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Microbiological oxidation of indole alkaloids
ARCHIVES
OF BIOCHEMISTRY
AND
Microbiological
BIOPHYSICS
79,
257-260
(1959)
Oxidation of Indole Alkaloids
Yen Hoong Loo and Mona Reidenberg From the Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana Received
May
16, 19.58
INTRODUCTION
Interest in the pharmacology of the RauwoZJiaalkaloids stimulated attempts to modify some of these compounds by microorganisms. This communication describes the isolation and characterization of a product obtained when the organism Streptomyces platen& (NRRL 2364) was grown in the presence of yohimbine. Data are presented which indicate that the oxidation product is lo-hydroxyyohimbine (I). A similar hydroxylation of HO
HICOOC-
indole compounds by microorganisms has been report’ed by Godtfredsen et al. (1) and Mitoma et al. (2). EXPERIMENTAL AND RESULTS Fermentation In a typical fermentation, 100 ml. of a 72.hr. culture of Streptomyces platensis (NRRL 2364) was used to inoculate a liter of medium, composed of 2% malt sirup, 1.5% soybean meal, 0.1% casein, 0.1% dried yeast, and 0.5% sodium chloride. The culture was grown at 29-3O’C. in Erlenmeyer flasks agitated on a rotary shaker. After 72 hr. growth, yohimbine (dissolved in 950/, ethanol) was added to give a final 257
258
LOO ASD REIDENBERG
concentration of 200 pg./ml. broth. The fermentation was allowed to proceed for 16 hr. and was then terminated by acidification of the whole culture to pH 2.5 with solid tartaric acid. Isolation and Purijkalim
of Oxidation Products
To the whole broth were added 0.5 g. ascorbic acid and an equal volume of methanol. The mixture was filtered, and the cell pad was washed three times with met.hanol. The combined filtrate and wsshings were concentrated in vacm to remove met,hanol. Three extractions with an equal volume of ether served to remove impurities. The aqueous layer was adjusted to pH 7 1vit.h solid NaHC03 , then to pH 8.5 wit,h concentrated F.X.,OH, and was saturated with K&l. The alkaloid fraction was then extracted into ethyl acetate. The extract was evaporated in vacua, and the residue was dissolved in acetone. A mixture of tartrates was obtained by acidificntion of the acetone solution to pH 2.5-3.0 with solid tartaric acid. Addition of 3 vol. ether precipitated 160 mg. solid. The salt, was dissolved in wat,er and was converted to the free base by adjusting the pH to 8.5 with concentraM NH&H. Chloroform was used to extract the free base. The extract was dried over anhydrous MgS04 and was t,hen concentrated to a smaller volume. Purification was efiecbed by chromatographing the solut,ion over 20 g. of activated alumina packed in chloroform in a column 1.2 cm. in diameter. The chromatogram was developed with chloroform, followed by mixtures of chloroform-met,hanol in volume ratios of 95:5, 90: 10, 80:20, and finally with methanol. Column cuts were examined for blue fluorescence under ultraviolet light, for the presence of alkaloid with a modified Dragendorff reagent (3), and for t,he presence of 5-hydroxyindolc compounds by the method of Udenfriend et al. (4). Chloroform eluted 80 mg. of unchanged yohimbinc, identified by infrared and ultraviolet absorption spectra as well as mixed melting point wit.h an authentic sample of yohimbine. The major oxidation product was found in the first chloroform-methanol (95:s) eluatc. From the peak cuts were isolated 70 mg. tartrate, dcc. 208-222°C.’ Crystallization of the tart,rate from acet,one raised the decomposit.ion point to 238°C. After recrystallization from acetone-methanol, t.he decomposition point remained const.ant at 242°C. The free base (I) was crystallized from aqueous methanol (dec. 180-185°C.). [aIf = +71 .I”
(ethanol). Traces of unidentified
oxidation
products were clutcd by increasing the
methanol concentration in the chloroform-methanol mixtures (90: 10 and 1 All melting points were taken on a Kofler microblock apparatus.
OXIDATION
OF
INDOLE
ALKALOIDS
259
80: 20). These fractions gave positive tests for alkaloid and 5-hydroxyindole compounds. Identifiation
of the Oxidation Product
The infrared mull spectrum of I shows peaks at 3.0, 5.85, 6.16, 6.27, 8.3, 8.6, 9.0, 10.0, and 10.35 p. In 66% dimethylformamide two groups are titrated, one with a pK of 7.2 and another with a pK of 13.5-11.0. The molecular weight determined by titration is 362 (calculated for monohydroxyyohimbine 370). The compound rea&s with 1-nitroso-2-napht’hol under the condit’ions described by Udenfriend et al. (4) to give a violet chromophore with a visible absorption curve resembling that described for 5-hydroxyindole compounds by these authors. log aM32omp= 3.90; 3.64. The ultraviolet absorption spectrum resembles t,hat of log a.k-540r,rp = 5-hydroxytryptamine and the yohimbine-t,ype alkaloid, sarpagine, whirh is hydroxylated at the lo-position. 5-Hydroxytryptamine (ethanol) : log aM224mC = 1.43; log ay275n,,,= 3.80. Sarpagine (methanol): log aM226mp= 4.54; log a,,,277,,,,,= 5.90. Oxidation product I (ethanol): log aM224,,rp= 4.37; log aMZ;8r,lP= C3.85; shoulders at 287 and 300 ml.r. In alkali the peak at 300 rnp shifts to 320, and this change may be reversed with acid. These data serve to identify the oxidation product (I) a,s lo-hydroxyyohimbine. En2 yme Specijicity Using the reaction with l-nitroso-2-naphthol to measure hydroxylation, the speeificit’y of the culture toward other substrates was tested. The following compounds were not converted to 5-hydroxyindoles: tryptophan, tryptamine, indole-3-acetic acid, gramine, the ergot alkaloids, and reserpine. Other indole alkaloids hydroxylated by this organism include rauwolscine, 3-epi-Lu-yohimbane, and alloyohimbine. The presence of 10e2 M KCN in the medium did not affect hydroxylation. Complete inhibition was observed when 1OP M p-nitrophenol or 1O-3 Jl S-hydroxyquinoline was added. ACKNOWLEDGMENT
The authors appreckte the cooperative efforts chemists of The Lilly Research Laboratories.
of the microbiologists
and physical
SUMMARY
The introduction of a hydroxyl group at the lo-position of yohimbine by a culture of Streptomyces platensis has been demonstrated.
260
LOO AND
REIDENBERG
REFERENCES 1. GODTFREDSEN, W. O., KORSBY, G., LORCK, H., AND VANOEDAL, S., Ezperientia 14, 88 (1958). 2. MITOMA, C., WEISSBACH, H., AND UDENFRIEND, S., Arch. Biochem. Biophys. 63, 122 (1956). 3. MUNIER, R., AND MADREBOEUF, M., Bull. SOC. chim. biol. 33, 846 (1951). 4. UDENFRIEND, S., WEISSBACH, H., AND CLARK, C. T., J. Biol. Chem. 216,337 (1955).
OF BIOCHEMISTRY
AND
Microbiological
BIOPHYSICS
79,
257-260
(1959)
Oxidation of Indole Alkaloids
Yen Hoong Loo and Mona Reidenberg From the Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana Received
May
16, 19.58
INTRODUCTION
Interest in the pharmacology of the RauwoZJiaalkaloids stimulated attempts to modify some of these compounds by microorganisms. This communication describes the isolation and characterization of a product obtained when the organism Streptomyces platen& (NRRL 2364) was grown in the presence of yohimbine. Data are presented which indicate that the oxidation product is lo-hydroxyyohimbine (I). A similar hydroxylation of HO
HICOOC-
indole compounds by microorganisms has been report’ed by Godtfredsen et al. (1) and Mitoma et al. (2). EXPERIMENTAL AND RESULTS Fermentation In a typical fermentation, 100 ml. of a 72.hr. culture of Streptomyces platensis (NRRL 2364) was used to inoculate a liter of medium, composed of 2% malt sirup, 1.5% soybean meal, 0.1% casein, 0.1% dried yeast, and 0.5% sodium chloride. The culture was grown at 29-3O’C. in Erlenmeyer flasks agitated on a rotary shaker. After 72 hr. growth, yohimbine (dissolved in 950/, ethanol) was added to give a final 257
258
LOO ASD REIDENBERG
concentration of 200 pg./ml. broth. The fermentation was allowed to proceed for 16 hr. and was then terminated by acidification of the whole culture to pH 2.5 with solid tartaric acid. Isolation and Purijkalim
of Oxidation Products
To the whole broth were added 0.5 g. ascorbic acid and an equal volume of methanol. The mixture was filtered, and the cell pad was washed three times with met.hanol. The combined filtrate and wsshings were concentrated in vacm to remove met,hanol. Three extractions with an equal volume of ether served to remove impurities. The aqueous layer was adjusted to pH 7 1vit.h solid NaHC03 , then to pH 8.5 wit,h concentrated F.X.,OH, and was saturated with K&l. The alkaloid fraction was then extracted into ethyl acetate. The extract was evaporated in vacua, and the residue was dissolved in acetone. A mixture of tartrates was obtained by acidificntion of the acetone solution to pH 2.5-3.0 with solid tartaric acid. Addition of 3 vol. ether precipitated 160 mg. solid. The salt, was dissolved in wat,er and was converted to the free base by adjusting the pH to 8.5 with concentraM NH&H. Chloroform was used to extract the free base. The extract was dried over anhydrous MgS04 and was t,hen concentrated to a smaller volume. Purification was efiecbed by chromatographing the solut,ion over 20 g. of activated alumina packed in chloroform in a column 1.2 cm. in diameter. The chromatogram was developed with chloroform, followed by mixtures of chloroform-met,hanol in volume ratios of 95:5, 90: 10, 80:20, and finally with methanol. Column cuts were examined for blue fluorescence under ultraviolet light, for the presence of alkaloid with a modified Dragendorff reagent (3), and for t,he presence of 5-hydroxyindolc compounds by the method of Udenfriend et al. (4). Chloroform eluted 80 mg. of unchanged yohimbinc, identified by infrared and ultraviolet absorption spectra as well as mixed melting point wit.h an authentic sample of yohimbine. The major oxidation product was found in the first chloroform-methanol (95:s) eluatc. From the peak cuts were isolated 70 mg. tartrate, dcc. 208-222°C.’ Crystallization of the tart,rate from acet,one raised the decomposit.ion point to 238°C. After recrystallization from acetone-methanol, t.he decomposition point remained const.ant at 242°C. The free base (I) was crystallized from aqueous methanol (dec. 180-185°C.). [aIf = +71 .I”
(ethanol). Traces of unidentified
oxidation
products were clutcd by increasing the
methanol concentration in the chloroform-methanol mixtures (90: 10 and 1 All melting points were taken on a Kofler microblock apparatus.
OXIDATION
OF
INDOLE
ALKALOIDS
259
80: 20). These fractions gave positive tests for alkaloid and 5-hydroxyindole compounds. Identifiation
of the Oxidation Product
The infrared mull spectrum of I shows peaks at 3.0, 5.85, 6.16, 6.27, 8.3, 8.6, 9.0, 10.0, and 10.35 p. In 66% dimethylformamide two groups are titrated, one with a pK of 7.2 and another with a pK of 13.5-11.0. The molecular weight determined by titration is 362 (calculated for monohydroxyyohimbine 370). The compound rea&s with 1-nitroso-2-napht’hol under the condit’ions described by Udenfriend et al. (4) to give a violet chromophore with a visible absorption curve resembling that described for 5-hydroxyindole compounds by these authors. log aM32omp= 3.90; 3.64. The ultraviolet absorption spectrum resembles t,hat of log a.k-540r,rp = 5-hydroxytryptamine and the yohimbine-t,ype alkaloid, sarpagine, whirh is hydroxylated at the lo-position. 5-Hydroxytryptamine (ethanol) : log aM224mC = 1.43; log ay275n,,,= 3.80. Sarpagine (methanol): log aM226mp= 4.54; log a,,,277,,,,,= 5.90. Oxidation product I (ethanol): log aM224,,rp= 4.37; log aMZ;8r,lP= C3.85; shoulders at 287 and 300 ml.r. In alkali the peak at 300 rnp shifts to 320, and this change may be reversed with acid. These data serve to identify the oxidation product (I) a,s lo-hydroxyyohimbine. En2 yme Specijicity Using the reaction with l-nitroso-2-naphthol to measure hydroxylation, the speeificit’y of the culture toward other substrates was tested. The following compounds were not converted to 5-hydroxyindoles: tryptophan, tryptamine, indole-3-acetic acid, gramine, the ergot alkaloids, and reserpine. Other indole alkaloids hydroxylated by this organism include rauwolscine, 3-epi-Lu-yohimbane, and alloyohimbine. The presence of 10e2 M KCN in the medium did not affect hydroxylation. Complete inhibition was observed when 1OP M p-nitrophenol or 1O-3 Jl S-hydroxyquinoline was added. ACKNOWLEDGMENT
The authors appreckte the cooperative efforts chemists of The Lilly Research Laboratories.
of the microbiologists
and physical
SUMMARY
The introduction of a hydroxyl group at the lo-position of yohimbine by a culture of Streptomyces platensis has been demonstrated.
260
LOO AND
REIDENBERG
REFERENCES 1. GODTFREDSEN, W. O., KORSBY, G., LORCK, H., AND VANOEDAL, S., Ezperientia 14, 88 (1958). 2. MITOMA, C., WEISSBACH, H., AND UDENFRIEND, S., Arch. Biochem. Biophys. 63, 122 (1956). 3. MUNIER, R., AND MADREBOEUF, M., Bull. SOC. chim. biol. 33, 846 (1951). 4. UDENFRIEND, S., WEISSBACH, H., AND CLARK, C. T., J. Biol. Chem. 216,337 (1955).