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A new metabolite of Streptomyces venezuelae: d -Threo-1-p-aminophenyl-2-dichloroacetamido-1,3-propanediol
ARCHIVES
OF
A New
BIOCHEMISTRY
AND
Meta bolite
BIOPHYSICS
159-163 (1963)
103,
of Streptomyces
veneruelae:
D-Three-l-p-Amino-
phenyl-2-Dichloroacetamido-1,3-Propanediol CHARLOTTE Prom
D. STRATTON
the Research Laboratories
AND
of Parke,
Received
MILDRED Davis
C. REBSTOCK
and Co., Ann 4rbor,
Michigan
July 8, 1963
Traces of aromatic nitro and amino compounds are formed in addition to chloramvenezzrelae phenicoll under normal conditions of antibiotic synthesis by Streptomvces in shake flasks or in 2Wgallon fermentors. The N-acetyl, N-propionyl, and N-butyrvl amides of p-nitrophenylserinol were identified by paper chromatography using colorimetric and microbiological methods of detection. n-Three-1-p-aminophenyl-2.dichloroacetamide-1,3-propanediol was isolated from fermentation tank beer. INTRODUCTION
Several approaches have been utilized to obtain information about the biosynthesis of chloramphenicol and the metabolism of the strains of Streptomyces producing it. Early efforts were directed toward finding conditions of fermentation conducive to the optimum production of the antibiotic (l-6). Some facts are known about the biosynthetic process from precursor studies utilizing labelled as well as nonradioactive materials, but the precise intermediates involved and the synthetic sequence are far from clear (7-11). Pertinent studies of C. G. Smith (12) have shown that Streptomyces SP. 3022, a chloramphenicol-producing strain, also produces N-acetyl, N-propionyl, and N-butyryl amides of p-nitrophenylserinol in addition to limited amounts of the above antibiotic when fermentation is carried out in media deficient in chloride ion. Dibromoacetyl and bromochloroacetyl derivatives were obtained as well as the above compounds when bromide ion was present at concentrations competitive with chloride ion. The present report deals with some results obtained in a study of the nature of the metabolites of Xtreptomyces venexuelae pro1 Chloramphenicol is the generic name of the antibiotic for which the trademark of Parke, Davis and Co. is Chloromycetin.
duced during “normal” antibiotic synthesis and concerns the formation of traces of aromatic nitro and aromatic amino compounds related to the antibiotic. The evidence of paper chromatographic studies suggests that certain of these compounds are the N-acetyl, N-propionyl, and probably N-butyryl amides of p-nitrophenylserinol which are apparently formed under “normal” conditions of fermentation, but at much lower levels than Smith found when the organism was deprived of chloride. D - Threo - I- p - aminophenyf - 2 - dichloroacetamido-1,3-propanediol was also isolated. The occurrence of the latter compound as a fermentation product of S. venezuelae has not been noted before although its synthesis was described some time ago (13, 14). The paper chromatographic studies reported by G. N. Smith and Worrel (15) suggest that the above compound may be a breakdown product of chloramphenicol when large numbers of cells of BaciZZus subtilis, B. mycoides, Eschekchia coli, or Proteus vulgaris are incubated with the antibiotic. MATERIALS
AND
METHODS
Fermentations were carried out in 50 ml. shake flasks2 and in a 2Wgallon fermenter% using the P.D. 04745 strain of Streptomyces uenezuelae as 2 We gratefully acknowledge our indebtedness to Dr. John Douros and Miss Georgia Senos and
159
STRATTON
160
AND
the inoculum. The medium consisted of lo/c glycerol, 1.75ojc special debittered yeast, 0.3yc B-Y fermentat,ion solubles % 250,0.57, sodium chloride, and 0.1% calcium carbonate, Shake flasks containing 55 ml. of medium were adjusted to pH 7.5, inoculated with 1 ml. of cell suspension, and agitated at 250 RPM (28°C.). After 24 hr. the first flask was harvested. Individual flasks were then harvested at 8.hr. intervals. When the fermentation was carried out in the 209gallon tank fermenter, sampling was started after 8 hr. and 500ml. aliquots were withdrawn at each 8-hr. interval thereafter until the conclusion of the experiment. The pH of all samples was noted and each was assayed against S. sonnei by the method of Joslyn and Galbraith (16). The formation of the antibiotic followed the pattern described in previous publications. During the early part of the fermentation, in both types of equipment the concentration increased rapidly, then levelled off and remained at about the same concentration during the remainder of the experiment. The maximum concentration of chloramphenicol in the shake flasks was 329 y per ml.; in the fermentation tank, 233 y per ml. The solids were removed by centrifugation and the clarified beers were chromatographed on paper strips using descending techniques and solvent systems known to separate many of the synthetic analogs of chloramphenicol having para nitrophenyl groups. Strips were run in duplicate or triplicate and examined by bioautography using plates seeded with E. coli, and/or B. subtilis and by the calorimetric method of Glaeko et al. (17). In certain cases the developed strip was cut lengt,hwise and half strips were examined by microbiological and chemical methods. EXAMINATION
OF BEERS
When strips spotted with concentrated beer centrifugates or filtrates were developed in most solvent systems, chloramphenicol appeared to be the only aromatic nitro compound present, When strips impregnated with glycerol3 were used and developed with an amyl acetate system, the presence of a trace of another aromatic nitro compound was noted. co-workers for carrying out the shake-flask fermentations and supplying materials for these studies, and to Dr. J. D. Howells and Mr. Wade Mumma for the 200-gallon fermenter preparations. 3 Whatman No. 1 strips were dipped in a mixture of 10% glycerol and 90% absol. methanol, blotted by laying on a sheet of Whatman paper, and then air dried.
REBSTOCH EXTRACTS
OF ACIDIFIED
BEERS
To facilitate the detection of trace amounts of aromatic nitro or amino compounds, ethyl acetate extracts were prepared for concentration. Since the volumes were small, the contents of 24-, 32-, and 40-hr. shake flasks were pooled for the extractions and consecutive shake-flask beers in groups of three were combined until the end of the series was reached. The fermentation tank aliquots were extracted separately. Samples were first extracted at pH 3.0 to transfer chloramphenicol and other neutral or acidic compounds which were soluble in ethyl acetate to the solvent phase. Concentrates were chromatographed on Whatman Ko. 1 strips in the following solvent systems: (a) ethylene dichloride saturated with water (developed 26 hr. or longer at 4’C.) ; (b) amyl acetate saturated with water (strips impregnated with glyceroP) ; (c) benzenemethanol-water, 1: 1: 2 (developed 48 hr.) ; (d) methyl isobutyl ketone saturated with water. The listed solvent systems are useful for separating chloramphenicol and simple acyl amides of p-nitrophenylserinol. Control strips spotted with synthetic samples of N-acetyl, N-propionyl, N-n-butyryl, Nisobutyryl, and N-monochloroacetyl amides were chromatographed simultaneously. Chloramphenicol travels faster than the above compounds in these systems. This consideration is important when strips are bioautographed since products which are present at markedly lower concentration than the antibiotic and which have low biological activity cannot be detected unless they are well separated from the antibiotic. Rf values are listed for the above compounds when systems (b) and (d) are used. In systems (a) and (c) the developing solvent was allowed to run off the strips and values calculated with reference to the position of chloramphenicol (see Table I). The developed strips were cut in half lengthwise and one part was examined calorimetrically and the other by standard bioautographic techniques. The faint zones of inhibition noted on E. coli or B. mbtilis plates coincided with red-purple zones characteristic of aromatic nitro compounds on the chemically treated strips. The zones were located
A NEW METABOLITE
OF STREPTOMYCES
in regions occupied on control strips by synthetic samples of N-acetyl, N-propionyl, and N-butyryl amides of nc-threo-p-nitrophenylserinol. The latter compounds have activity of 14, 13.4, and 15.0% of chloramphenicol when assayed against S. sonnei by the standard turbidimetric technique (16). Although the compounds in question were not, isolated as crystalline entities, their chromatographic identification is validated by the fact that of the many nitrophenyl analogs of chloramphenicol which have been chromatographed in these systems, the above compounds are the only ones with appreciable microbiological activity which occupy the positions in question in solvent systems listed above. The identity of the butyramide is in question because the “normal” isomer cannot be separated from the “iso” analog under these conditions. Monochloroacetamide can be detected using solvent system (a) which separates it from the butyramides. There was no evidence for the presence of the former compound. The above amides appeared at, the same time as chloramphenicol during the course of the fermentation. Their concentrations increased during the period of increasing antibiotic production and levelled off when chloramphenicol concentration plateaued. However, under the above fermentation conditions these compounds were only minor metabolites of S. venexuelae when compared to chloramphenicol and could be demonstrated only if concentrated solvent extracts were chromatographed. EXTRACTS
OF ALKALINE
BEERS
In order to examine the fermentation products of S. venezuelae for the presence of basic aromatic nitro or amino compounds the acidic aqueous residues from the ethyl acetate extractions were adjusted to pH 9.0 and the ethyl acetate extraction was repeated. The concentrated ethyl acetate extracts were then chromatographed in the solvent systems: (e) butanol saturated with 3 % aqueous ammonia and (f) butanol saturated with 3% aqueous acetic acid. Duplicate strips were run or single developed strips were cut in two as described above. One strip or half strip was treated by the
VENEZUELAE
161
TABLE I OF N-ACYI,
CHROMATOGRAPHY DERIVATIVES OF P-NITROPHENYLSERINOL Rj values Amide Sys5m
Dichloroacetyl Monochloroacetyl Acetyl Propionyl n-Butyryl Isobutyryl
0.88 0.65 0.16 0.38 0.61 0.64
Sygm
0.86 0.79 0.47 0.69 0.82 0.82
l&n values Sy;t;m a
1.0 0.62 0.08 0.24 0.48 -
Sy;t;m c
1.0 0.54 0.07 0.26 0.64 O.G9
calorimetric procedure of Glazko et al. (17) as usual. The second strip was treated the same except that the initial reduction step which converts nitro to amino was omitted. Aromatic nitro and amino compounds are readily distinguished since only the amino compounds can give a positive reaction. Three aromatic amino compounds appeared at various stages of the fermentation when the pH 9 extracts of the fermentation tank were analyzed. Two of these occurred early and had Rf's of 0.68 and 0.80 when strips containing samples were chromatographed in the butanol, acetic acid system. The fast moving compound disappeared after 80 hr. and the other was no longer detectable after 104 hr. A third compound having Rf 0.55 appeared in the 56-hr. sample and gradually accumulated during the remainder of the fermentation period. It, was isolated from the harvested beer and identified as D-threop - aminophenyl - 2 - dichloroacetamido-I ,3propanediol first by chromatography, then by preparation of a crystalline derivative. ISOLATION OF l-p-AMINOPHENYL-2DICHLOROACETAMIDO-1,3PROPANEDIOL
The beer (122 gal.) obtained on harvesting the 200-gallon fermenter was adjusted to pH 3.0 and extracted with ethyl acetate.4 The aqueous phase was adjusted to pH 9.0 and
re-extracted.
The
latter
extract
was
evaporated and 33.6 g. of dark tarry product 4 We are much indebted to Dr. S. A. Fusari and Mr. Neil Willmer for making the large scale extractions at pH 3 and 9 and concentrating these to convenient volumes for our studies.
162
STRATTON TABLE
AND
II
PAPER CHROMATOGRAPHK COMPARISON OF DTHXEO -1-p - AMINOPHENYL 2 - UICHLORUACETAMIDO-l,$PROPANEDIOJ> AlVD pH 9.0 EXTRACTED MATERIAL Rf values Solvent system
Acetone-acetic acid-water (50:3:47) Benzene-met)hanol-water (1:1:2) Benzene sat’d. with water Water sat’d. with benzene Ethyl acetate sat’d. with water Water sat’d. with ethyl acetate Methyl isobutyl ketone sat’d. with water Butanol sat’d. with 3yc acetic acid
pH 9 extract
Aminwhwl compound
0.78
0.77
0.02
0.01
0.00 0.76 0.53
0.00 0.77 0.49
0.80
0.79
0.58
0.57
0.53
0.53
was obtained. Trituration with n-heptane reduced the weight to 20 g. Appreciable quantities of chloramphenicol were found to be present and it was obvious that the large scale pH 3.0 extraction was not complete. The residue was reconstituted in water at pH 3 and extracted with ethyl acetate; then readjusted to pH 9 and again extracted. The evaporated basic extract now yielded 1.35 g. of product showing absorption in the ultraviolet characteristic of aromatic amines. The maximum at X241, E:, 307, in ethanolic NaOH, shifted to below X220 on acidification. The similarity of the absorption spectrum of this material to that of l-p-aminophenyl-:!-dichloroacetamido-1,3-propanediol (h,,,. 236, E! , 370, in aqueous NaOH) was noted. A paper chromatographic comparison of the two materials in a variety of solvent systems showed the amino aromatic compounds had identical Rf’s and were chromatographically indistinguishable (see Table II). D-ThIY?O - 1 - p - aminophenyl - 2 - dichloroacetamido-1 ,3-propanediol is difficult to purify in the free base form or as the hydrochloride salt when contaminated with other organic products, but forms crystalline Schiff bases with benzaldehyde or p-nitro-
REBSTOCK
benzaldehyde which are more easily handled. The latter derivative was prepared by treating 65 mg. of the pH 9 extracted amine, dissolved in 2 ml. of absolute methanol, with 30 mg. p-nitrobenzaldehyde. The product was recrystallized for analysis from aqueous ethanol. There was no melting point depression with an authentic sample of the Schiff base of Dp-nitrobenzaldehyde three - 1 - p - aminophenyl - 2 dichloro acetamido-1,3-propanediol (m.p., 171-172” C.), and the I.R. spectra were identical. Found: C, 50.54, 50.77; H, 4.13, 4.14; K, 9.87. Required: C, 50.71; H, 4.02; N, 9.86. The above aminophenyl chloramphenicol related product was found in the fermentation tank beer but not in shake-flask fermentations. Its appearance late in the fermentation was noted above. EVIDENCE
FOR
OTHER
METABOLITES
Trace amounts of other aromatic nitro and amino compounds are also formed. The aryl amine products appeared mainly when the fermentation was carried out in the 200-gallon fermenter. At least two acidic aromatic nitro acids occur in shake flask beers (Rf’s 0.55 and 0.60; system, butanol sat’d. with 3% aqueous ammonia). The slower moving zone may be p-nitrobenzoic acid. Gottlieb et al. (8) reported chromatographic evidence for the formation of this compound as a C&labelled metabolite when X. venezuelae was grown in the presence of C&p-nitrophenylserinol. The same group noted that none of the above compound was metabolized to chloramphenicol but labelled N-acetyl p-nitrophenylserinol was produced. These results suggest that traces of p-nitrophenylserinol formed as a side reaction product or as a breakdown product of the antibiotic may be serving as a precursor in the formation of the unhalogenated amides. If this be the case, any p-nitrophenylserinol which is formed must be metabolized immediately since no evidence for its presence in beer samples could be found at any stage of the fermentation. In the pH 3 extracts of the fermentation tank aliquots the presence of two aromatic amines was noted. Because of their peculiar extraction patterns, these compounds are
A NEW
METABOLITE
OF STREPTOMYCES
probably amphoteric in nature. When the above extracts were chromatographed in a variety of solvent systems, the amines could not be distinguished from p-aminobenzoic acid and anthranilic acid. The latter compounds are well-known microbial metabolites and their occurrence in preparations of this kind would not be unexpected. ACKNOWLETlGMENT We gratefully acknowledge the contributions of the following individuals to these studies: Dr. John Ehrlich for his support and encouragement, Dr. Robert Hans and Mrs. Margaret Galbraith for microbiological assays, Dr. John R. Douros and Miss Georgia Senos for shake-flask preparations, Dr. J. D. Howells and Mr. Wade Mumma for fermentation-tank preparations, and Dr. S. A. Fusari and Mr. Neil Willmer for the large scale extractions and concentrations of fermentation tank beer. REFERENCES 1. EHRLICH, J., GOTTLIEB, D., BURKHOLDER, P. R., ANDERSON, L. E., AND PRIDHAM, T. Cr., J. Bacterial. 66, 467 (1948). 2. SMITH, R. M., JOSLYN, D. A., GRUHZIT, 0. M., MCLEAN, I. W., JR., PENNER, M. A., AND EHRLICH, J., J. Bacterial. 66, 425 (1948). 3. Ovaas, .J. E., EHRLICH, J., .~ND SMITH, R. M., Znd. Eng. Chem,. 42, 1775 (1950).
VENEZUELAE
163
4. LEGATOR, M., AND GOTTLIEB, D., Antibiot. Chemotherapy 3, 809 (1953). 5. MATSUOKA, M., YAGISHITA, Ii., AND UMEZAWA, H., Japan. J. Med. Sci. Biol. 6, 161 (1953). 6. GALLICCHIO, v., BND GOTTLIEB, D., Mycologia 60, 490 (1958). 7. GOTTLIEB, D., CARTER, H. E., LEGATOR, M., AND GALLICCHIO, I-., J. Bacterial. 68, 243 (1954). 8. GOTTLIEB, D., ROBBINS, P. W., AND CARTER, H. E., J. Bacterial. 72, 153 (1956). 9. GOTTLIEB, II., CARTER, H. E., ROBBINS, P. W., AND BURG, R. W., J. Bacterial. 84, 888 (1962). 10. Y.~GISHITA, K., AND UMEZAW~, H., Japan. J. Med. Sci. Biol. 3, 289 (1950). 11. WANG, E. L., IzAw.~, M., MIURA, T., AND UMEZAWA, H., J. Antibiotics (Tokyo) Ser. A. 12, 81 (1959). 12. SMITH, C. G., J. Bacterial. 76, 577 (1958). 13. SGLLIVAN, M. J., Parke, Davis and Co. U.S. Patent 2,568,571, Sept. 18, 1951. 14. SHEMYAKIN, E. M., BAMDAS, E. I., J7INOGRADOVA, M. G., KARAPETYAN, M. G., KOLOSOV, M. N., KHOKHLOV, M. G., SHVETSOV, Yu. B., AND SHCHUKINA, L. R., Dokl. Akad. Nauk. U.S.S.R. 86, 565 (1952). 16. SMITH, G. N., AND WORREL, C. S., Arch. Biochem. Biophys. 28, 232 (1950). 16. JOSLYN, D. A., AND GALBRAITH, M., 3. Bacteriol. 69, 711 (1950). 17. GL~ZKO, A. J., WOLF, L. M., AND DILL, w. A., Arch. Biochem. Biophys. 23, 411 (1949).
OF
A New
BIOCHEMISTRY
AND
Meta bolite
BIOPHYSICS
159-163 (1963)
103,
of Streptomyces
veneruelae:
D-Three-l-p-Amino-
phenyl-2-Dichloroacetamido-1,3-Propanediol CHARLOTTE Prom
D. STRATTON
the Research Laboratories
AND
of Parke,
Received
MILDRED Davis
C. REBSTOCK
and Co., Ann 4rbor,
Michigan
July 8, 1963
Traces of aromatic nitro and amino compounds are formed in addition to chloramvenezzrelae phenicoll under normal conditions of antibiotic synthesis by Streptomvces in shake flasks or in 2Wgallon fermentors. The N-acetyl, N-propionyl, and N-butyrvl amides of p-nitrophenylserinol were identified by paper chromatography using colorimetric and microbiological methods of detection. n-Three-1-p-aminophenyl-2.dichloroacetamide-1,3-propanediol was isolated from fermentation tank beer. INTRODUCTION
Several approaches have been utilized to obtain information about the biosynthesis of chloramphenicol and the metabolism of the strains of Streptomyces producing it. Early efforts were directed toward finding conditions of fermentation conducive to the optimum production of the antibiotic (l-6). Some facts are known about the biosynthetic process from precursor studies utilizing labelled as well as nonradioactive materials, but the precise intermediates involved and the synthetic sequence are far from clear (7-11). Pertinent studies of C. G. Smith (12) have shown that Streptomyces SP. 3022, a chloramphenicol-producing strain, also produces N-acetyl, N-propionyl, and N-butyryl amides of p-nitrophenylserinol in addition to limited amounts of the above antibiotic when fermentation is carried out in media deficient in chloride ion. Dibromoacetyl and bromochloroacetyl derivatives were obtained as well as the above compounds when bromide ion was present at concentrations competitive with chloride ion. The present report deals with some results obtained in a study of the nature of the metabolites of Xtreptomyces venexuelae pro1 Chloramphenicol is the generic name of the antibiotic for which the trademark of Parke, Davis and Co. is Chloromycetin.
duced during “normal” antibiotic synthesis and concerns the formation of traces of aromatic nitro and aromatic amino compounds related to the antibiotic. The evidence of paper chromatographic studies suggests that certain of these compounds are the N-acetyl, N-propionyl, and probably N-butyryl amides of p-nitrophenylserinol which are apparently formed under “normal” conditions of fermentation, but at much lower levels than Smith found when the organism was deprived of chloride. D - Threo - I- p - aminophenyf - 2 - dichloroacetamido-1,3-propanediol was also isolated. The occurrence of the latter compound as a fermentation product of S. venezuelae has not been noted before although its synthesis was described some time ago (13, 14). The paper chromatographic studies reported by G. N. Smith and Worrel (15) suggest that the above compound may be a breakdown product of chloramphenicol when large numbers of cells of BaciZZus subtilis, B. mycoides, Eschekchia coli, or Proteus vulgaris are incubated with the antibiotic. MATERIALS
AND
METHODS
Fermentations were carried out in 50 ml. shake flasks2 and in a 2Wgallon fermenter% using the P.D. 04745 strain of Streptomyces uenezuelae as 2 We gratefully acknowledge our indebtedness to Dr. John Douros and Miss Georgia Senos and
159
STRATTON
160
AND
the inoculum. The medium consisted of lo/c glycerol, 1.75ojc special debittered yeast, 0.3yc B-Y fermentat,ion solubles % 250,0.57, sodium chloride, and 0.1% calcium carbonate, Shake flasks containing 55 ml. of medium were adjusted to pH 7.5, inoculated with 1 ml. of cell suspension, and agitated at 250 RPM (28°C.). After 24 hr. the first flask was harvested. Individual flasks were then harvested at 8.hr. intervals. When the fermentation was carried out in the 209gallon tank fermenter, sampling was started after 8 hr. and 500ml. aliquots were withdrawn at each 8-hr. interval thereafter until the conclusion of the experiment. The pH of all samples was noted and each was assayed against S. sonnei by the method of Joslyn and Galbraith (16). The formation of the antibiotic followed the pattern described in previous publications. During the early part of the fermentation, in both types of equipment the concentration increased rapidly, then levelled off and remained at about the same concentration during the remainder of the experiment. The maximum concentration of chloramphenicol in the shake flasks was 329 y per ml.; in the fermentation tank, 233 y per ml. The solids were removed by centrifugation and the clarified beers were chromatographed on paper strips using descending techniques and solvent systems known to separate many of the synthetic analogs of chloramphenicol having para nitrophenyl groups. Strips were run in duplicate or triplicate and examined by bioautography using plates seeded with E. coli, and/or B. subtilis and by the calorimetric method of Glaeko et al. (17). In certain cases the developed strip was cut lengt,hwise and half strips were examined by microbiological and chemical methods. EXAMINATION
OF BEERS
When strips spotted with concentrated beer centrifugates or filtrates were developed in most solvent systems, chloramphenicol appeared to be the only aromatic nitro compound present, When strips impregnated with glycerol3 were used and developed with an amyl acetate system, the presence of a trace of another aromatic nitro compound was noted. co-workers for carrying out the shake-flask fermentations and supplying materials for these studies, and to Dr. J. D. Howells and Mr. Wade Mumma for the 200-gallon fermenter preparations. 3 Whatman No. 1 strips were dipped in a mixture of 10% glycerol and 90% absol. methanol, blotted by laying on a sheet of Whatman paper, and then air dried.
REBSTOCH EXTRACTS
OF ACIDIFIED
BEERS
To facilitate the detection of trace amounts of aromatic nitro or amino compounds, ethyl acetate extracts were prepared for concentration. Since the volumes were small, the contents of 24-, 32-, and 40-hr. shake flasks were pooled for the extractions and consecutive shake-flask beers in groups of three were combined until the end of the series was reached. The fermentation tank aliquots were extracted separately. Samples were first extracted at pH 3.0 to transfer chloramphenicol and other neutral or acidic compounds which were soluble in ethyl acetate to the solvent phase. Concentrates were chromatographed on Whatman Ko. 1 strips in the following solvent systems: (a) ethylene dichloride saturated with water (developed 26 hr. or longer at 4’C.) ; (b) amyl acetate saturated with water (strips impregnated with glyceroP) ; (c) benzenemethanol-water, 1: 1: 2 (developed 48 hr.) ; (d) methyl isobutyl ketone saturated with water. The listed solvent systems are useful for separating chloramphenicol and simple acyl amides of p-nitrophenylserinol. Control strips spotted with synthetic samples of N-acetyl, N-propionyl, N-n-butyryl, Nisobutyryl, and N-monochloroacetyl amides were chromatographed simultaneously. Chloramphenicol travels faster than the above compounds in these systems. This consideration is important when strips are bioautographed since products which are present at markedly lower concentration than the antibiotic and which have low biological activity cannot be detected unless they are well separated from the antibiotic. Rf values are listed for the above compounds when systems (b) and (d) are used. In systems (a) and (c) the developing solvent was allowed to run off the strips and values calculated with reference to the position of chloramphenicol (see Table I). The developed strips were cut in half lengthwise and one part was examined calorimetrically and the other by standard bioautographic techniques. The faint zones of inhibition noted on E. coli or B. mbtilis plates coincided with red-purple zones characteristic of aromatic nitro compounds on the chemically treated strips. The zones were located
A NEW METABOLITE
OF STREPTOMYCES
in regions occupied on control strips by synthetic samples of N-acetyl, N-propionyl, and N-butyryl amides of nc-threo-p-nitrophenylserinol. The latter compounds have activity of 14, 13.4, and 15.0% of chloramphenicol when assayed against S. sonnei by the standard turbidimetric technique (16). Although the compounds in question were not, isolated as crystalline entities, their chromatographic identification is validated by the fact that of the many nitrophenyl analogs of chloramphenicol which have been chromatographed in these systems, the above compounds are the only ones with appreciable microbiological activity which occupy the positions in question in solvent systems listed above. The identity of the butyramide is in question because the “normal” isomer cannot be separated from the “iso” analog under these conditions. Monochloroacetamide can be detected using solvent system (a) which separates it from the butyramides. There was no evidence for the presence of the former compound. The above amides appeared at, the same time as chloramphenicol during the course of the fermentation. Their concentrations increased during the period of increasing antibiotic production and levelled off when chloramphenicol concentration plateaued. However, under the above fermentation conditions these compounds were only minor metabolites of S. venexuelae when compared to chloramphenicol and could be demonstrated only if concentrated solvent extracts were chromatographed. EXTRACTS
OF ALKALINE
BEERS
In order to examine the fermentation products of S. venezuelae for the presence of basic aromatic nitro or amino compounds the acidic aqueous residues from the ethyl acetate extractions were adjusted to pH 9.0 and the ethyl acetate extraction was repeated. The concentrated ethyl acetate extracts were then chromatographed in the solvent systems: (e) butanol saturated with 3 % aqueous ammonia and (f) butanol saturated with 3% aqueous acetic acid. Duplicate strips were run or single developed strips were cut in two as described above. One strip or half strip was treated by the
VENEZUELAE
161
TABLE I OF N-ACYI,
CHROMATOGRAPHY DERIVATIVES OF P-NITROPHENYLSERINOL Rj values Amide Sys5m
Dichloroacetyl Monochloroacetyl Acetyl Propionyl n-Butyryl Isobutyryl
0.88 0.65 0.16 0.38 0.61 0.64
Sygm
0.86 0.79 0.47 0.69 0.82 0.82
l&n values Sy;t;m a
1.0 0.62 0.08 0.24 0.48 -
Sy;t;m c
1.0 0.54 0.07 0.26 0.64 O.G9
calorimetric procedure of Glazko et al. (17) as usual. The second strip was treated the same except that the initial reduction step which converts nitro to amino was omitted. Aromatic nitro and amino compounds are readily distinguished since only the amino compounds can give a positive reaction. Three aromatic amino compounds appeared at various stages of the fermentation when the pH 9 extracts of the fermentation tank were analyzed. Two of these occurred early and had Rf's of 0.68 and 0.80 when strips containing samples were chromatographed in the butanol, acetic acid system. The fast moving compound disappeared after 80 hr. and the other was no longer detectable after 104 hr. A third compound having Rf 0.55 appeared in the 56-hr. sample and gradually accumulated during the remainder of the fermentation period. It, was isolated from the harvested beer and identified as D-threop - aminophenyl - 2 - dichloroacetamido-I ,3propanediol first by chromatography, then by preparation of a crystalline derivative. ISOLATION OF l-p-AMINOPHENYL-2DICHLOROACETAMIDO-1,3PROPANEDIOL
The beer (122 gal.) obtained on harvesting the 200-gallon fermenter was adjusted to pH 3.0 and extracted with ethyl acetate.4 The aqueous phase was adjusted to pH 9.0 and
re-extracted.
The
latter
extract
was
evaporated and 33.6 g. of dark tarry product 4 We are much indebted to Dr. S. A. Fusari and Mr. Neil Willmer for making the large scale extractions at pH 3 and 9 and concentrating these to convenient volumes for our studies.
162
STRATTON TABLE
AND
II
PAPER CHROMATOGRAPHK COMPARISON OF DTHXEO -1-p - AMINOPHENYL 2 - UICHLORUACETAMIDO-l,$PROPANEDIOJ> AlVD pH 9.0 EXTRACTED MATERIAL Rf values Solvent system
Acetone-acetic acid-water (50:3:47) Benzene-met)hanol-water (1:1:2) Benzene sat’d. with water Water sat’d. with benzene Ethyl acetate sat’d. with water Water sat’d. with ethyl acetate Methyl isobutyl ketone sat’d. with water Butanol sat’d. with 3yc acetic acid
pH 9 extract
Aminwhwl compound
0.78
0.77
0.02
0.01
0.00 0.76 0.53
0.00 0.77 0.49
0.80
0.79
0.58
0.57
0.53
0.53
was obtained. Trituration with n-heptane reduced the weight to 20 g. Appreciable quantities of chloramphenicol were found to be present and it was obvious that the large scale pH 3.0 extraction was not complete. The residue was reconstituted in water at pH 3 and extracted with ethyl acetate; then readjusted to pH 9 and again extracted. The evaporated basic extract now yielded 1.35 g. of product showing absorption in the ultraviolet characteristic of aromatic amines. The maximum at X241, E:, 307, in ethanolic NaOH, shifted to below X220 on acidification. The similarity of the absorption spectrum of this material to that of l-p-aminophenyl-:!-dichloroacetamido-1,3-propanediol (h,,,. 236, E! , 370, in aqueous NaOH) was noted. A paper chromatographic comparison of the two materials in a variety of solvent systems showed the amino aromatic compounds had identical Rf’s and were chromatographically indistinguishable (see Table II). D-ThIY?O - 1 - p - aminophenyl - 2 - dichloroacetamido-1 ,3-propanediol is difficult to purify in the free base form or as the hydrochloride salt when contaminated with other organic products, but forms crystalline Schiff bases with benzaldehyde or p-nitro-
REBSTOCK
benzaldehyde which are more easily handled. The latter derivative was prepared by treating 65 mg. of the pH 9 extracted amine, dissolved in 2 ml. of absolute methanol, with 30 mg. p-nitrobenzaldehyde. The product was recrystallized for analysis from aqueous ethanol. There was no melting point depression with an authentic sample of the Schiff base of Dp-nitrobenzaldehyde three - 1 - p - aminophenyl - 2 dichloro acetamido-1,3-propanediol (m.p., 171-172” C.), and the I.R. spectra were identical. Found: C, 50.54, 50.77; H, 4.13, 4.14; K, 9.87. Required: C, 50.71; H, 4.02; N, 9.86. The above aminophenyl chloramphenicol related product was found in the fermentation tank beer but not in shake-flask fermentations. Its appearance late in the fermentation was noted above. EVIDENCE
FOR
OTHER
METABOLITES
Trace amounts of other aromatic nitro and amino compounds are also formed. The aryl amine products appeared mainly when the fermentation was carried out in the 200-gallon fermenter. At least two acidic aromatic nitro acids occur in shake flask beers (Rf’s 0.55 and 0.60; system, butanol sat’d. with 3% aqueous ammonia). The slower moving zone may be p-nitrobenzoic acid. Gottlieb et al. (8) reported chromatographic evidence for the formation of this compound as a C&labelled metabolite when X. venezuelae was grown in the presence of C&p-nitrophenylserinol. The same group noted that none of the above compound was metabolized to chloramphenicol but labelled N-acetyl p-nitrophenylserinol was produced. These results suggest that traces of p-nitrophenylserinol formed as a side reaction product or as a breakdown product of the antibiotic may be serving as a precursor in the formation of the unhalogenated amides. If this be the case, any p-nitrophenylserinol which is formed must be metabolized immediately since no evidence for its presence in beer samples could be found at any stage of the fermentation. In the pH 3 extracts of the fermentation tank aliquots the presence of two aromatic amines was noted. Because of their peculiar extraction patterns, these compounds are
A NEW
METABOLITE
OF STREPTOMYCES
probably amphoteric in nature. When the above extracts were chromatographed in a variety of solvent systems, the amines could not be distinguished from p-aminobenzoic acid and anthranilic acid. The latter compounds are well-known microbial metabolites and their occurrence in preparations of this kind would not be unexpected. ACKNOWLETlGMENT We gratefully acknowledge the contributions of the following individuals to these studies: Dr. John Ehrlich for his support and encouragement, Dr. Robert Hans and Mrs. Margaret Galbraith for microbiological assays, Dr. John R. Douros and Miss Georgia Senos for shake-flask preparations, Dr. J. D. Howells and Mr. Wade Mumma for fermentation-tank preparations, and Dr. S. A. Fusari and Mr. Neil Willmer for the large scale extractions and concentrations of fermentation tank beer. REFERENCES 1. EHRLICH, J., GOTTLIEB, D., BURKHOLDER, P. R., ANDERSON, L. E., AND PRIDHAM, T. Cr., J. Bacterial. 66, 467 (1948). 2. SMITH, R. M., JOSLYN, D. A., GRUHZIT, 0. M., MCLEAN, I. W., JR., PENNER, M. A., AND EHRLICH, J., J. Bacterial. 66, 425 (1948). 3. Ovaas, .J. E., EHRLICH, J., .~ND SMITH, R. M., Znd. Eng. Chem,. 42, 1775 (1950).
VENEZUELAE
163
4. LEGATOR, M., AND GOTTLIEB, D., Antibiot. Chemotherapy 3, 809 (1953). 5. MATSUOKA, M., YAGISHITA, Ii., AND UMEZAWA, H., Japan. J. Med. Sci. Biol. 6, 161 (1953). 6. GALLICCHIO, v., BND GOTTLIEB, D., Mycologia 60, 490 (1958). 7. GOTTLIEB, D., CARTER, H. E., LEGATOR, M., AND GALLICCHIO, I-., J. Bacterial. 68, 243 (1954). 8. GOTTLIEB, D., ROBBINS, P. W., AND CARTER, H. E., J. Bacterial. 72, 153 (1956). 9. GOTTLIEB, II., CARTER, H. E., ROBBINS, P. W., AND BURG, R. W., J. Bacterial. 84, 888 (1962). 10. Y.~GISHITA, K., AND UMEZAW~, H., Japan. J. Med. Sci. Biol. 3, 289 (1950). 11. WANG, E. L., IzAw.~, M., MIURA, T., AND UMEZAWA, H., J. Antibiotics (Tokyo) Ser. A. 12, 81 (1959). 12. SMITH, C. G., J. Bacterial. 76, 577 (1958). 13. SGLLIVAN, M. J., Parke, Davis and Co. U.S. Patent 2,568,571, Sept. 18, 1951. 14. SHEMYAKIN, E. M., BAMDAS, E. I., J7INOGRADOVA, M. G., KARAPETYAN, M. G., KOLOSOV, M. N., KHOKHLOV, M. G., SHVETSOV, Yu. B., AND SHCHUKINA, L. R., Dokl. Akad. Nauk. U.S.S.R. 86, 565 (1952). 16. SMITH, G. N., AND WORREL, C. S., Arch. Biochem. Biophys. 28, 232 (1950). 16. JOSLYN, D. A., AND GALBRAITH, M., 3. Bacteriol. 69, 711 (1950). 17. GL~ZKO, A. J., WOLF, L. M., AND DILL, w. A., Arch. Biochem. Biophys. 23, 411 (1949).