightl.y Active
NH2

7.

SH
2-Aminobenzenet,hiol
SH

N=C

C=--S

CH3-C

II

It is perhaps noteworthy that the highly active

thiadiazole (no. 13) has structural features resembling
a condensation product derived from the activitypromoting group (I).

Total Activity
(Tetracycline

Equiva-

8.

-x/

[VOL. 7

CH-C-CH3
CH3

2-Mercapt,o-4, 6, 6-trimet hyl1, 3, 6-thiazine

CHLORINATION INHIBITORS IN BIOSYNTHESIS

19591

TABLE 1-Continued
Level

Compound

9.

H2C-N
J C-SH

H2b

Total
Activity
(Tetracycline
Equivalents)

Tetracycline

0.003

Ug1/ml
3070

%
65

0.005

2935

50-55

ACKNOWLEDGMENTS

2-Mercapto-2-thiazoline
Moderately Active

l0.

The study described above gives no definite clue

as to the mechanism by which these compounds affect
the biosynthesis. The presence of the sulfhydryl group
in I raises the possibility of participation in an oxidation-reduction type of reaction. Since the chloride
ion must be oxidized before it can be incorporated
into chlortetracycline, these compounds may work by
interfering with this process.

H3C-C-N
H3C-I-S

0.003
0.005

2470
2030

65
80-85

The authors wish to thank Mr. D. L. Johnson for

carrying out the paper chromatographic studies and
Mrs. Elinor Hillers for her assistance in carrying out
the fermentation studies.

SUMMARY
2-Mercapto-4,5-dimethylthiazole
N

11.

0.003
C-SH

3888
2160
2805

50-55
65
80

0.004
0.006

2600
3380

93-95

0.004

2360

90-95

0.004

0.006

2-Mercaptobenzothiazole

Very Active
N

12.

C-SH

90

0
2-Benzoxazolethiol
13.

Hs_t

t-SH
N

REFERENCES
ARISHMA, M., SEKIZAWA, Y., SAKAMATO, J. M., MIWA, K.,
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J. Agr. Chem. Soc., Japan, 30, 407-409.
DOERSCHUK, A. P., MCCORMICK, J. R. D., GOODMAN, J. J.,
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1956 The halide metabolism of Streptomyces aureofaciens
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2,5-Dimercapto-1,3,4-thiadiazole
14.

A variety of compounds were studied to determine

their effect on the biosynthesis of tetracycline by
Streptomyces aureofaciens in the presence of chloride.
A number of compounds were found to inhibit chlorination, increasing markedly the ratio of tetracycline
to chlortetracycline formed. These compounds had
certain structural features in common and the relationship between structure and function was pointed
out.

0.006

1510

90-95

C-SH
NH

2-Mereaptobenzimidazole

Differences in structure at sites other than the activity-promoting group can affect the over-all activity. Comparison of compound 9 with 10 and 11
with 14 demonstrates influences of relatively small
differences.

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