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Transethylation in antibiotic biosynthesis I. An ethyl homolog of oxytetracycline
PRELIMINARY NOTEb
447
The oxidation of D P N H was not enhanced by histamine This supportb the evidence 7 indicating t h a t the active center of xanthine oxidase for the oxidation of purlnes is different from t h a t for D P N H Acknowledgement ~s due to Dr E OHMURA and Dr M YONEDA of the Takeda Research Laboratory, Osaka, for donating xanthine oxldase preparations in the earl 5 period of this study, and also to technical assistance of Mrs K SUGIYAMA Thanks are due to Professor H YAMASAKI for his interest and encouragement throughout these experiments
Okayama Umvers~ty Medical School, Department of Pharmacology, SUBURO MURAOK~ Okayama (Japan) MURAOKA, M [NOUN AND H YAMASAKI, Nature, 19o (1961) 532 ]!¢[URAOKA, ~V~ SUGIYAMA AND H YAMASAKI, Nature, in t h e press YAMASAKI, S MURAOKA, ~{ SUGIYAMA AND K ENDO, Japan J Pharmacol , i i (1961) 54 E PRICE, l~I C OTEY AND P PLESNNR, in S P COLOWICK AND N O KAPLAN, Methods ~n Enzymologv, Vol 2, A c a d e m i c Press, Inc, N e w Y o r k , 1955, p 448 5 E G BALL, J Bzol Chem , 128 (1939) 51 6 H M KALCKAR, J B,ol Chem, 167 (1947) 429 7 E C DE RENZO, Advances zn Enz'vmol, 17 (1956) 293 1 S 2G 3H 4r
Received April I6th, 1962 B~ochzrn B*ophys Acta, 60 (1962) 445-447
Transethylation in antibiotic biosynthesis I. An ethyl homolo9 of oxytetracycline Evidence has been published t h a t ethionlne can participate in reactions analogous to t r a n s m e t h y l a t l o n S-Adenosylethionlne has been isolated from yeast cells grown m the presence of ethlonlne 1 This substance participates In a transethylatlon reaction with L-homocysteine to form ethionme in an enzyme system *n wtro, derived from Torulopsm ut,hs 2 Transethylatlon to form N-ethylglycocyamine was observed with pig-liver homogenate 3 A similar reaction has been inferred ~n wvo as the ethyl group of ethlonlne is Incorporated Into the creatIne and choline of rat tissues 4 Methvl groups in several antibiotics are derived from transmethylatlon reactions Interference with transmethylatlon by Streptomyces wr,d,fac,ens, a chlortetracychne producer, through the use of methxonme antagonists or methionme-dependent mutantb, results in the formation of 6-demethylchlortetracychnea The report that the three methyl groups of oxytetracychne are derived from methlonine 8 makeb it a t t r a c t i v e to study similarly the effects of possible Interference with transmethylation m an oxytetracychne-producmg orgamsm as well as the possible participation of ethionine in a transethylation reaction Streptomvces rzmosus, strain MA-558, was the parent culture used in these studies We prepared from this culture a series of ultraviolet light-induced m u t a n t s shoulng metabohc lesions at varmus steps in methionIne synthesis Since similar results were obtained with a series of these auxotiophs, our discussion will be limited to MA-558, the parent culture, and to MA-935, an auxotroph blocked between cystelne and cystathIonme These cultures were tested with and without methionlne analogs using B~ochzrn B*ophvs Acta, 60 (1962) 4 4 7 - 4 4 9
448
PRELIMINARY NOTE~
the experimental conditions and assay methods described previously s with the exception of the fermentation medium This medium contaaned 20 g glycerol, 15 g Pharmame&a*, and 30 mg ZnSO 4 7H20 per liter of distilled water The results are summarized m Table I Under the experimental condltmns used, MA-935 makes only oxytetracychne except when the medium IS supplemented with DL-ethlonine In the presence of ethlonlne, an additional ultraviolet hght-fluorescmg, bioactlve compound is produced Methlomne dependence l~ not necessary for the formation of this product because MA-558, a prototroph, also make~ it in the presence of ethlonme TABLE I EFFECT
OF
METHIONINE
AN~kLOGS ON
TETRACYCLINE
FORMATION
Products
Culture No
Supplement (too l~g/ml)
Total brotk potency (per ccnt of control)
MA-935
None (control) DL-Ethlonlne DL-~-Methylmethlonme DL-Methlonme sulfone S-Methylcysteme S-Benzvlcysteme S-Benzylhomocysteme DL-C~s-Crotvlglycme
ioo 4° I oo IOO 60 80 i oo I oo
-7 + + + + + + +
None (control) nL-Ethlonlne
ioo 45
+ +
MA-558
Oxytetrac~dmc
~ew product
+
+
Butanol extracts of ethlonlne-supplemented fermentation broths were compared to the known tetracychnes In four chromatographic systems and the new compound was found to be different from any known tetracycline avaalable to us The moblhty of the new compound m the chromatograms suggested it was not 6-demethyloxytetracycline The fact that it was produced only in the presence of ethlonlne suggested that perhaps an ethyl group was being incorporated mto the oxytetracychne molecule This hypothesis was tested by addition of [I-14C-ethyllethlonlne to fermentation media The isolated new product was found to be strongly labeled with no labeling occurring in the isolated oxytetracychne concurrently produced Incorporation of ethyl from ethlonlne into the new compound prompted the testing of other possible ethyl donors,, e, S-ethyl-L-cystelne, DL-e-methylethionine, DL-ethlonlne sulfone and DL-ethlonlne sulfoxlde In no instance did addition of these compounds result In formation of the new product nor did these compounds show the Inhibition of over-all antibiotic productivity caused by ethlonme Apparently, the specific ethlonme structure is required for transethylatIon, perhaps by competitive inhibition of transmethylatIon Both D and L-ethlonine are effective, but not equally so, on a weight basis The new product was isolated by butanol extraction of acidified filtered broth The butanol extract was purified by back-extraction mto water at pH 2 o followed by chromatography on Whatman 3MM paper using an ethyl a c e t a t e - o 05 M phosphate buffer system (pH 4 7) The eluate from paper was precipitated as the b a r i u m * A cotton-seed protein n u t r i e n t f r o m Traders Oil Mill C o , Traders P r o t e m Dlvlsxon, F o r t W o r t h , Texas (U S A ) Bzoch~m Bzoph~s 4cta, 6o (1962) 447-449
449
PRELIMINARY NOTES
magnesium complex, then decomposed with acid and the compound finally isolated via a 2o-tube counter-current extraction usmg a b u t a n o l - o oi N HC1 system Ultraviolet absorption and fluorescence spectra showed the new compound to be a tetracychne very similar to oxytetracychne Rapid destruction of the typical tetracycline ultraviolet-absorption spectrum of the new compound in both alkali and acid indicated the presence of a tertiary hydroxyl at C-6 A kinetic study with model compounds showed the new tetracycline to have a stability similar to, but not identical with, oxytetracychne When heated overnight at 60 ° in o 5 N HC1, the new tetracycline degraded to an apo 7 rather than an anhvdro product indicating the presence of a hydroxyl at C-5 Radioactive material was Isolated from fermentations supplemented with [I-14Cethyl]ethIonlne This was found to be single-component material when chromatographed in several solvent systems with the ultraviolet fluorescence, bioactlvity, and radioactivity coinciding Alkaline hydrolysisa of this labeled tetracycline showed all of the radioactivity to be associated with the volatile almne liberated on degradation This Indicated an ethyl substitution on the anuno group at C-4 The structure of the volatile amine was deterrmned to be methylethylalmne by the chromatographic method of BRENNER et al o and by nuclear-magnetic-resonance spectroscopy Thus, the new tetracycline is N-methylethyl oxytetracycllne This new tetracycline has activity ~n wtro comparable to oxytetracychne against a wide spectrum of bacteria In preliminary ~tudles an v,vo, it proved to be half a~ active as oxytetracychne in protecting mice from Staphylococcus aureus and D,plococcus p n e u m o m a e infections This is the first recognition of an ethyl-transfer reaction to produce a N-ethyl homolog of a known antibiotic Ethyl transfer may be a generahzed process in antibiotic biosynthesis Ethyl transfer to yield an 0-ethyl homolog of griseofulvln l~ reported in a companion publication from this laboratory We wish to thank Drs E O GTAPLEYand A K MILLERfor the evaluation of the new tetracycline ,n wtro and zn v,vo EUGENE L D U L A N E Y IRVING P U T T E R
Merck Sharp and Dohme Research Laboratories, D , w s , o n of Merck and Co, I n c , Rahway, N J (U S A )
DOROTHY D R E S C H E R LOUIS CHAIET WILLIAM J FRANK J
MILLER
WOLF
DAVID H E N D L I N 1 F SCHLENK AND J A TILLOTSON, J Bzol Chem , 2o6 (1954) 687 2 L "v~ PARKS, J B , o l Chem , 232 (1958) 109 a H TuPpY AND K D u s , Monatsh C h e m , 89 (1958 ) 318 J A STEKOL ~ND K WEISS, J B~ol C h e m , 185 (195o) 577 5 D HENDLIN, E L DULANEY, D DRESCHER, T COOK AND L CHAIET, Bzoch*m B*ophvs Acta, 58 (1962) 635 e j F SNELL, A J BIRCH AND P L THOMSON, J A m Chem Soc , 82 (196o) 2402 7 F A HOCHSTEIN, C R STEPHENS,L H CONOVER, P P REGNA, 1~ PASTERNACK,P N GORDON, P" J PILGRIM, K J BRUNINGS AND 1R B WOODWARD, J A m Chem S o c , 75 (1953) 5455 8 R PASTERNACK, A BAVLEY, 1R L WAGNER, F A HOCHSTEIN, P P REGNA AND K J BRUNINGS, J A m Chem Soc , 74 (1952) 1926 J M BRENNER AND 1R H KENTEN, Bzochem J , 49 (1951 ) 651
Received Max, ist, 1962 B,och~m Bzophvs Acta, 60 (1962) 4 4 7 - 4 4 9
447
The oxidation of D P N H was not enhanced by histamine This supportb the evidence 7 indicating t h a t the active center of xanthine oxidase for the oxidation of purlnes is different from t h a t for D P N H Acknowledgement ~s due to Dr E OHMURA and Dr M YONEDA of the Takeda Research Laboratory, Osaka, for donating xanthine oxldase preparations in the earl 5 period of this study, and also to technical assistance of Mrs K SUGIYAMA Thanks are due to Professor H YAMASAKI for his interest and encouragement throughout these experiments
Okayama Umvers~ty Medical School, Department of Pharmacology, SUBURO MURAOK~ Okayama (Japan) MURAOKA, M [NOUN AND H YAMASAKI, Nature, 19o (1961) 532 ]!¢[URAOKA, ~V~ SUGIYAMA AND H YAMASAKI, Nature, in t h e press YAMASAKI, S MURAOKA, ~{ SUGIYAMA AND K ENDO, Japan J Pharmacol , i i (1961) 54 E PRICE, l~I C OTEY AND P PLESNNR, in S P COLOWICK AND N O KAPLAN, Methods ~n Enzymologv, Vol 2, A c a d e m i c Press, Inc, N e w Y o r k , 1955, p 448 5 E G BALL, J Bzol Chem , 128 (1939) 51 6 H M KALCKAR, J B,ol Chem, 167 (1947) 429 7 E C DE RENZO, Advances zn Enz'vmol, 17 (1956) 293 1 S 2G 3H 4r
Received April I6th, 1962 B~ochzrn B*ophys Acta, 60 (1962) 445-447
Transethylation in antibiotic biosynthesis I. An ethyl homolo9 of oxytetracycline Evidence has been published t h a t ethionlne can participate in reactions analogous to t r a n s m e t h y l a t l o n S-Adenosylethionlne has been isolated from yeast cells grown m the presence of ethlonlne 1 This substance participates In a transethylatlon reaction with L-homocysteine to form ethionme in an enzyme system *n wtro, derived from Torulopsm ut,hs 2 Transethylatlon to form N-ethylglycocyamine was observed with pig-liver homogenate 3 A similar reaction has been inferred ~n wvo as the ethyl group of ethlonlne is Incorporated Into the creatIne and choline of rat tissues 4 Methvl groups in several antibiotics are derived from transmethylatlon reactions Interference with transmethylatlon by Streptomyces wr,d,fac,ens, a chlortetracychne producer, through the use of methxonme antagonists or methionme-dependent mutantb, results in the formation of 6-demethylchlortetracychnea The report that the three methyl groups of oxytetracychne are derived from methlonine 8 makeb it a t t r a c t i v e to study similarly the effects of possible Interference with transmethylation m an oxytetracychne-producmg orgamsm as well as the possible participation of ethionine in a transethylation reaction Streptomvces rzmosus, strain MA-558, was the parent culture used in these studies We prepared from this culture a series of ultraviolet light-induced m u t a n t s shoulng metabohc lesions at varmus steps in methionIne synthesis Since similar results were obtained with a series of these auxotiophs, our discussion will be limited to MA-558, the parent culture, and to MA-935, an auxotroph blocked between cystelne and cystathIonme These cultures were tested with and without methionlne analogs using B~ochzrn B*ophvs Acta, 60 (1962) 4 4 7 - 4 4 9
448
PRELIMINARY NOTE~
the experimental conditions and assay methods described previously s with the exception of the fermentation medium This medium contaaned 20 g glycerol, 15 g Pharmame&a*, and 30 mg ZnSO 4 7H20 per liter of distilled water The results are summarized m Table I Under the experimental condltmns used, MA-935 makes only oxytetracychne except when the medium IS supplemented with DL-ethlonine In the presence of ethlonlne, an additional ultraviolet hght-fluorescmg, bioactlve compound is produced Methlomne dependence l~ not necessary for the formation of this product because MA-558, a prototroph, also make~ it in the presence of ethlonme TABLE I EFFECT
OF
METHIONINE
AN~kLOGS ON
TETRACYCLINE
FORMATION
Products
Culture No
Supplement (too l~g/ml)
Total brotk potency (per ccnt of control)
MA-935
None (control) DL-Ethlonlne DL-~-Methylmethlonme DL-Methlonme sulfone S-Methylcysteme S-Benzvlcysteme S-Benzylhomocysteme DL-C~s-Crotvlglycme
ioo 4° I oo IOO 60 80 i oo I oo
-7 + + + + + + +
None (control) nL-Ethlonlne
ioo 45
+ +
MA-558
Oxytetrac~dmc
~ew product
+
+
Butanol extracts of ethlonlne-supplemented fermentation broths were compared to the known tetracychnes In four chromatographic systems and the new compound was found to be different from any known tetracycline avaalable to us The moblhty of the new compound m the chromatograms suggested it was not 6-demethyloxytetracycline The fact that it was produced only in the presence of ethlonlne suggested that perhaps an ethyl group was being incorporated mto the oxytetracychne molecule This hypothesis was tested by addition of [I-14C-ethyllethlonlne to fermentation media The isolated new product was found to be strongly labeled with no labeling occurring in the isolated oxytetracychne concurrently produced Incorporation of ethyl from ethlonlne into the new compound prompted the testing of other possible ethyl donors,, e, S-ethyl-L-cystelne, DL-e-methylethionine, DL-ethlonlne sulfone and DL-ethlonlne sulfoxlde In no instance did addition of these compounds result In formation of the new product nor did these compounds show the Inhibition of over-all antibiotic productivity caused by ethlonme Apparently, the specific ethlonme structure is required for transethylatIon, perhaps by competitive inhibition of transmethylatIon Both D and L-ethlonine are effective, but not equally so, on a weight basis The new product was isolated by butanol extraction of acidified filtered broth The butanol extract was purified by back-extraction mto water at pH 2 o followed by chromatography on Whatman 3MM paper using an ethyl a c e t a t e - o 05 M phosphate buffer system (pH 4 7) The eluate from paper was precipitated as the b a r i u m * A cotton-seed protein n u t r i e n t f r o m Traders Oil Mill C o , Traders P r o t e m Dlvlsxon, F o r t W o r t h , Texas (U S A ) Bzoch~m Bzoph~s 4cta, 6o (1962) 447-449
449
PRELIMINARY NOTES
magnesium complex, then decomposed with acid and the compound finally isolated via a 2o-tube counter-current extraction usmg a b u t a n o l - o oi N HC1 system Ultraviolet absorption and fluorescence spectra showed the new compound to be a tetracychne very similar to oxytetracychne Rapid destruction of the typical tetracycline ultraviolet-absorption spectrum of the new compound in both alkali and acid indicated the presence of a tertiary hydroxyl at C-6 A kinetic study with model compounds showed the new tetracycline to have a stability similar to, but not identical with, oxytetracychne When heated overnight at 60 ° in o 5 N HC1, the new tetracycline degraded to an apo 7 rather than an anhvdro product indicating the presence of a hydroxyl at C-5 Radioactive material was Isolated from fermentations supplemented with [I-14Cethyl]ethIonlne This was found to be single-component material when chromatographed in several solvent systems with the ultraviolet fluorescence, bioactlvity, and radioactivity coinciding Alkaline hydrolysisa of this labeled tetracycline showed all of the radioactivity to be associated with the volatile almne liberated on degradation This Indicated an ethyl substitution on the anuno group at C-4 The structure of the volatile amine was deterrmned to be methylethylalmne by the chromatographic method of BRENNER et al o and by nuclear-magnetic-resonance spectroscopy Thus, the new tetracycline is N-methylethyl oxytetracycllne This new tetracycline has activity ~n wtro comparable to oxytetracychne against a wide spectrum of bacteria In preliminary ~tudles an v,vo, it proved to be half a~ active as oxytetracychne in protecting mice from Staphylococcus aureus and D,plococcus p n e u m o m a e infections This is the first recognition of an ethyl-transfer reaction to produce a N-ethyl homolog of a known antibiotic Ethyl transfer may be a generahzed process in antibiotic biosynthesis Ethyl transfer to yield an 0-ethyl homolog of griseofulvln l~ reported in a companion publication from this laboratory We wish to thank Drs E O GTAPLEYand A K MILLERfor the evaluation of the new tetracycline ,n wtro and zn v,vo EUGENE L D U L A N E Y IRVING P U T T E R
Merck Sharp and Dohme Research Laboratories, D , w s , o n of Merck and Co, I n c , Rahway, N J (U S A )
DOROTHY D R E S C H E R LOUIS CHAIET WILLIAM J FRANK J
MILLER
WOLF
DAVID H E N D L I N 1 F SCHLENK AND J A TILLOTSON, J Bzol Chem , 2o6 (1954) 687 2 L "v~ PARKS, J B , o l Chem , 232 (1958) 109 a H TuPpY AND K D u s , Monatsh C h e m , 89 (1958 ) 318 J A STEKOL ~ND K WEISS, J B~ol C h e m , 185 (195o) 577 5 D HENDLIN, E L DULANEY, D DRESCHER, T COOK AND L CHAIET, Bzoch*m B*ophvs Acta, 58 (1962) 635 e j F SNELL, A J BIRCH AND P L THOMSON, J A m Chem Soc , 82 (196o) 2402 7 F A HOCHSTEIN, C R STEPHENS,L H CONOVER, P P REGNA, 1~ PASTERNACK,P N GORDON, P" J PILGRIM, K J BRUNINGS AND 1R B WOODWARD, J A m Chem S o c , 75 (1953) 5455 8 R PASTERNACK, A BAVLEY, 1R L WAGNER, F A HOCHSTEIN, P P REGNA AND K J BRUNINGS, J A m Chem Soc , 74 (1952) 1926 J M BRENNER AND 1R H KENTEN, Bzochem J , 49 (1951 ) 651
Received Max, ist, 1962 B,och~m Bzophvs Acta, 60 (1962) 4 4 7 - 4 4 9