Beitrage zur Tabakforschung International· Volume 9 ·No. 5 ·December 1978

Transformation of Tobacco Alkaloids* by T. Kisaki, S. Maeda, A. Koiwai, Y. Mikami, T. Sasaki and H. Matsushita Central Research Institute, The Japan Tobacco and Salt Public Corporation, Yokohama, Japan

This is a review of the work on the transformation of We found another rearrangement reaction of the oxide. tobacco alkaloids which has been carried out at the Upon acyl halides' or anhydrides' being reacted with Japan Tobacco and Salt Public Corporation in the past nicotine-N' -oxide by triturating it with a glass roq in a ten years. glass flask, the oxide was spontaneously rearranged giv­ Figure 1 shows the transformation of N-methylpyrro­ ing an N-acylated butylamine derivative [IV]. When lidines and N-methylpiperidine oxides. Nicotine oxide [11] this reaction occurs under cooled conditions, then a near­ is easily formed from nicotine under natural conditions, theoretical yield of N' -acylated pseudooxynicotine can and it is stable at room temperature. Rayburn {1) has be obtained. When the N'-methylmyosmine [V] was shown that the oxide is rearranged to 2-methyl-6-(3'­ heated under vacuum, nicotyrine [VI], N-methylnico­ pyridyl)tetrahydro-1,2-oxazine [Ill] by heating. tinamide [VII], other pyridine compounds and a large

• Presented at the 6th International Tobacco Scientific Congress (Coresta) held in Tokyo, Japan, in November 1976.

Figure 1. Reaction of nlcotlne-N'-oxlde.

o-9 + (0) ~ . I CHJ o-90CH3 11 ~v•. ~ N <(. CHJ i~ vacuum llMJ '~-~ C-NHCH3 lit CH3 V erN Vlt

Reaction of N-methylpyrrolldlne and N-methylplperldlne oxide.

0 Q 11 I ~0 (R-C)20 CHJ -:,0 R-c 'X Q 0I ~0 I CH3 CH3

1: nicotine 11: nlcotlne-N'-oxlde IV: N-acyi-N-methyl-4-oxo-4-(3'-pyrldyl)butylamlne VI: nlcotyrlne Ill: 2-methyl-6-(3'-pyrldyl)tetrahydro-1;2-oxazlne V: N'-methylmyosmlne VII: N-methylnlcotlnamlde

308 amount of resin were obtained. This is very suggestive from N' -methylmyosmine. When a nicotine salt solution of natural nicotine degradation. Nicotine-N'-oxide [11] was heated at 200 °C under vacuum in an autoclave is formed during fermentation, but it is stable at room to determine the manner of initial pyrolytic degradation temperature. However, a nucleophilic attack on the oxide of nicotine, nornicotine was obtained as a major product [11] would probably occur during fermentation, so that together with metanicotine and dihydrometanicotine. In the formed unstable N' -methylmyosmine [V] would be all the detected degradation products, the C-N bonds of transformed to give various further transformed com­ the pyrrolidine ring moiety were cleaved (5, 6). pounds. This rearrangement reaction of the N'-oxide [11] In the tobacco plant, nicotine has been regarded as a could be used for the preparation of L\1-pyrrolines or secondary metabolite. However, dealkylation of nic­ L\1-piperideines (2, 3). N' -methylmyosmine is regarded otine commonly occurs in most wild species and some as a key compound in nicotine degradation. However, cultivars. We tried to establish a structural minimum there has been no information confirming the structure requirement of the dealkylation system of tobacco alka­ of so-called "N'-methylmyosmine". We obtained N'­ loids in tobacco plants, using a number of derivatives of methylmyosmine (b.p. 90-93 °C, 2 mmHg) by reflux­ tobacco alkaloids and leaves of the Cherry Red strain ing pseudooxynicotine with benzene and confirmed the of the Bright yellow variety of Nicotiana tabacum L., as structure of 1-methyl-2-(3'-pyridyl)-2-pyrroline by com­ Dawson (7) has worked using a limited number of to­ paring with 2-methyl-1-(2'-methyl-6'-cyclohexene-1'-yl)­ bacco alkaloid derivatives. Experimental leaves were pre­ pyrrolidine by means of lH-NMR (18). pared by grafting the tobacco scion on a tomato stock. Frankenburg and eo-workers (4) have demonstrated a The substrates were fed from the end of the petiole of number of degradation products of nicotine in cigar to­ the leaves and analyzed. The substrates in the feeding bacco. We conducted an experiment on nicotine aeration experiment were prepared by chemical synthesis or isola­ to determine the oxidation of nicotine by air at room tion from natural sources. As shown in Table 1, among temperature. A number of products were identified, as compounds analogous to nicotine, both R- and S-nic­ seen in Fig. 2. A large part of the products was nicotine­ otine (pKa 1 = 7.9, pKa 2 = 3.1), N'-methylanabasine, N' -oxide. N'-Methylmyosmine was first identified as a nicotine-N' -oxide (being reduced to nicotine) and 6- degradation product. Most compounds, except cotinine hydroxynicotine were demethylated. No nicotyrine and nicotine-N' -oxide, are supposedly derived from N'­ (pKa 1 = 4.9) and cotinine (pKa 1 = 4.5) resulted in the methylmyosmine. Nicotinic acid must be derived from demethylated compound. From these result~ it was in­ N-methylnicotinamide which has in turn been derived ferred that the methylated nitrogen atom is required to

Figure 2. Air oxidation and low-temperature pyrolysis of nicotine.

0 N air a-?N~ CH3 I ~0N 11 CH3 N Ill o.-9CH3

30 °C, 4 weeks N I 0 er-9N IV CH3 o:-0N V o-flN VI

oC-CH3 11 o-COOH NHCH3 NH3 N 0 N VII VIII 3days

ll~H3 .. ) ~ c er9N IX a-?N X CH3 N VI ~ o-0 lN~ HN: N V XI CH3

1: cotlnlne IV: N"-methylmyosmlne VII: 3-pyrldylmethyl ketone 11: nlcotlne-N'-oxlde V: myosmlne VIII: nicotinic acid X: dlhydrometanlcotlne Ill: nlcotyrlne VI: 3-pyrldylpropyl ketone IX: nornlcotlne XI: metanlcotlne

309 Table 1. Demethylatlon of tobacco alkaloid analogues In tobacco leaves.

Substrata Product Substrata Product

(partially Py--CJ racemic) L-~ H none ~ ..N JJ '!~CH,,

OL-Py--C:J (0-domlnant) I CH, o-q HO N CH, HOo-9 N

Py-0 none N I none CH,

L-Py-Oo none I CH, none

OL-Py-0 Py-0 (0-domlnant) I H CH3 none L-Py-CJ L-Py--CJ Py--Q l"'llo I H none CH3 CH, (partially racemic)

L-Py_()._ none none CH3

none o--7N CH, PyD 0 NH I none CH, none PyJf] H,c 1 I none CH, Py -,P,.N,..) o" I none CH, Dl-Py-r:J HO NH I none DL-Py-CJ CH, I none CO I Pyn .. CH, N-CH, I none Dl-Py-0 CH, none I CHO CH, CH, CH3 CH,

Dl-Py ~ ) N ) (D-dominant) H N Py...J..... I H H CH, Py-Q H

Py- CH2 CH, 'N""" I CH, Figure 3. Minimum requirement of the structure for de- Figure 5. Transformation of anabasine In Nlcotlana taba­ alkylation of nicotine analogues In tobacco leaves. cum.

D-domlnant In R Ef> R' ~ 0 ()"' ~~- . N. tabacum L ,N: and H CH. N cPN o+N 'R" Anabasine Anabaseine R- or :1= R' R- or :1= R" further transformed in the plant. The metabolite was identified as anabaseine by isolation (12). The trans­ be ionized for demethylation under physiological con­ formation pathway of anabasine in tobacco leaves is ditions. The anionic site would be present in the de­ shown in Fig. 5. alkylation enzyme system. No alicyclic amine such as We have noticed the presence of a fairly large amount pyrrolidine or piperidine was necessarily required for the of an isatin-positive alkaloid (I-A) other than nornic­ dealkylation substrate. An appropriate distance (approx. otine in the Cherry Red strain of converter-type tobacco 4.16 A) between the nitrogen atom in the pyridine and (14), as shown in the paper chromatogram (Fig. 6). I-A the nitrogen atom to be dealkylated is required. The was isolated from Cherry Red leaves of Nicotiana taba­ dealkylation was not confined to methyl groups. Experi­ cum L., Bright yellow, by the sequential procedure of mentally, we could work out the structural minimum methanol extraction, cation exchange chromatography, requirement of the dealkylation system in tobacco leaves partition chromatography using tert-amylalcohol-acetate as shown in Fig. 3 (8). buffer and HPLC. I-A was isolated in a resinous form. Tobacco alkaloids are secondary products which have This was crystallized by treatment with chloroform and been regarded as metabolically inert substances except purified by recrystallization from chloroform and liquid their N-methyl group. The N-methyl group of nicotine chromatography [m.p. 66-68 °C (decomposition), is fairly well metabolized. L-Nicotine was demethylated [a]~ = -83.80° (c = 1.08 in H20)]. A high-pressure to give partially racemized L-nornicotine (9). We proved further transformation of nornicotine and anabasine in liquid chromatogram of the methanol extract of tobacco is shown in Fig. 7. Color test, hydrolysis, .and spectral tobacco leaves by feeding optically inactive ones and by data (lH- and lSC-NMR, MS, IR and UV}'sllggest that measuring the optical activity of recovered alkaloids. As seen in Table 2, the optically inactive alkaloids were transformed into optically active compounds. Nicotine Table 2. Transformation of nicotine and nornlcotlne In Nlcotlana tabacum L (Cherry Red) leaves. was predominantly demethylated in D-form, and nor­ nicotine was predominantly dehydrogenated to myos­ DL-nicotine feeding DL-nornicotlne feeding mine in L-form (10, 11, 12). The transformation path­ 4 4 way of nicotine in tobacco leaves is shown in Fig. 4. hours la1& weeks [al& Recently, myosmine was also confirmed as a metabolite in the leaves by Leete and his eo-workers (13). 0 0 0 0 Anabasine is a principal alkaloid in Nicotiana glauca L. 64 -4.57 2 +0.98 and also a common minor alkaloid in N icotiana tab a­ 112 -6.76 4 +2.62 cum L. The anabasine isolated from the root of Nico­ 160 -10.12

tiana glauca L. showed a little levo rotation and the one Nicotine and nornlcotlne were recovered at the Indicated time after in the leaf showed dextrorotation (Table 3). These facts feeding CL-nicotine and DL-nornlcotlne. suggest that anabasine in Nicotiana glauca L. is first bio­ synthesized in the racemic form and the D-enantiomer Table 3. Transformation of anabasine In Nlcotlana glauca is predominantly degraded 1in the root, wher_eas the L­ L. and Nlcotlana tabacum L. enantiomer is predominantly degraded in the leaf. The stereospecificity of the degradation of anabasine in the Anabasine tal ~4 leaves of Nicotiana tabacum L. was shown in the feed­ ing experiments to be of reverse order. The variation of in N. glauca L.* 88days 115 days 145 days stereospecificity of anabasine degradation remains to be explained. The optical rotation increased from 0 to -2.9 Root -2.09 -0.23 -3.91 after a week (Table 3). This suggests that anabasine is Stalk -2.13 +0.28 -1.12 Leaf -1.02 +1.36 +2.12 Figure 4. Transformation of nicotine and nornlcotlne In Nlcotlana tabacum (Cherry Red) leaves. in 1)1. tabacum L.** 0 hours 116 hours 164 hours Leaf 0.00 -2.08 -2.90

~D D-domlnant • Anabasine was Isolated from the root, stalk and leaf of Nlcotlana glauca L. at .the Indicated time after sowing. lN~ bH. •• Anabasine was recovered at the Indicated time after feeding DL· Nicotine Nornicotine Myosmine anabasine;

311 Figure 8. Paper chromatogram of methanol extract of Figure 8. Structure of the lsatln-posltlve alkaloid (1-A). Cherry Red strain of converter-type tobacco. Samples were developed In descending order on Whatman No. 1 HOCV~~H dried after impregnation with 0.~ N sodium acetate in the solvent system of the upper layer [tert-amyl alcohol • 0.2 N sodium acetate (pH 5.6) - 1 : 1)

Col or development: 1. p-aminobenzoic acid I BrCN 2. !satin Shadowed spots were positive with the !satin reagent. ~ 1-(1' -2' (S)-nornicotl no)-1-deoxy-('1-D-fructofu ran os I de

Figure 9. Changes of alkaloid content In the course ol 0 flue-curing. 0 The isatin-posltive alkaloid (1-A) was determined by liquid chromato­ 8 graphy, nicotine and nornlcotlne by gas chromatography.

80

1-A and 70 nicotine 6' Jl £.. I 60 ~ Anatalllne 0 ' / .a Anatablne \/ ..:;; I' c. Anabasine i 10 / \ 50 .,E 0 1- I ,' I "' ,,l 40 5 ,, '.... ,, Nornicotlne , 0 --·----.. _;,..---·------·---.0 30 --..1/l------"' 0 ~ua~~--~~--~-----L----~--~ 0 20 40 60 80 100 120 hours

----- 1 temperature Proline - · -1:::. · - · : nornlcotlne X - - 1 lsatln-posltlve alkaloid (1-A) -- 0 -- 1 nicotine Cherry Rod Normal

Figure 10. High-pressure liquid chromatogram of micro· Figure 7. High-pressure liquid chromatography of meth· blal metabolltes of 2'(S)·nlcotlne-1'·N-oxlde In 4 days' anol extract of Cherry Red tobacco. culture. WATERS HPLC ALC 201 WATERS HPLC ALC 201 column: ...-Bondapak c18 (4 X 300 mm) column: ~~-Bondapak c18 (4 X 300 mm) elution conditions: 50% methanol (2 ml/ m in) elution conditions: 0.02 M NH4HC03 :methanol (1 : 1) (2 ml/ min)

c: e0 /"4-(3'-pyridyl)-4-oxo-butyric acid 0 i nicotine-N'-oxide > :::> li' pseudooxynicotine ---/'--J~ 16 1B 20 0 0 2 3 4 5 6 7 10 15 20 25 Retention time (mlnr Retention time (mln)

312 Figure 11. Cell growth and microbial degradation of 2'(8)-nlcotlne-1'-N-oxlda and S-nlcotlne. Culture medium: 2 g of nlcotlne-N'-oxlde or nicotine; 1 g, KHoPO•; 0.5 g, MgSO• • 7 HoO; 0.3 ml of 1 °/o Feso. solution; 0.3 ml of 1 °/o CaCio solution; trace of Mnso, in 1 llter of distilled water (pH 6.4). Analysis was carried out by HPLC. Bacteria: Arthrobacter globfformls Jrs•-a·· (shaking the culture at 26 OC)

Nicotlne-N'-oxlde Nicotine

10

e=: OJ 'i 9 .2. ,g:

E 8 :I 'i E .5 c !, ___.__ .g 2 cI! 8c 8

.-"')(... , ]r ' ' ' \

0 2 4 0 2 4 6 6 10 12 Days Days

--e-: cell growth - D-: 2'(S)-nlcotlne-1'-N-oxlde --X--: pseudooxynicotlne - /::;.- : s-nlcotlne the structure of the compound in the solid state is 1-(1'- opment of Cherry Red tobacco leaves during the curing 2' (S)-nornicotino )-1-deoxy-~ D-fructofuranoside (Fig. 8). and redrying process. The infra-red absorption spectrum of this compound Finally, the bacterial transformation of nicotine oxide is was identical to that of the one synthesized from $-nor­ described. Nicotine oxide is known to be formed during nicotine and D-glucose in the presence of malic acid. It fermentation. Along with the memical degradation of ·must be formed from glucose and nornicotine via N'-D­ this compound, microbial degradation might occur dur­ glucopyranosyl-2'(S)-nornicotine by Amadori-rearrange­ ing fermentation. The bacteria degrading the oxide and ment. the manner of the degradation of this compound are not Fig. 9 shows the rise and fall of the alkaloid content yet known. We isolated the bacteria JTS*-5 and JTS-6 during flue-curing of Cherry Red tobacco. As the curing whim degrade the oxide from "Nambu" Japanese cigar temperature of tobacco leaves was increased, nicotine tobacco leaves. Both strains were identified as Arthro­ decreased first. Nornicotine increased first and then de­ bacter globiformis by morphological, physiological and creased. 1-(1 '-2'(S)-nornicotino )-1-deoxy-~-D-fructofura­ biomemical comparison with the type strain ATCC 8010 noside increased up to 15 mg I g dry weight, whim indi­ (15). The guanine and cytosine content of the DNA in cates a non-enzymatic process. It has been reported that the bacteria also supported the identification. the converter-type tobacco appears in a frequency of Fig. 10 shows a liquid mromatogram of the degradation 0.8 Ofo in natural badt mutation. Therefore, the crop of product of 2'(5)-nicotine-1'-N-oxide. Pseudooxynicotine nicotine-:-type tobacco must be contaminated with the was isolated as a principal product. 4-0xo-4-(3'-pyri­ converter-type tobacco at that rate. The converter-type dyl)butyric acid was also identified as one of the prod­ tobacco is generally regarded as a tobacco with light but ucts. As shown in Fig. 11, after a 3-day culture period unpleasant taste. No isolated compound gave an un­ almost all of the oxide was degraded and the pseudo­ pleasant taste. As a flavouring agent for tobacco, on the oxynicotine had accumulated to a maximum level. The contrary, it had an improving effect on inferior tasting maximum rate of the cell multiplication came a little tobacco. 1-(1'-2'(S)-nornicotino)-1-deoxy-~-D-fructofu­ earlier. On the other hand, nicotine was degraded slowly. ranoside was easily broken down and displayed a red Degradation products of nicotine by the bacteria were bladt color in the presence of moist air after isolation. This must be one of the reasons for the red color devel- • The Japan Tobacco and Salt Public Corporation.

313 Figure 12. Degradation pathway of nicotine and nlcotlne-N'-oxlde by Arthrobacter globlformls JTS*-6. ~ -~~ )HOo-9N7 ~ ) 0~ )... lN.J ~ 7r HO o-9N cH CH3 1 CH3 CH3 3 H 11 Ill IV

) ~OOH lN) ~ - --)~···

V VI VII VIII

1: nicotine Ill: 6-hydroxy-N'-methylmyosmlne VI: N'-methylmyosmlne 11: 6-hydroxynlcotlne IV: N-methyl-4-oxo-4-(3'-pyrldonyl)butylamine VII: 3-pyrldyi-N-methylamlnopropyl ketone V: nicotine-N'-oxlde VIII: 4-(3'-pyrldyl)-4-oxobutyrlc acid • Japan Tobacco and Salt Public Corporation

6-hydroxynicotine and 6-hydroxy-N'-methylmyosmine, SUMMARY but not pseudooxynicotine (16). In this organism the Chemical transformation: In air oxidation of nicotine at degradation of nicotine and nicotine-N'-oxide follows room temperature, N'-methylmyosmine, which is sup­ different pathways: nicotine through 6-hydroxynicotine, posed to be an active intermediate of degradation, coti­ nicotine-N'-oxide through N'-methylmyosmine (Fig. 12). nine, nicotine-N'-oxide, nicotyrine, myosmine, 3-pyridyl­ W ada and Y amasaki (17) of our Corporation have al­ propyl ketone, 3-pyridylmethyl ketone, nicotinic acid, ready demonstrated the nicotine degradation pathway methylamine and ammonia were isolated. N'-Methyl­ through pseudooxynicotine by the micro-organism iso­ myosmine was first characterized by 1H-NMR. When lated from a tobacco field. The micro-organisms that we N'-methylmyosmine was heated, N-methylnicotinamide have worked with evidently degraded nicotine-N'-oxide and nicotyrine were obtained in addition to a large directly to pseudooxynicotine without reducing it back to amount of polymerized resinous substances. 2'(5)-nic­ nicotine. TheN'-oxides ofS- andR-nicotine have 2 stereo­ otine-1'-N-oxide was rearranged to acetyl pseudooxynic­ isomers each with respect to the nitrogen atom: 1'(S)- otine by reaction with acetyl chloride or acetyl anhydride. 2'(S)-trans and 1'(R)-2'(S)-cis; 1'(R)-2'(R)-cis and 1'(S)- This rearrangement could be generally useful for the prep­ 2'(R)~trans, as seen in Fig. 13. The usual forms are: 1'(S)- aration of ~ 1 -pyrrolines or ~1-piperideines. When appro­ 2'(S)-trans and 1'(R)-2'(S)-cis. By this micro-organism priate acetyl groups were used, the products were effec­ 2'(R)-nicotine-1'-N-oxide was more efficiently degraded tive in improving tobacco taste. than2'(S)-nicotine-1'-N-oxide. When the mixture of1'(S)- 2'(S)-trans and 1'(R)-2'(S)-cis isomers of 2'(5)-nicotine- Phytochemical transformation: Transformation of alka­ 1'-N-oxide was metabolized for 7 days, the [a]D-value loids in the tobacco plant was investigated by measuring changed from +30° to +90° (Fig. 14), which indicates that this micro-organism degraded the 1'(R)-2'(S)-cis Figure 14. Time course of microbial degradation of dla- isomer more efficiently than the 1'(S)-2'(S)-trans isomer. stereomers. Culture condition: 10 ml broth In Erlenmeyer flask (shaking the culture at 28 oc). Inoculation: 2.2 X 107 cells I ml. Figure 13. Configurations of the four possible nlcotlne-N'· oxides. 100 +100" J;} .W H@~H• Rcfe\H, 1' (S)-2' (S)-trans 1'(R)-2'(R)-cls ~50 ~ <.> Q) a:

,-Q H'Q H J e RI CH, 0 CH•'oe 0 1'(R)-2'(S)-cls 1'(5)-2' (R)-trans 0 4 7 Days --0--: 1'(8)-nlcotlne-1'-N-oxide -- X --: 1'(R)-nlcotlne-1'-N-oxide R: o- - . - e - . - : [a} ~ (N HCI) of the mixture

314 their optical rotatory power, from which it was pre­ kaloide mit sekundaren Amingruppen wie Anabasin und sumed that nicotine is biosynthesized in the S-form. Anatabin liegen in der razemisc:hen Form vor. Aus The nornicotine formed in the leaves is synthesized from Cherry-Red-Tabak wurde 1-(1'-2'(5)-Nornikotin)-1-~­ S-nicotine, but the one formed in the root is synthesized D-fructofuranosid (Schmelzpunkt: 66-68 °C), ein Um­ in the racemic form, indicating a route different from that setzungsprodukt von Nornikotin, zum ersten Male iso­ found in the leaves. Secondary amine alkaloids such as liert. Die Struktur konnte physikodtemisch und schlieB­ anabasine and anatabine are in the racemic form. From lich durch Synthese bestii.tigt werden. Der Gehalt an die­ Cherry Red tobacco, a transformation product of nor­ ser Verbindung nahm wahrend der ROhrentrocknung nicotine, 1-(1 '-2'(S)-nornicotino )-1-~-D-fructofuranoside deutlidt zu, insbesondere am Ende des Trocknungspro­ (m.p. 66-68 °C), was isolated for the first time. The zesses beim Austrodmen des Blattgutes, was auf die Ent­ structure was confirmed physico-c:hemically and finally by stehung in einem nidttenzymatischen ProzeB sc:hlieBen synthesis. This compound increased markedly during cur­ IaBt. ing, especially at the drying stage, suggesting formation Mikrobielle Umwandlung: 2'(5)-Nikotin-1'-N-oxid, das through a non-enzymatic process. haufigste natiirlic:he Oxidationsprodukt des Nikotins, Microbial transformation: 2'(5)-nicotine-1'-N-oxide, wurde durdt Bakterien abgebaut, die auf der Oberfl:iche which is the most common natural oxidation product des Tabakblattes und im Ackerboden des Tabakfeldes of nicotine, was degraded by bacteria abundant on the reic:hlich vorkommen. Die isolienen Mikroorganismen ge­ tobacco leaf surface and in the tobacco field soil. The hOren zum Genus Arthrobacter. Die Abbauroute war isolated micro-organisms belong to genus Arthrobacter. folgende: Nikotin-N'-oxid -+ N'-Methylmyosmin (Aus­ Degradation pathway was: nicotine-N'-oxide ->- N'­ beute: 60-70 °/o) -+ 4-0xo-4-(3'-pyridyl)-buttersiure. methylmyosmine (60 Ofo-70 O/o yield) ->- 4-oxo-4- Nikotin hingegen wurde langsam auf einem anderen Weg (3' -pyridyl)butyric acid, whereas nicotine degraded abgebaut: S-Nikotin->- 6-Hydroxynikotin-+ 6-Hydroxy­ slowly by a different route: S-nicotine -+ 6-hydroxy­ N'-methylmyosmin. Die Bakterien zersetzten keine der nicotine -+ 6-hydroxy-N'-methylmyosmine. No analog­ untersuchren analogen und homologen Oxide. Im Vergleidt ous and homologous oxides tested were degraded by the zu 1'(S)-2'(S)-Nikotin-l'-N-oxid wurde 1'(R)-2'(S)­ bacteria. 1'(R)-2'(S)-Nicotine-1'-N-oxide was preferen­ Nikotin-1'-N-oxid vorzugsweise abgebaut. tially degraded, compared to l'(S)-2'(5)-nicotine-1'-N­ oxide. R£SUM£

ZUSAMMENFASSUNG Transformation chimique: A !'oxidation a !'air de la ni­ cotine 3. temperature ambiante, on a isole les composes Chemische Umwandlung: Bei der Oxidation des Nikotins suivants: la N'-methylmyosmine (consid&ee comme an der Luft bei Raumtemperatur wurden folgende Ver­ interm&Haire actif de degradation), la cotinine, le ni­ bindungen isoliert: N'-Methylmyosmin (das fiir ein ak­ cotine-N'-oxide, la nicotyrine, la myosmine,la 3-pyridyl­ tives intermedi1ires Abbauprodukt gehalten wird), Coti­ propylclitone, la 3-pyridylmethyldtone, l'acide ni­ nin, Nikotin-N'-oxid, Nikotyrin, Myosmin, 3-Pyridyl­ cotinique, la methylamine et !'ammoniac. La N'-methyl­ propylketon, 3-Pyridylmethylketon, Nikotinsiiure, Me­ myosmine a ete identi6ee pour la premiere fois par 1H­ thylamin und Ammoniak. N'-Methylmyosmin wurde NMR. En c:hauffant la N'-mc!thylmyosmine, on a obtenu dabei dun:h 1H-NMR zum ersten Male identifiziert. Bei de la N-methylnicotinamide et de la nicotyrine en plus dem Erhitzen von N'-Methylmyosmin wurden neben d'une grande quantite de substances rlisineuses poly­ einer groBen Menge polymerisiener harzartiger Substan­ merislies. Par reaction avec le dtlorure d'acetyle ou zen N-Methylnikotinamid und Nikotyrin erhalten. !'anhydride acerique, on a obtenu le rearrangement du 2'(5)-Nikotin-1'-N-oxid wurde bei Reaktion mit Acetyl­ 2'(S)-nicotine-1'-N-oxide en pseudo-oxynicotineacetique. dtlorid oder Acetylanhydrid zu Acetylpseudooxynikotin Ce rearrangement pourrait @tre utile en general pour la umgelagert. Diese Umlagerung kOnnte allgemein von Nut­ synthese des 6 1-pyrrolines ou .6.1-piperideines. Lors de zen fiir die Synthese von dt-Pyrrolinen oder .6.t-Pipe­ !'utilisation de groupes aceriques approprilis, ces substan­ rideinen sein. Bei Verwendung geeigneter Acetylgruppen ces se sont revelees efficaces pour !'amelioration du goilt erwiesen sich diese Substanzen als wirksam hinsichtlic:h du tabac. der Verbesserung des Tabakgeschmacks. Transformation phytochimique: On a examine la trans­ Phytochemische Umwandlung: Als Ergebnis der Unter­ formation des alcaloides dans la plante du tabac en suc:hung der Umwandlung der Alkaloide in der Tabak­ mesurant leur pouvoir rotatoire. On en a deduit que la pflanze durc:h Messung ihres RotationsvermOgens wird nicotine est bio-synthetisee dans la forme S. La nor­ angenommen, daB Nikotin sic:h in lebendem Gewebe in nicotine qui se forme dans les feuilles est syntherislie a der S-Form bildet. Das in den BHittern entstehende Nor­ partir de la $-nicotine, mais celle qui se forme dans les nikotin bildet sic:h aus S-Nikotin; jenes Nornikotin je­ racines est synrhetisee sous la forme racemique, in­ dodl, das in den Wurzeln entsteht, bildet sidt in der diquant ainsi un metabolisme different de celui des razemisc:hen Form, wodurc:h sich ein Stoffwedtselweg an­ feuilles. Des alcaloides a fonction amine secondaire zeigt, der von. dem in den Bl1ittern versc:hieden ist. AI- comme !'anabasine et l'anatabine ont ere trouves sous

315 la forme racem1que. Un produit de transformation de 5. Wada, E., i. Kisaki, K. Saito: Arch. Bioc:hem. Bio­ la nornicotine, le 1·{1'-2'(S}-nornicotine)-1·P·D-fruc­ phys, 79 (1959), 124. tofuranoside (point de fusion 66-68 °C), a ece isote 6. Kisaki, i., Y. "Matsubara, E. iamaki: J. Agr. Chem. pour la premi~re fois dans le tabac Cherry Red. La Soc. (Japan) 36 (1962), 374. structure en a CtC con6rmCe par des m6thodes physico­ 7. Dawson, R. F.: Advances in Enzymol. 8 (1948), 203. chimiques et 6nalement par synthhe. La quantite de ce 8. Kisaki, i., E. iamaki: J. Agr. Chem. Soc. (Japan) compose a augmence significativement pendant le «Cur­ 38 (1964), 549. ing~, particuli~rement au stade du sCchage, suggCrant 9. Kisaki, i., E. iamaki: Arch. Bioc:hem. Biophys. 92 ainsi une formation par un procede non enzymatique. (1961), 351. 10. Kisaki, T., E. Tamaki: Arch. Biochem. Biophys. 94 Transformation microbienne: Le 2'(5)-nicotine-1'-N· (1961), 252. oxide, qui est le produit d'oxidation naturel de la ni­ 11. Kisaki, T., E. Tamaki: Agr. Bioi. Chem. 28 (1964), cotine le plus rCpandu, a ere dCgrade par des baccCries 492. abondantes la surface des feuilles de tabac et dans a 12. Kisaki, T., E. Tamaki: Phytochemistry 5 (1966), le sol des c:hamps de tabac. Les micro-organismes isoiCs 293. appartiennent au genre Arthrobacter. La degradation 13. Leete, E., C. R. Miles: Phytochemistry 13 (1974), passe par les stades suivants: nicotine-N'-oxide --+ N'­ 1853. methylmyosmine (rendement 60 - 70 °/o) --+ acide 14. Kisaki, T., A. Koiwai, Y. Mikami, H. Matsushita: 4-oxo-4-(3'-pyridyl) butyrique. La degradation de la Agr. Bioi. Chem. 43 (1979), in press. · nicotine, par contre, est lente et passe par les Ccapes 15. Maeda, S., S. Uchida, T. Sasaki, Y. Mikami, T. Ki­ suivantes: $-nicotine...... ,_ 6-hydroxynicotine--+ 6·hydroxy­ saki: Abst. 1976 Seasonal meeting of the Agr. Chem. N'-methylmyosmine. Aucun des oxides analogues et Soc. (Japan), 50, N60. homologues examines n'a Cte degrade par les bactCries. 16. Maeda, S., S. Uc:hida, T. Kisaki: Agr. Bioi. Chem. Le 1'(R)-2'(S)·nicotine-1'-N-oxide a ece dCgrade de 42 (1978), 1455. preference au 1'(S)-2'(5)-nicotine-1 '-N-oxide. 17. Wada, E., K. Yamasaki: J. Am. Chem. Soc. 76 (1954), 155. REFERENCES 18. Maeda, S., H. Matsushita, Y. Mikami, T. Kisaki: 1. Rayburn, C. H., W. R. Haran, H. R. Hanmer: J. Agr. Bioi. Chem. 42 (1978), 2177. Am. Chem. Soc. 72 (1950), 1721. 2. Kisaki, i., M. lida, E. Wada: Bull. Agr. Chem. Soc. 23 (1959), 454. Authors' address: 3. Kisaki, i., M. Iida, E. Wada: Bull Agr. Chem. Soc. 24 {1960), 719. The Japan Tobacco and Salt Public Corporation, 4. Frankenburg, W. G., A. M. Gottsc:ho: Ind. Eng. Central Research Institute, 6-2 Umegaoka, Chem. 44 (1952), 301. Midori-ku, Yokohama, Kanagawa, 227, Japan.

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