APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1979, p. 836-839
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N-Demethylation of Nicotine and Reduction of Nicotine-1'-NOxide by Microsporum gypseum ROBERT D. SINDELAR, JOHN P. ROSAZZA, AND CHARLES F. BARFKNECHT* College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242
Received for publication 6 August 1979
Several microorganisms were examined for their abilities to convert S-nicotine into nornicotine. Five microorganisms including Microsporum gypseum (ATCC 11395) produced nornicotine and three unknown metabolites. M. gypseum efficiently reduced nicotine-1'-N-oxide to nicotine, but no nornicotine was obtained when the N-oxide was used as substrate.
S-Nicotine (Fig. la) elicits a number of pharmacological responses in mammalian systems (26). Our investigations of the cardiovascular effects of nicotine necessitated a supply of Snornicotine (Fig. lb). Nornicotine is not readily available commercially, and its stereochemistry and purity are questionable (18). Literature methods for the preparation of S-nornicotine are complicated by side reactions and low yield (14, 21). Although microbial degradation of nicotine and tobacco alkaloids has been studied in detail by a number of groups (15, 17, 27, 30), little emphasis was given to the N-demethylation reaction. Since microbial N-demethylation of several alkaloids occurs (28), we attempted to develop microbial systems which could provide the required nornicotine. This report describes results obtained when selected cultures were examined for their abilities to achieve N-demethylation of nicotine, and describes the metabolic fate of nicotine-l'-N-oxide (Fig. lc), a possible intermediate in the N-demethylation reaction. MATERIALS AND METHODS Chromatographic procedures. Thin-layer chromatography (TLC) was performed on 0.25-mm-thick layer plates of silica gel GF254 or 1.0-mm-thick layer plates of silica gel G (Analtech). Plates were ovenactivated for 15 min at 130°C before use. Solvent systems used in developing TLC plates were (A) CHCl3-MeOH-58% NH40H (60:10:1) (13); (B) C6H6ethanol (3:1); (C) CHCl3-MeOH-glacial acetic acid (60:10:1) (13). Visualization involved viewing developed plates under short (254 nm) and long (365 nm) wavelength ultraviolet light. The plates were later sprayed with Dragendorff reagent (5), followed by a solution of polyphosphoric acid-water (1:1). Chemicals. Nicotine (Eastman) was treated with phenyl isocyanate to remove secondary amine contaminants (25) and fractionally distilled. Nicotine-l'N-oxide and nicotine-i, l'-di-N-oxide were synthesized by the method of Craig and Purushothaman (8). Myosmine was prepared according to Brandange and Lind-
blom (7) and reduced to racemic nornicotine with NaBH4 (4). N. Castagnoli (Medical Center, University of California, San Francisco) supplied a sample of 4',4'dideuteronornicotine. Cotinine was prepared by a literature method (24). All compounds were chromatographically pure and were fully characterized by nuclear magnetic resonance and mass spectral methods. Fermentation procedures. Cultures used in this study are maintained in the University of Iowa College of Pharmacy culture collection and are stored in sealed screw-cap tubes at 4°C in a refrigerator. Organisms were grown in a medium of the following composition: soybean meal (5 g), glucose (20 g), yeast extract (5 g), NaCl (5 g), K2HPO4 (5 g), and distilled water to 1.0 liter; pH was adjusted to 7.0 with 5 N HCI. Media were sterilized in an autoclave at 121°C for 15 min. Fermentations were conducted in cotton-plugged Erlenmeyer flasks containing one-fifth of their volume of medium. A standard two-stage fermentation protocol was used for all incubations (3). The surface growth from fresh agar slants of microorganisms was suspended in sterile medium, and the resulting vegetative or spore suspension was used to inoculate firststage cultures. First-stage cultures were incubated with shaking at 250 rpm at 27°C on rotary shakers for 72 h, during which time cultures attained maximum growth. Actively growing stage I cultures were then used to inoculate stage II cultures, which were incubated for 24 h before receiving nicotine. Substratecontaining flasks were incubated with shaking, and samples (5 ml) were withdrawn at 24, 72, 96, 120 and 144 h. Substrate added to sterile medium and fermentation blanks containing no substrate served as controls. Large-scale fermentation of Microsporum gypseum. Stage II cultures of M. gypseum were grown in a total of 2.4 liters of medium held in 12 1.0-liter flasks. Repeated serial transfer of M. gypseum cultures from slant to slant resulted in diminishing ability of the organism to produce metabolites. Therefore, M. gypseum cultures which had been transferred no more than two times were used for experiments. Nicotine (100 mg in 0.8 ml of dimethyl formamide) was added to each culture flask. Formation of metabolites was followed by TLC (solvent system A). Fermentations were harvested at 168 h, and the contents of all flasks
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were pooled, adjusted to pH 10.0 with aqueous K2CO3, and exhaustively extracted with CHCL3. The extract was dried (Na2SO4) and evaporated in vacuo to give a crude extract (0.9 g). The crude extract was adsorbed as a band on 1.0-mm-thick layer plates of silica gel G and was developed with solvent system A. Each separated band was scraped from the plate, extracted with CHC13, and concentrated in vacuo. Low-resolution mass spectral data were obtained on the band corresponding to nornicotine on a Finnigan 3200 spectrometer. mle (percent relative abundance) was 148 (26), 147 (37), 120 (26), 119 (100), and 70 (5); TLC Rf values were 0.44 (system A), 0.06 (system B), and 0.03 (system C).
RESULTS Preliminary small-scale fermentation experiments were conducted with eight cultures which had previously demonstrated N-demethylating ability with a variety of alkaloid substrates. Six of the microorganisms examined produced four different metabolites of nicotine (Table 1). The total amount of nicotine converted to metabolites was estimated to be less than 20% by TLC. Not all metabolites were produced simultaneously, and their order of appearance was II (96 h), I and III (120 h), and IV (144 h). Metabolite IV was not always formed. A preparative scale fermentation was performed with M. gypseum to obtain sufficient quantities of metabolites for isolation and structure elucidation. Metabolite II was detected after 72 h. Little increase in conversion to metabolites was observed after 120 h, and fermentations were harvested and pooled 168 h after substrate addition. After extraction, 0.9 g of crude metabolites was isolated. The largest portion of the crude extract was unmetabolized nicotine. Chromatographic comparison of the mixture of metabolites showed that metabolite III had the same Rf as synthetic nornicotine and dideuteronornicotine. The mixture of nicotine metabolites was separated by preparative TLC. An electron impact mass spectrum of isolated metabolite III was identical to that of synthetic nornicotine and that reported in the literature (12) (observed [percent relative abundance]:) 148 [26], 147 [37], 120 [26], 119 [100], and 70 [5]; literature values [11]: 148 [25], 147 [35], 120 [30], 119 [100], and 70 [85]). The fragmentation pattern of dideuteronornicotine corresponded well with that of metabolite III (for dideuteronornicotine, observed [percent relative abundance]: 150 [29], 149 [36], 120 [30], 119 [100], and 72 [25]). This metabolite exhibited one spot when cochromatographed (systems A, B, and C) with synthetic nornicotine and dideuteronornicotine. The data collected for metabolite III are consistent for nornicotine. Unequivocal identifi-
N-DEMETHYLATION OF NICOTINE
837
6 I
Fig. laX=N-CH3 bX=N- H cX=N-CH3 0 FIG. 1. S-Nicotine (a), S-nornicotine (b), and nicotine-l'-N-oxide (c).
cation of the other metabolites was not possible due to low yield. Resting cell cultures of M. gypseum were examinedYfor their abilities to produce metabolites (11). Cells were incubated with 0.1 M phosphate buffer (pH 7.0) plus 0.25% glucose and with buffer only. The glucose concentration used was that which normally remained in 24-h-old stage II cultures. The fermentation containing glucose converted nicotine to metabolites I and II 72 h after addition of substrate, whereas the same metabolites were formed in the fermentation without glucose 48 h after substrate addition. Metabolites III and IV were not detected. These results suggested a possible catabolite repressive effect for glucose in this transformation reaction. Since nicotine-l'-N-oxide is a plausible intermediate in the N-demethylation of the alkaloid, the N-oxide was used as the substrate in a number of experiments. M. gypseum cell homogenates (Sorvall Omni-Mixer, 10,000 rpm, 75-ml cannister) reductively converted more than 80% of nicotine-1'-N-oxide to nicotine after 12 h, and the reaction went almost to completion in 24 h when the N-oxide was added to 10,000 x g supernatants. The cellular debris fraction also produced nicotine in greater than 50% yield (estimated yields are based on visual evaluation of TLC plates). A trace amount of metabolite I was detected after 36 h. Nicotine was identified as the metabolite of the N-oxide by TLC (systems A, B, and C) comparison with comercially available nicotine. The electron impact mass spectrum of the isolated metabolite was identical to that of a nicotine standard and to one reported in the literature (11) (metabolite [percent rela-
838
APPL. ENVIRON. MICROBIOL.
SINDELAR, ROSAZZA, AND BARFKNECHT TABLE 1. Production of nicotine metabolites
Rf values of metabolitesb Culture
Collection no.a
I (0.02) +
II
(0.10)
III (0.44)
IV
(0.62)
ATCC 8688 + Cunninghamella blakesleena NRRL 3665 + + + Cunninghamella echinulata ATCC 11395 + + + Microsporum gypseum + ATCC 13378 + + + Sepedonium chyrosperum + + + + UI 1158 Streptomyces griseus + ATCC 25466 Streptomyces lincolnensis + + -ATCC 13865 Streptomyces platenses NRRL 3529 Streptomyces punipulas ATCC, American Type Culture Collection, Rockville, Md.; NRRL, Northern Regional Research Laboratories, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Ill.; UI, University of Iowa, College of Pharmacy Culture Collection, Iowa City. b Rf when TLC plates were developed with solvent system A. a
tive abundance]: 162 [18], 161 [15], 133 [36], 84 [100]; nicotine standard: 162 [22], 161 [21], 133 [32], 84 [100]; literature values [11]: 162 [17], 161 [17], 133 [32], 84 [100]). DISCUSSION Microbial transformations were explo;ed as a possible means of converting nicotine to nornicotine. Microorganisms previously found to accomplish N-demethylations in our laboratories and those reported in the literature to have potential for N-dealkylation were selected (1, 2,
otine) when nornicotine was used as a substrate in growing cultures. Large (19) proposed three possible pathways for the N-dealkylation of 30 amines by microorganisms. One of these involves a monooxygenase system which converts a tertiary amine to the secondary amine through an intermediary tertiary amine-N-oxide. The possibility that nicotine-1'-N-oxide was formed as an intermediate in the N-demethylation of nicotine by M. gypseum was examined. Unexpectedly, the N-oxide was efficiently reduced back to nicotine in cellfree systems. Controls consisting of N-oxide suspended in sterile phosphate buffer (pH 6.8) and in dimethyl formamide (substrate addition solvent) showed no reduction to nicotine even after 240 h. When nicotine-1'-N-oxide was added to growing cultures of M. gypseum, no reduction was evident until 168 h, when cell lysis was visible and the N-oxide was reduced to nicotine in greater than 80% yield. Microbial reduction of tertiary amine-N-oxides has been reported for pyridine-N-oxide (22) and trimethylamine-Noxide (29). Interest in this type of reduction derives from the anaerobic reduction of nicotine1'-pxode in vitro with mammalian cells (6) and its reduction in the gastrointestinal tract of humans (16). Microbial N-demethylation of nicotine was observed, but this reaction is not of preparative value. This is the first report of the microbial reduction of nicotine-l'-oxide, although the Noxide has been examined as a substrate for bacterial metabolism (20).
23). Of the cultures examined, only Streptomyces lincolnensis and S. punipalus failed to metabolize nicotine at all. All metabolites appeared late in the fermentations; although four metabolites (I to IV) were formed, no microorganism yielded a single compound; and at least three organisms produced all metabolites. M. gypseum was selected for preparative scale incubations in an attempt to obtain nicotine metabolites I to IV. Chromatographic and mass spectral evidence supports the structure of metabolite III as nornicotine. However, the other three metabolites could not be unequivocally identified. The low conversion of nicotine to its metabolites afforded only enough of metabolite III to obtain an interpretable mass spectrum. TLC comparisons (systems A, B, and C) with synthetic nicotine derivatives suggested that metabolite II was probably nicotine-l'-N-oxide. However, metabolite II did not give the characteristic color reaction when the TLC plates were detected with Konigs reaction (12). Metabolite I had chromatographic mobilities (systems A, B, ACKNOWLEDGMENTS and C) nearly identical to those of synthetic We thank N. Castagnoli (Medical Center, University of nicotine-1,1'-N-oxide. Metabolite IV was deSan Francisco) for the gift of dideuteronornicotine tected last and produced in the lowest yield of California, and J. Browne for assistance in the synthesis of TLC standall compounds. It did not appear to be formed ards. by further metabolism of metabolite III (nornicThis study was supported in part by Public Health Service
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N-DEMETHYLATION OF NICOTINE
grant GM-22365 from the National Institute of General Medical Sciences.
of the carbon-nitrogen bond cleavage by chloroformate esters. Chimia 28:665-666. Iizuka, H., and A. Naito. 1967. Microbial transformations of steroids and alkaloids, p. 271-272. University of Tokyo Press, Tokyo. Jenner, P., J. W. Gorrod, and A. H. Beckett. 1973. The absorption of nicotine-l'-N-oxide and its reduction in the gastro-intestinal tract in man. Xenobiotica 3: 341-349. Kieslich, K. 1976. Microbial transformations of nonsteroid cyclic compounds, p. 205-210. George Thieme, Stuttgard. Kisaki, T., and E. Tamaki. 1961. Phytochemical studies on the tobacco alkaloids. 1. Optical rotatory power of nornicotine. Arch. Biochem. Biophys. 92:351-355. Large, P. J. 1971. The oxidative cleavage of alkyl-nitrogen in microorganisms. Xenobiotica 1:457-467. Maeda, S., S. Uchida, and T. Kisaki. 1978. Microbial degradation of nicotine-N'-oxide. I. Degradation products. Agric. Biol. Chem. 42:1455-1460. Marion, L. 1950. The pyridine alkaloids, p. 245. In R. Manske and H. Holmes (ed.), The alkaloids-chemistry and physiology, vol. I. Academic Press, New York. May, A. 1957. Bioreduction of pyridine-N-oxide. Enzymologia 18:142-144. Mitscher, L. A., W. W. Andres, G. 0. Morton, and E. L. Patterson. 1968. Microbiological transformation of 6,14-endo-ethenotetrahydrothebaine alkaloids. Experientia 24:133-134. Picot, A., and X. Lusinchi. 1975. The formation of lactams by selective oxidation of cyclic tertiary amines with bromine under basic conditions. Synthesis, p. 109111. Sauer, J. C. 1963. Laurone, p. 561. In N. Rabjohn (ed.), Organic synthesis, collective vol. 4. J. Wiley & Sons, New York. Schievelbein, H., and R. Eberhardt. 1972. Cardiovascular actions of nicotine and smoking. J. Natl. Cancer Inst. 48:1785-1794. Schmidt, J. 1966. Uber nikotinabbau durch mikroorganismen in modellversuchem. Beitr. Tabakforsch. 3:539-
15. 1.
2.
3.
4. 5. 6. 7.
8. 9.
LITERATURE CMD Argoudelis, A. D., J. H. Coats, D. J. Mason, and 0. K. Sebek. 1969. Microbial transformation of antibiotics. III. Conversion of clindamycin to 1'-demethylcindamycin and clindamycin sulfoxide by Streptomyces species. J. Antibiot. Ser. A 22:309-314. Bellet, P., and T. V. Truong. 1970. La N-denmthylajmaline. Ann. Pharm. Fr. 28:245-249. Betts, R. E., D. E. Walters, and J. P. Rosazza. 1974. Microbial transformations of antitumor compounds. 1. Conversion of acronycine to 9-hydroxyacronycine by Cunninghamella echinulata. J. Med. Chem. 17:599602. Billman, J., and A. Diesing. 1957. Reduction of Schiff bases with sodium borohydride. J. Org. Chem. 22:10681070. Bobbitt, J. M. 1963. Thin-layer chromatography, p. 93. Reinhold, New York. Booth, J., and E. Boyland. 1971. Enzymatic oxidation of (-)-nicotine by guinea-pig tissues in vitro. Biochem. Pharmacol. 20:407-415. Brandange, S., and L. Lindblom. 1976. N-Vinyl as N-H protecting group. A convenient synthesis of myosmine. Acta Chem. Scand. Sect. B 30:93. Craig, J. C., and K. Purushothaman. 1970. An improved preparation of tertiary amine-N-oxides. J. Org. Chem. 35:1721-1722. Davis, P. J., and J. P. Rosazza. 1976. Microbial transformations of natural antitumor agents. 2. Studies with d-tetrandrine and laudanosine. J. Org. Chem. 41:2548-
16.
17. 18.
19. 20. 21. 22.
23.
24.
25.
2551. 10. DeMoss, R. D., and R. C. Bard. 1957. Physiological and biochemical technics, p. 175-176. In H. J. Conn (ed.), Manual of microbiological methods. McGraw-Hill, New York. 11. Duffield, A. M., H. Budzikiewicz, and C. Djerassi. 1965. Mass spectrometry in structural and stereochemical problems. LXXII. A study of the fragmentation processes of some tobacco alkaloids. J. Am. Chem. Soc. 87:2926-2932. 12. Fejer-Kossey, 0. 1967. The separation of ten tobacco alkaloids by thin-layer chromatography. J. Chromatogr. 31:592-593. 13. Hodgson, E., E. Smith, and F. Guthrie. 1965. Two dimensional thin layer chromatography of tobacco alkaloids and related compounds. J. Chromatogr. 20:176177. 14. Hootele, C., and J. P. Lenders. 1974. Stereochemistry
26. 27.
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544. 28. Smith, R., and J. P. Rosazza. 1975. Microbial systems for study of the biotransformations of drugs. Biotechnol. Bioeng. 17:785-814. 29. Unemoto, T., M. Hayashi, K. Miyaki, and M. Hayashi. 1965. Intracellular localization and properties of tri-
methylamine-N-oxide reductase in Vibrio parahaemolyticus. Biochim. Biophys. Acta 110:319-328. 30. Vining, L. 1969. Microbial transformation of alkaloids, p. 720-721. In D. Perlman (ed.), Fermentation advances. Academic Press, New York.
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N-Demethylation of Nicotine and Reduction of Nicotine-1'-NOxide by Microsporum gypseum ROBERT D. SINDELAR, JOHN P. ROSAZZA, AND CHARLES F. BARFKNECHT* College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242
Received for publication 6 August 1979
Several microorganisms were examined for their abilities to convert S-nicotine into nornicotine. Five microorganisms including Microsporum gypseum (ATCC 11395) produced nornicotine and three unknown metabolites. M. gypseum efficiently reduced nicotine-1'-N-oxide to nicotine, but no nornicotine was obtained when the N-oxide was used as substrate.
S-Nicotine (Fig. la) elicits a number of pharmacological responses in mammalian systems (26). Our investigations of the cardiovascular effects of nicotine necessitated a supply of Snornicotine (Fig. lb). Nornicotine is not readily available commercially, and its stereochemistry and purity are questionable (18). Literature methods for the preparation of S-nornicotine are complicated by side reactions and low yield (14, 21). Although microbial degradation of nicotine and tobacco alkaloids has been studied in detail by a number of groups (15, 17, 27, 30), little emphasis was given to the N-demethylation reaction. Since microbial N-demethylation of several alkaloids occurs (28), we attempted to develop microbial systems which could provide the required nornicotine. This report describes results obtained when selected cultures were examined for their abilities to achieve N-demethylation of nicotine, and describes the metabolic fate of nicotine-l'-N-oxide (Fig. lc), a possible intermediate in the N-demethylation reaction. MATERIALS AND METHODS Chromatographic procedures. Thin-layer chromatography (TLC) was performed on 0.25-mm-thick layer plates of silica gel GF254 or 1.0-mm-thick layer plates of silica gel G (Analtech). Plates were ovenactivated for 15 min at 130°C before use. Solvent systems used in developing TLC plates were (A) CHCl3-MeOH-58% NH40H (60:10:1) (13); (B) C6H6ethanol (3:1); (C) CHCl3-MeOH-glacial acetic acid (60:10:1) (13). Visualization involved viewing developed plates under short (254 nm) and long (365 nm) wavelength ultraviolet light. The plates were later sprayed with Dragendorff reagent (5), followed by a solution of polyphosphoric acid-water (1:1). Chemicals. Nicotine (Eastman) was treated with phenyl isocyanate to remove secondary amine contaminants (25) and fractionally distilled. Nicotine-l'N-oxide and nicotine-i, l'-di-N-oxide were synthesized by the method of Craig and Purushothaman (8). Myosmine was prepared according to Brandange and Lind-
blom (7) and reduced to racemic nornicotine with NaBH4 (4). N. Castagnoli (Medical Center, University of California, San Francisco) supplied a sample of 4',4'dideuteronornicotine. Cotinine was prepared by a literature method (24). All compounds were chromatographically pure and were fully characterized by nuclear magnetic resonance and mass spectral methods. Fermentation procedures. Cultures used in this study are maintained in the University of Iowa College of Pharmacy culture collection and are stored in sealed screw-cap tubes at 4°C in a refrigerator. Organisms were grown in a medium of the following composition: soybean meal (5 g), glucose (20 g), yeast extract (5 g), NaCl (5 g), K2HPO4 (5 g), and distilled water to 1.0 liter; pH was adjusted to 7.0 with 5 N HCI. Media were sterilized in an autoclave at 121°C for 15 min. Fermentations were conducted in cotton-plugged Erlenmeyer flasks containing one-fifth of their volume of medium. A standard two-stage fermentation protocol was used for all incubations (3). The surface growth from fresh agar slants of microorganisms was suspended in sterile medium, and the resulting vegetative or spore suspension was used to inoculate firststage cultures. First-stage cultures were incubated with shaking at 250 rpm at 27°C on rotary shakers for 72 h, during which time cultures attained maximum growth. Actively growing stage I cultures were then used to inoculate stage II cultures, which were incubated for 24 h before receiving nicotine. Substratecontaining flasks were incubated with shaking, and samples (5 ml) were withdrawn at 24, 72, 96, 120 and 144 h. Substrate added to sterile medium and fermentation blanks containing no substrate served as controls. Large-scale fermentation of Microsporum gypseum. Stage II cultures of M. gypseum were grown in a total of 2.4 liters of medium held in 12 1.0-liter flasks. Repeated serial transfer of M. gypseum cultures from slant to slant resulted in diminishing ability of the organism to produce metabolites. Therefore, M. gypseum cultures which had been transferred no more than two times were used for experiments. Nicotine (100 mg in 0.8 ml of dimethyl formamide) was added to each culture flask. Formation of metabolites was followed by TLC (solvent system A). Fermentations were harvested at 168 h, and the contents of all flasks
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were pooled, adjusted to pH 10.0 with aqueous K2CO3, and exhaustively extracted with CHCL3. The extract was dried (Na2SO4) and evaporated in vacuo to give a crude extract (0.9 g). The crude extract was adsorbed as a band on 1.0-mm-thick layer plates of silica gel G and was developed with solvent system A. Each separated band was scraped from the plate, extracted with CHC13, and concentrated in vacuo. Low-resolution mass spectral data were obtained on the band corresponding to nornicotine on a Finnigan 3200 spectrometer. mle (percent relative abundance) was 148 (26), 147 (37), 120 (26), 119 (100), and 70 (5); TLC Rf values were 0.44 (system A), 0.06 (system B), and 0.03 (system C).
RESULTS Preliminary small-scale fermentation experiments were conducted with eight cultures which had previously demonstrated N-demethylating ability with a variety of alkaloid substrates. Six of the microorganisms examined produced four different metabolites of nicotine (Table 1). The total amount of nicotine converted to metabolites was estimated to be less than 20% by TLC. Not all metabolites were produced simultaneously, and their order of appearance was II (96 h), I and III (120 h), and IV (144 h). Metabolite IV was not always formed. A preparative scale fermentation was performed with M. gypseum to obtain sufficient quantities of metabolites for isolation and structure elucidation. Metabolite II was detected after 72 h. Little increase in conversion to metabolites was observed after 120 h, and fermentations were harvested and pooled 168 h after substrate addition. After extraction, 0.9 g of crude metabolites was isolated. The largest portion of the crude extract was unmetabolized nicotine. Chromatographic comparison of the mixture of metabolites showed that metabolite III had the same Rf as synthetic nornicotine and dideuteronornicotine. The mixture of nicotine metabolites was separated by preparative TLC. An electron impact mass spectrum of isolated metabolite III was identical to that of synthetic nornicotine and that reported in the literature (12) (observed [percent relative abundance]:) 148 [26], 147 [37], 120 [26], 119 [100], and 70 [5]; literature values [11]: 148 [25], 147 [35], 120 [30], 119 [100], and 70 [85]). The fragmentation pattern of dideuteronornicotine corresponded well with that of metabolite III (for dideuteronornicotine, observed [percent relative abundance]: 150 [29], 149 [36], 120 [30], 119 [100], and 72 [25]). This metabolite exhibited one spot when cochromatographed (systems A, B, and C) with synthetic nornicotine and dideuteronornicotine. The data collected for metabolite III are consistent for nornicotine. Unequivocal identifi-
N-DEMETHYLATION OF NICOTINE
837
6 I
Fig. laX=N-CH3 bX=N- H cX=N-CH3 0 FIG. 1. S-Nicotine (a), S-nornicotine (b), and nicotine-l'-N-oxide (c).
cation of the other metabolites was not possible due to low yield. Resting cell cultures of M. gypseum were examinedYfor their abilities to produce metabolites (11). Cells were incubated with 0.1 M phosphate buffer (pH 7.0) plus 0.25% glucose and with buffer only. The glucose concentration used was that which normally remained in 24-h-old stage II cultures. The fermentation containing glucose converted nicotine to metabolites I and II 72 h after addition of substrate, whereas the same metabolites were formed in the fermentation without glucose 48 h after substrate addition. Metabolites III and IV were not detected. These results suggested a possible catabolite repressive effect for glucose in this transformation reaction. Since nicotine-l'-N-oxide is a plausible intermediate in the N-demethylation of the alkaloid, the N-oxide was used as the substrate in a number of experiments. M. gypseum cell homogenates (Sorvall Omni-Mixer, 10,000 rpm, 75-ml cannister) reductively converted more than 80% of nicotine-1'-N-oxide to nicotine after 12 h, and the reaction went almost to completion in 24 h when the N-oxide was added to 10,000 x g supernatants. The cellular debris fraction also produced nicotine in greater than 50% yield (estimated yields are based on visual evaluation of TLC plates). A trace amount of metabolite I was detected after 36 h. Nicotine was identified as the metabolite of the N-oxide by TLC (systems A, B, and C) comparison with comercially available nicotine. The electron impact mass spectrum of the isolated metabolite was identical to that of a nicotine standard and to one reported in the literature (11) (metabolite [percent rela-
838
APPL. ENVIRON. MICROBIOL.
SINDELAR, ROSAZZA, AND BARFKNECHT TABLE 1. Production of nicotine metabolites
Rf values of metabolitesb Culture
Collection no.a
I (0.02) +
II
(0.10)
III (0.44)
IV
(0.62)
ATCC 8688 + Cunninghamella blakesleena NRRL 3665 + + + Cunninghamella echinulata ATCC 11395 + + + Microsporum gypseum + ATCC 13378 + + + Sepedonium chyrosperum + + + + UI 1158 Streptomyces griseus + ATCC 25466 Streptomyces lincolnensis + + -ATCC 13865 Streptomyces platenses NRRL 3529 Streptomyces punipulas ATCC, American Type Culture Collection, Rockville, Md.; NRRL, Northern Regional Research Laboratories, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Ill.; UI, University of Iowa, College of Pharmacy Culture Collection, Iowa City. b Rf when TLC plates were developed with solvent system A. a
tive abundance]: 162 [18], 161 [15], 133 [36], 84 [100]; nicotine standard: 162 [22], 161 [21], 133 [32], 84 [100]; literature values [11]: 162 [17], 161 [17], 133 [32], 84 [100]). DISCUSSION Microbial transformations were explo;ed as a possible means of converting nicotine to nornicotine. Microorganisms previously found to accomplish N-demethylations in our laboratories and those reported in the literature to have potential for N-dealkylation were selected (1, 2,
otine) when nornicotine was used as a substrate in growing cultures. Large (19) proposed three possible pathways for the N-dealkylation of 30 amines by microorganisms. One of these involves a monooxygenase system which converts a tertiary amine to the secondary amine through an intermediary tertiary amine-N-oxide. The possibility that nicotine-1'-N-oxide was formed as an intermediate in the N-demethylation of nicotine by M. gypseum was examined. Unexpectedly, the N-oxide was efficiently reduced back to nicotine in cellfree systems. Controls consisting of N-oxide suspended in sterile phosphate buffer (pH 6.8) and in dimethyl formamide (substrate addition solvent) showed no reduction to nicotine even after 240 h. When nicotine-1'-N-oxide was added to growing cultures of M. gypseum, no reduction was evident until 168 h, when cell lysis was visible and the N-oxide was reduced to nicotine in greater than 80% yield. Microbial reduction of tertiary amine-N-oxides has been reported for pyridine-N-oxide (22) and trimethylamine-Noxide (29). Interest in this type of reduction derives from the anaerobic reduction of nicotine1'-pxode in vitro with mammalian cells (6) and its reduction in the gastrointestinal tract of humans (16). Microbial N-demethylation of nicotine was observed, but this reaction is not of preparative value. This is the first report of the microbial reduction of nicotine-l'-oxide, although the Noxide has been examined as a substrate for bacterial metabolism (20).
23). Of the cultures examined, only Streptomyces lincolnensis and S. punipalus failed to metabolize nicotine at all. All metabolites appeared late in the fermentations; although four metabolites (I to IV) were formed, no microorganism yielded a single compound; and at least three organisms produced all metabolites. M. gypseum was selected for preparative scale incubations in an attempt to obtain nicotine metabolites I to IV. Chromatographic and mass spectral evidence supports the structure of metabolite III as nornicotine. However, the other three metabolites could not be unequivocally identified. The low conversion of nicotine to its metabolites afforded only enough of metabolite III to obtain an interpretable mass spectrum. TLC comparisons (systems A, B, and C) with synthetic nicotine derivatives suggested that metabolite II was probably nicotine-l'-N-oxide. However, metabolite II did not give the characteristic color reaction when the TLC plates were detected with Konigs reaction (12). Metabolite I had chromatographic mobilities (systems A, B, ACKNOWLEDGMENTS and C) nearly identical to those of synthetic We thank N. Castagnoli (Medical Center, University of nicotine-1,1'-N-oxide. Metabolite IV was deSan Francisco) for the gift of dideuteronornicotine tected last and produced in the lowest yield of California, and J. Browne for assistance in the synthesis of TLC standall compounds. It did not appear to be formed ards. by further metabolism of metabolite III (nornicThis study was supported in part by Public Health Service
VOL. 38, 1979
N-DEMETHYLATION OF NICOTINE
grant GM-22365 from the National Institute of General Medical Sciences.
of the carbon-nitrogen bond cleavage by chloroformate esters. Chimia 28:665-666. Iizuka, H., and A. Naito. 1967. Microbial transformations of steroids and alkaloids, p. 271-272. University of Tokyo Press, Tokyo. Jenner, P., J. W. Gorrod, and A. H. Beckett. 1973. The absorption of nicotine-l'-N-oxide and its reduction in the gastro-intestinal tract in man. Xenobiotica 3: 341-349. Kieslich, K. 1976. Microbial transformations of nonsteroid cyclic compounds, p. 205-210. George Thieme, Stuttgard. Kisaki, T., and E. Tamaki. 1961. Phytochemical studies on the tobacco alkaloids. 1. Optical rotatory power of nornicotine. Arch. Biochem. Biophys. 92:351-355. Large, P. J. 1971. The oxidative cleavage of alkyl-nitrogen in microorganisms. Xenobiotica 1:457-467. Maeda, S., S. Uchida, and T. Kisaki. 1978. Microbial degradation of nicotine-N'-oxide. I. Degradation products. Agric. Biol. Chem. 42:1455-1460. Marion, L. 1950. The pyridine alkaloids, p. 245. In R. Manske and H. Holmes (ed.), The alkaloids-chemistry and physiology, vol. I. Academic Press, New York. May, A. 1957. Bioreduction of pyridine-N-oxide. Enzymologia 18:142-144. Mitscher, L. A., W. W. Andres, G. 0. Morton, and E. L. Patterson. 1968. Microbiological transformation of 6,14-endo-ethenotetrahydrothebaine alkaloids. Experientia 24:133-134. Picot, A., and X. Lusinchi. 1975. The formation of lactams by selective oxidation of cyclic tertiary amines with bromine under basic conditions. Synthesis, p. 109111. Sauer, J. C. 1963. Laurone, p. 561. In N. Rabjohn (ed.), Organic synthesis, collective vol. 4. J. Wiley & Sons, New York. Schievelbein, H., and R. Eberhardt. 1972. Cardiovascular actions of nicotine and smoking. J. Natl. Cancer Inst. 48:1785-1794. Schmidt, J. 1966. Uber nikotinabbau durch mikroorganismen in modellversuchem. Beitr. Tabakforsch. 3:539-
15. 1.
2.
3.
4. 5. 6. 7.
8. 9.
LITERATURE CMD Argoudelis, A. D., J. H. Coats, D. J. Mason, and 0. K. Sebek. 1969. Microbial transformation of antibiotics. III. Conversion of clindamycin to 1'-demethylcindamycin and clindamycin sulfoxide by Streptomyces species. J. Antibiot. Ser. A 22:309-314. Bellet, P., and T. V. Truong. 1970. La N-denmthylajmaline. Ann. Pharm. Fr. 28:245-249. Betts, R. E., D. E. Walters, and J. P. Rosazza. 1974. Microbial transformations of antitumor compounds. 1. Conversion of acronycine to 9-hydroxyacronycine by Cunninghamella echinulata. J. Med. Chem. 17:599602. Billman, J., and A. Diesing. 1957. Reduction of Schiff bases with sodium borohydride. J. Org. Chem. 22:10681070. Bobbitt, J. M. 1963. Thin-layer chromatography, p. 93. Reinhold, New York. Booth, J., and E. Boyland. 1971. Enzymatic oxidation of (-)-nicotine by guinea-pig tissues in vitro. Biochem. Pharmacol. 20:407-415. Brandange, S., and L. Lindblom. 1976. N-Vinyl as N-H protecting group. A convenient synthesis of myosmine. Acta Chem. Scand. Sect. B 30:93. Craig, J. C., and K. Purushothaman. 1970. An improved preparation of tertiary amine-N-oxides. J. Org. Chem. 35:1721-1722. Davis, P. J., and J. P. Rosazza. 1976. Microbial transformations of natural antitumor agents. 2. Studies with d-tetrandrine and laudanosine. J. Org. Chem. 41:2548-
16.
17. 18.
19. 20. 21. 22.
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24.
25.
2551. 10. DeMoss, R. D., and R. C. Bard. 1957. Physiological and biochemical technics, p. 175-176. In H. J. Conn (ed.), Manual of microbiological methods. McGraw-Hill, New York. 11. Duffield, A. M., H. Budzikiewicz, and C. Djerassi. 1965. Mass spectrometry in structural and stereochemical problems. LXXII. A study of the fragmentation processes of some tobacco alkaloids. J. Am. Chem. Soc. 87:2926-2932. 12. Fejer-Kossey, 0. 1967. The separation of ten tobacco alkaloids by thin-layer chromatography. J. Chromatogr. 31:592-593. 13. Hodgson, E., E. Smith, and F. Guthrie. 1965. Two dimensional thin layer chromatography of tobacco alkaloids and related compounds. J. Chromatogr. 20:176177. 14. Hootele, C., and J. P. Lenders. 1974. Stereochemistry
26. 27.
839
544. 28. Smith, R., and J. P. Rosazza. 1975. Microbial systems for study of the biotransformations of drugs. Biotechnol. Bioeng. 17:785-814. 29. Unemoto, T., M. Hayashi, K. Miyaki, and M. Hayashi. 1965. Intracellular localization and properties of tri-
methylamine-N-oxide reductase in Vibrio parahaemolyticus. Biochim. Biophys. Acta 110:319-328. 30. Vining, L. 1969. Microbial transformation of alkaloids, p. 720-721. In D. Perlman (ed.), Fermentation advances. Academic Press, New York.
