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Biosynthesis of the nicotine alkaloids in Nicotiana glutinosa
ARCHIVES
OF
BIOCHEMISTRY
ANII
Interrelationships
among Nicotine,
WILLIAM of Chemistry
45-53 (19%)
112,
of the Nicotine
Biosynthesis
Department
BIOPHYSICS
Alkaloids Nornicotine,
L. ALWORTH
AND
in Nicofiana Anabasine,
HENRY
and Lawrence Radiation Laboratory, Berkeley, California Received
April
glufinosa and Anatabine’
RAPOPORT University
of California,
15, 1965
Biosyntheses from WO2 carried out with both intact Nicotiana glutinosa plants and seedlings have demonstrated that nornicotine is not the precursor of nicotine in either the root or the aerial portions of the plant. This conclusion contrasts with that reached on the basis of 3H incorporations into the alkaloids of Nicotiana rustica. The most probable relationship, based on WOZ biosyntheses and supporting some previous conclusions, is that nornicotine arises by demethylation of nicotine in both the root and aerial portions. The relative specific activities of anabasine and anatabine after the 14COt biosyntheses indicated that anabasine is not the precursor of anatabine. In the case of nicotine, nornicotine, anatabine, and anabasine, higher specific activities were noted in the samples isolated from the root than in those isolated from the aerial portions. This shows that these four alkaloids all can be formed in the Nicotiana root, and furthermore, since the l4C in these experiments first enters into the metabolism of the aerial portion of the plants, these results indicate that the N. glutinosa root is the most active site of formation of each of these alkaloids. Finally, a comparison of the specific activities of anabasine, anatabine, and nicotine suggests that if a precursor-product relationship exists among these alkaloids it must. be nicotine -+ anatabine or anabasine, or both.
The biosynthesis of the Nicotiana, alkaloids has been more extensively studied than that of any other family of alkaloids, and the results and conclusions of these studies have been summarized in several comprehensive reviews (l-4). In addition to the work described in these reviews, which has resulted in some degree of understanding of the biosynthetic pathways to nicotine and anabasine, a large number of experiments have been carried out which bear upon possible biosynthetic interrelationships among the various Nicotiana alkaloids. A clear understanding of these interrelationships has not been gained, however, since interpretations of the per-
tinent investigations have been complicated by certain details of the experimental procedures. Several different Nicotiana species have been used in these investigations, and certain of the observations, therefore, may be due to species differences. Furthermore, several of the pertinent studies (5-7) were carried out with Nicotiana. plants which had been decapitated by the removal of the flower buds and some of the leaves (topping and suckering). This modification of the plant was carried out because it is known to stimulate nicotine alkaloid synthesis (8). Since it has been found that the relative amounts of nicotine and nornicotine in the N. glutinosa and N. tabacum plant are altered by this decapitation procedure (8), and since there is no guarantee that the synthesis of the nicotine alkaloids is not altered qualitatively as well as quantitatively by this procedure, it would seem to
1 Sponsored in part by the United States Atomic Energy Commission. 2 Public Health Service Predoctoral Research Fellow of the National Institute of General Medicsl Sciences. 45
46
ALWORTH
AND
be potentially dangerous in experiments where interrelationships among the various nicotine alkaloids are being investigated. Finally, in several of the experiments (6, 9-18) the alkaloids were extracted only after the plant material had been dried. Several instances of an apparent conversion of nicotine into nornicotine during the drying of Nicotiana plant material have been noted (8, 19-al), and experiments involving use of 15N have shown that the conversion of nicotine into nornicotine is only one of several transformations occurring during the drying process (21). In consideration of these observations, data pertaining to the relationship between nicotine and nornicotine which were obtained by examining the alkaloid content of dried plants must be viewed with extreme skepticism. Our studies of the biosyntheses of the nicotine alkaloids from 14C02 were undertaken, in part, to investigate various possible interrelationships among nicotine, nornicotine, anabasine, and anata.bine. Experimental procedures were therefore designed to eliminate those features discussed above which had weakened the interpretations of previous studies. Our experiments were performed with a single species, Nicotiana glutinosa, which has a suitable distribution of the various Nicotiana alkaloids (8, 22). The plants used were not topped, grafted, or modified in any manner and were frozen in liquid nitrogen immediately after the biosynthesis experiments. The alkaloids were then isolated from the still frozen fresh plant material. Root and aerial fractions were treated separately since other studies, in particular grafting experiments, had indicated that the leaf and root carried out independent and different processes of alkaloid biosynthesis. (23, 24). The kinetics of 14C02 incorporation were instrumental in establishing the sequence thebaine + codeine + morphine in Papaver somniferum (25). It was felt that a similar examination of the rate of incorporation of 14C02 into the alkaloids of N. glutinosa might indicate at least the possible interrelationships among these alkaloids. The rate of labeling of various portions of the nicotine molecule led to several interesting observations which have been reported pre-
RAPOPORT
viously (26, 27). In this paper, the rates of incorporation of 14C02 into nornicotine (II), anabasine (III), and anatabine (IV) are reported and compared with those found previously for nicotine (I) isolated from the same biosyntheses. These relative rates of labeling are discussed in terms of possible interrelationships among the alkaloids of Nicotiana
glutinosa. --
I, R =CH, II, R = H
/A
I’ -‘1
III
IV
METHODS The method of growing the N. glutinosa plants used in these biosynthetic experiments, exposing them to 14C02 for the desired period of time, and isolating and purifying the various alkaloids separately from the root and aerial portions, has been described in detail previously (26, 27). The N. glutinosa seedlings used in separate biosynthetic experiments reported here were grown in a Petri dish on filter paper (Whatman No. 1) which was dampened with half-strength nutrient solution (28). The seeds were germinated and the seedlings were grown under regular laboratory lighting with a slow stream of air passing over the germinating seeds to maintain aerobic conditions. The seedlings were exposed to %OZ under the conditions described for expt. XI, i.e., the CO* concentration was maintained at the normal air level throughout the exposure period (27). The alkaloids were extracted from 8 gm of fresh N. glutinosa seedlings; 250 ml of 5ooj, aqueous acetone was used in the same manner as described for the root and aerial portions of the mature plants. The relative concentrations of the various Nicotiana alkaloids in the root and aerial portions of the N. glutinosa plants were determined by gas-liquid chromatography (g.1.c.) as previously described (26). For those alkaloids below the limit of mass detection during the fractionation of the crude alkalid samples, i.e., anabasine in both the root and aerial samples and nornicotine and anatabine in the majority of the root samples, the original g.1.c. collections were made by retention times. Aliquots of the alkaloids collected in this manner were reinjected into a more sensitive g.1.c. apparatus, and peaks corresponding to the respective alkaloid were observed. The integrated areas of the peaks were compared with a plot of peak
BIOSYNTHESIS
OF NICOTINE
area versus mass that had been prepared by injecting known amounts of a standard solution of the alkaloid in question and the amount of alkaloid collected by g.1.c. was calculated. These values, corrected for the collection efficiency, gave the relative amounts of those alkaloids which did not show a visible peak during the initial g.1.c. fractionation. The g.l.c.-proportional counting technique that had been utilized to determine the specific activity of the nicotine samples and described in detail previously (26) was also used to determine the specific activities of the other alkaloids. However, the apparatus was not that described previously, but was modified in design so as to be more suitable for the investigation of microgram samples,3 and 0.25 pg of nicotine could be easily detected. The minimum specific activity that could be detected in the case of the nicotine alkaloids was OJ pC per millimole. The g.1.c. column used was a polybutylene glyco1 column of the type previously described (5foot X $-inch column, 10% polybutylene glycol on 60/80 KOH treated firebrick) (26). When operated at 180-200”, with an argon flow of 60 ml per minute, nicotine, nornicotine, anabasine, and anatabine were satisfactorily resolved by this column, and the identical retention times of the isolated radioactive samples and of standard samples of these alkaloids were used as the criteria of identification. In addition certain representative samples of each alkaloid were chromatographed on paper (phosphate buffered paper developed with water saturated t-amyl alcohol) (26) versus the standard alkaloid samples and in each case alkaloid spots with R,‘s identical to those of the standards were noted. The iv-methyl analysis was performed on the radioactive nicotine sample obtained from the 5day-old N. glzhtinosa seedlings after the alkaloid material was diluted with 50 ~1 of nicotine and purified by g.1.c. The Herzig-Meyer determination was carried out and the methyltriethylammonium iodide was isolated and purified as previously described (27). Liquid scintillation counting of the ammonium iodide salt indicated that less than 1% of the total nicotine activity was located in the N-methyl group. RESULTS
The 2-3-month-old N. glutinosa plants used for the biosyntheses were each found to contain from 1.5 to 7.5 mg of nicotine in 3 This apparatus was designed and assembled by Dr. R. 0. Martin of this laboratory, and will be described in a forthcoming publication.
47
ALKALOIDS TABLE
I
RELATIVE ALKALOID CONTENT OF NICOTIANA QLUTINOSA Alkaloid
Nicotine Nornicotine Anabasine Anatabine
Determination by g.1.c. Root
Aerial
14 2 1 6
15 9 1 6
Previous analysis (8) Root Aerial 28 1 1
5 16 1
the roots and 4.7-43 mg of nicotine in the aerial portions. The relative alkaloid content of N. gZutinosa is presented in Table I along with an analysis of a mature plant reported by Tso and Jeffrey (8). Although these workers failed to identify anatabine as a component, others (22) have reported that the concentration of anatabine in the roots of this species is greater than that of nornicotine. Because of dilution effects, these relative pool sizes are important in determining the relative specific activities found within each plant portion after short term biosyntheses from 14C02. The specific activities of nornicotine, anabasine, and anatabine isolated from N. glutinosa after biosyntheses from 14C0, are listed in Table II along with those previously determined for nicotine. In those cases where no activity could be detected in the alkaloid samples, the upper limits of the specific activities, as estimated from the amount of sample examined, are listed. DISCUSSION
Nicotine-Nornicotine
Relationships
The majority of the investigations concerning the interrelationships among the Nicotiana alkaloids have dealt with the possible relationships between nicotine and nornicotine. The results of these investigations are summarized below. (a) In mature N. glutinosa and glauca the major amount of nornicotine seems to be formed in the aerial portions by demethylation of nicotine translocated from the roots. This was concluded from studies of the alkaloid content of grafted tomato and N. glutinosa plants (23). The results obtained from
48
ALWORTH
AND TABLE
SPECIFIC ACTIVITIES OF NICOTIANA Exposure (hours)
Expt.”
ft
4
IX ;
6
XI ;
6
II ;
6
VIII
12 xii III
;
12
ALKALOIDS
RAPOPORT II ISOLATED
AFTER
Specific Activities
BI~SYNTHESES
FROM ‘GO,
(pC/mmole)
Nicotine
Nornicotine
0.14 4.9 0.9 5.9 0.4 7.4 3.2 54.6 2.6 41.4 40.7 152
0.1* 1.2b O.lb 2.9 O.lb 3.7 lb 25 -
None None None
lib 4 10
None
Anabasine
46 8.26 2b detected detected detected 8b 74 2.6 3.9 detected 26.4
Anatabine
4b 8.2 1
2.3 73 2.4 5 36 63
a The biosynthesis experiments listed here refer to those previously described (26, 27); Roman numeral designates the chronological sequence, and the capital letters refer to the plant portions: A to the aerial portions (leaves and stems); R to the roots. b No activity peak was noted. The values reported are upper limits estimated from the amounts of sayple injected.
stddies of these grafted plants were consistent with the previous observations that nicotine was formed in the root of the n’icotiana plant (24) and further indicated that nornicotine was produced only in the Nicotiana leaf at the expense of translocated nicotine. Experiments involving the use of isotopically labeled nicotine have also shown a conversion of nicotine to nornicotine by the Nicotiana plant. Radioactive nornicotine, as well as radioactive nicotine, was isolated 4 days after N. glauca shoots were supplied with nicotine-14C (9). In other experiments (5), nicotine enriched equally in both heterocyclic rings with 15N was fed hydroponically to an intact N. glauca plant. After 3 weeks’ metabolism, 80% of the 15K found in the alkaloid fraction was recovered as nornicotine. Since the nornicotine isolated was found to be equally labeled with 15N in both rings, this suggested that the nicotine was converted directly to nornicotine. While these experiments with isotopically labeled nicotine indicate a conversion of nicotine to n?rnicotine in N. glauca, it should be emphasized that in N. rustica, a species which contains nicotine as the major alkaloid, the results were totally different (5). When 15N nicotine was supplied to N. rustica in an identical manner to that used in the experi-
ments with N. glauca, no conversion to nornicotine was observed. On the other hand, when 15N-enriched nornicotine was supplied to N. rustica, nearly half of the 15N excess present in the isolated alkaloids was found in the form of nicotine. Other experiments using excised N. glutinosa leaves that had been obtained from grafted plants with tomato roots and therefore originally contained negligible amounts of nicotine and nornicotine, demonstrated that 22 % of the l-nicotine supplied to the leaf could be recovered as nornicotine after 2 days’ metabolism (10). (b) Studies involving the use of 15N-enriched nutrients indicate that nomicotine can also arise independently of nicotine in Nicotiana plants. When grafted plants consisting of tomato scions on N. tabacum root stocks were exposed to nutrient solutions containing 20 atom % excess 15N for 4 weeks, the excess 15N in the root nornicotine (9.38%) was higher than the l5S excess in the shoot nornicotine (7.49%), and both were higher than the excess in the root nicotine (6.43 %) or the shoot nicotine (.i.30%) (21). When intact N. glutinosa plants were exposed to 15N-enriched nutrients t’hroughout their entire growth period, the nornicotine isolated also was found to have a greater 15N
BIOSYNTHESIS
OF NICOTINE
content than did the nicotine, although plants exposed to 15N-enriched nutrients only during the final stages of growth showed a greater 15N content in the nicotine (6). Since the transformation of nicotine into nornicotine would not involve a change in the amount of nitrogen in the molecule, the higher 15N content in the nornicotine isolated in these insta,nces (6, 21) indicates that the nornicotine was derived independently of the nice tine. (c) The rate of incorporation of 3H from tritiuted water into nornicotine b?~N. rustica i-s more rapid than the rate of 3H incorporation into nicotine. Using topped N. rustica plants, a species in which nicotine accounts for about 95 % of the total alkaloids, Tso and Jeffrey (7) found that 30 minutes after exposure times of 0.5, 2.5, and 70 hours, the highest 3H specific activity was not found in nicotine but in nornicotine. The ratios of the specific activities of nicotine to nornicotine were 0.48, 0.75, and 0.88, respectively. The authors concluded the 3H was being incorporated into nornicotine and the nornicotine-3H was then being slowly converted into nicotine, that is, that the nornicotine was an actual precursor of the nicotine. (d) Nicotine and nornicotine take part in transmethylation reactions within the Nicotiana pla.nt with various methyl donors. The Nmethyl-14C group of nicotine can be transferred to choline (29) ; also the S-methyl group of methionine can be transferred intact to become the N-methyl group of nicotine (11). Most recently it has been reported that when nornicotine-15N and L-methionine--methyl-14C were supplied to tobacco plants, nicotine labeled with both 15N and 14Cwas isolated (30). In connection with these observations, however, it should be noted that a myriad of nicotine methyl precursors have been established including formic acid (12), formaldehyde (13), serine (13), methionine (11, 12), choline (31), betaine (32), glycolic acid (33), and glycine (34), and that under comparable conditions formaldehyde-14C (13) was found to be a somewhat better precursor of the N-methyl group of nicotine than was methioninemethyl-14C (12). (e) During the air drying of Nicotiana,
ALKALOIDS
49
changes in alkaloid content and relative concentrations occur. As noted in the introduction one such change is a decrease in the nicotine content and a corresponding increase in the nornicotine content (8, 21). In previous papers dealing with the rate of labeling of the nicotine molecule from 14COz (26, 27), we reported that the rate of labeling of the N-methyl group of the nicotine molecule was surprisingly slow. In the majority of cases the percent,age of the activity located in the N-methyl group was between 1 and 5 % (whereas the statistical value would be 10%) and the maximum incorporation noted was only 8.2 % after twelve hours exposure to 14C02. This means that when the specific activities of nicotine and nornicotine listed in Table I are compared, one is essentially comparing amounts of incorporation into the two heterocyclic rings. With this fact in mind, a comparison of the values in Table II allows us to eliminate several of the theoretically possible nicotine-nornicotine interrelationships. Contrary to the conclusions reached from the rate of incorporation of 3H into nicotine and nornicotine (7) the 14Cspecific activities of nicotine and nornicotine listed in Table I indicate that nornicotine is not the precursor of nicotine in either the root or the aerial portions of N. glutinosa. Even after 12 hours’ exposure to an atmosphere of 14C02 (expt. III), the specific activity of the nornicotine in both the aerial and root portions of the plant were found to be several times less than the specific activity of the corresponding nicotine samples. Similar results were observed in the nicotine and nornicotine isolated from t’he root after biosyntheses runs in which “CO2 was added to maintain a normal CO2 concentration (expt. X). When 12C02was added, however, the lower resulting specific activities prevented meaningful comparisons of samples from the aerial portions. In the case of the nornicotine samples obtained from 14C02 exposures of less than 12 hours, the g.l.c.proportional counting determinations also showed that the nornicotine specific activities were less than the corresponding nicotine specific activities, although the slow rate of incorporation of 14C02 into nornico-
50
ALWORTH
AND RAPOPORT
tine prevented as definitive a comparison as was possible in the case of the twelve hour biosyntheses. Since an insignificant amount of 14C is incorporated into the N-methyl of nicotine under the conditions of our experiments, the observations that activity from 14C02 is incorporated into nicotine more rapidly than into nornicotine clearly show that nornicotine is not the precursor of nicotine in N. glutinosa. The opposite conclusion reached after studying the relative rates of 3H incorporation by N. rustica (7) may be explained by several factors. First, the N. rustica plants used had been topped and suckered, a modification which may be affecting the relative rates of alkaloid synthesis. Second, in view of the fact that about 95 % of the alkaloid content of N. rustica is nicotine, the failure to separate the root and aerial portions of the plants may have resulted in the active root nicotine being diluted by a large pool of nicotine from the aerial portions. This could have led to a large amount of nicotine of lower specific activity than was found in the smaller amount of nornicotine. Third, as was pointed out previously, the experiments involving the metabolism of 15N-enriched alkaloids by Nicotiana (5) indicated that the nicotine-nornicotine relationships of N. rustica might not be typical. Thus, although nicotine was converted readily to nornicotine by N. gluuca, in N. rustica only the opposite transformation was observed. Fourth, the contrasting results may truly represent different relative rates of incorporation of 3H and l4C into nicotine and nornicotine. While the nicotine isolated from Nicotiana has been found to be pure levorotatory, the nornicotine isolated has always been found to be a mixture of enantiomers (14). Some experimental evidence indicates that the conversion of nicotine to nornicotine by Nicotiana may involve a change in stereochemistry at C-2’ (14-16). Such a change could indicate that the conversion takes place via a dehydro compound such as myosmine or N-methylmyosmine. If this were the case, under the experimental conditions used, 3H would have been incorporated into nornicotine from tritiated water after the formation of the nicotine-nornicotine carbon
skeleton. Therefore the 3H incorporations, the sites of which were not determined, need not be consistent with the relative rate of formation of the carbon skeletons of these two alkaloids as indicated by the rates of 14C02 incorporation. It has been observed that while nornicotine seemed to be derived independently of nicotine at the earlier stages of growth of N. glutinosa, at later stages the relative labeling from 15N indicated that the nornicotine could be derived largely from nicotine (6). The N. glutinosa used for the experiments listed in Table I were from 2 to 3 months old. The observation that the plants were just beginning to bud and the fact that they contained comparable am0unt.s of nicotine and nornicotine in their aerial portions indicated that they were at a relatively early growth stage (8). Nevertheless, to test if our conclusions concerning the nicotine-nornicotine relationship had been unduly influenced by the age of the N. glutinosa plants used, an additional biosynthesis was carried out with five day old seedlings. About 8 gm (fresh weight) of N. glutinosa plants, 5 days from seeding, was exposed for 2 hours to 14C02. When the alkaloid fraction was isolated from these seedlings in the usual manner and examined in the g.l.c.-proportional counting apparatus, activity was detected in nicotine but not in nornicotine. Activity was readily detected in 20 pg of nicotine, but no activity could be detected in about 2.8 pg of nornicotine. As an upper limit, t,herefore, the nornicotine could have had a specific activity seven times that of nicotine. However, the amount of nicotine isolated from the 5-day seedlings was 30 times the amount of nornicotine isolated. Considering the relative pool sizes, therefore, in order for nornicotine to be the precursor of nicotine in these 5-day-old N. glutinosa seedlings, the specific activity of the nornicotine would have had to be at least 30 times that of nicotine after a short exposure to 14C02.This specific activity would have been over four times the estimated upper limit of the relative nornicotine activity. When the radioactive nicotine isolated from these 5-day-old seedlings was degraded, less than 1% of the incorporated label was found to be located in
BIOSYNTHESIS
OF NICOTINE
the N-methyl group. This labeling pattern was consistent with that observed after 14C02 biosynt,heses performed with mature N. glutinosa (26, 27) and further indicated that the direct comparison of nicotine and nornicotine specific activities made in the above discussion is valid. The relative rate of 14C incorporation from 14C02 into the alkaloids of 5-day-old N. glutinosa seedlings therefore leads to the same conclusion reached from 14Cincorporation into nicotine and nornicotine in 2-3-month-old plants; namely, that nornicotine is not the precursor of nicotine. Although the proposed schemes for the biosynthesis of the pyrrolidine ring of nicotine (27, 35) lead to a Alpyrroline intermediate which, when coupled with the pyridine ring precursor would lead to nornicotine, these schemes would not be altered to a significant extent by adding the N-methyl function at an ea.rlier stage. In view of the fact that the grafting experiments (23) indicated that nicotine was the precursor of the majority of the nornicotine in the aerial portions of N. glutinosa, it is tempting to propose that the root and aerial nornicotine in our N. glutinosa are arising by demethylation of the root and aerial nicotine, respectively. The relative specific activities listed in Table II would be consistent with this nicotine-nornicotine relationship. Experiments involving the use of 15N enriched nutrients, however, have indicated that the nornicotine of N. glutinosa can be derived independently of the nicotine (6, 21). Since the determined specific activities of Table II would also be consistent with the formation of nicotine and nornicotine independently from different precursors, or from a common precursor if the rate of nicotine formation from the precursor is many times that of nornicotine formation, additional experiments will be necessary before the relationship can be finally resolved and the results of both the 14C and the 15N incorporations reconciled. Whatever the nicotine-nornicotine relationship might be, the values listed in Table II clearly indicate that the nornicotine found in the roots of our N. glutinosa was not derived from translocation of nornicotine from the aerial portions of the plants. Thus, the relative specific activities of the
51
ALKALOIDS
nornicotine derived from the root and aerial portions of the plants after 6 (expts. IX, XI, and II) and 12 hours (expt. III) exposure to 14C02 clearly indicate that the nornicotine in the root is‘ ‘d&rived independently of the nornicotine in the aerial portions of the plant, since the root nornicotine has a higher specific activity than the aerial nornicotine. This observation is consistent with experiments mentioned previously that were carried out with grafted N. tabacum plants (21), where the relative rate of 15N incorporation also indicated that nornicotine in the Nicotiana root was not derived from translocation of the nornicotine found in the aerial portions of the grafted plants. Anabasine-Anatabine
Relationships
Only a few experiments have been reported in which the possible interrelationships between anabasine and anatabine and their relationship to nicotine have been investigated. The conclusions that can be derived from these experiments are summarized below. (a) Considerable amounts of anabasine are formed independently in the aerial portions of Nicotiana plants. Investigations involving grafted N. glauca plants showed that anabasine was formed both in the root and aerial portions of the Nicotiana plants (36, 37). The fact that excised N. glauca leaves were capable of anabasine synthesis was also demonstrated (38) by the isolation of uniformly labeled anabasine 51 hours after the leaf was exposed to 14C02.While these results suggested that the biosynthesis of anabasine and nicotine are separate processes, it has been proposed that in addition, a transformation of nicotine into anabasine was possible in the aerial portions of N. glauca (17, 39, 40). (b) Experiments have indicated an interrelationship may exist between nicotine and nornicotine, and anabasine in Nicotiana. When 15N was supplied to N. rustica in the form of anabasine, it was largely recovered in the form of nicotine (5). The 15N supplied in the form of nornicotine was recovered in both nicotine and anabasine, while the 15N supplied to these plants in the form of nicotine was recovered as such. In the case of N. glauca, however, 15N supplied to the
52
ALWORTH
AND RAPOPORT
plants in the form of nicotine was recovered in anabasine and in nornicotine. In contrast to the 15N experiments, no conversion of nicotine-W into anabasine was observed in the N. glauca shoot after 4 days (9). Fina,lly, in one case, N. glauca, when supplied with a radioactive pyrrolidine ring precursor, began forming radioactive nicotine, nornicotine, and anabasine (18). These results could be obtained only during months when the plants were in a dormant state; during months when the plants were growing vigorously only negative results were obtained and other experiments (41) carried out with excised N. glauca root cultures which were producing nicotine and anabasine concurrently indicated that it was impossible to divert the alkaloid biosynthesis from one to the other. These observations, therefore, although indicating that some relationship between nicotine and anabasine may exist in the Nicotiana plant cannot be satisfactorily explained. (c) Experimental results indicate that anabasine is not serving as the precursor of anatabine. Thus in the experiments involving the re-feeding of W-enriched alkaloids to N. rustica, no conversion of anabasine to anatabine was noted (5). Furthermore, in the studies involving the incorporation of 3H into the alkaloids of N. rustica (7), the specific activity of anata,bine after exposure times of 0.5 or 2.5 hours was greater than that of anabasine, indicating again that anabasine is not t’he precursor of anatabine. In view of the confusing experimental results that have appeared in the literature dealing with the possible relationships of anabasine and anatabine to nicotine, it is unfortunate that the anabasine and anatabine specific activities determined after biosyntheses from 14C02 could not have led to definite conclusions as was possible in the case of nornicotine and nicotine. However, the relative specific activities determined for anabasine and anatabine and listed in Table II are such that only tenta,tive conclusions are justified. In every case where the specific activities of both the anabasine and anatabine samples could be determined, the specific activity of anatabine was either equal to, or greater than, the specific activity of t,he corresponding anabasine samples. This was true both in the root (III,, Xx,
and IIR) and in the aerial (X, and IX,) samples. If anabasine were the precursor of anatabine, the relative pool sizes (1 anabasine: 6 anatabine) are such that the specific activity of anabasine should have been at least six times that of anatabine after short term exposures to 14C02. The values listed in Table I show that this was not the case and therefore the results of biosyntheses from 14C02 are consistent with other experiments which have indicated that anabasine is not the precursor of anatabine. The specific activities listed in Table II are consistent either with the independent syntheses of anabasine and anatabine from 14C02 at rat,es comparable to their relative pool sizes, or with the formation of anabasine from anatabine at a fairly rapid rate. Although i4C first enters the aerial portions of the Nicotiana plants during the biosyntheses from 14C02,the values in Table I show that the specific activity of the root anatabine is higher than the specific activity of the corresponding aerial anatabine sa.mples. This observation indicates that the Nicotiana root is the site of the most rapid formation of anatabine, just as has been observed in the case of nornicotine (Table II) and previously in the case of nicotine (26). The observation, however, that this same relationship appears to be true also in the case of anabasine (expts. II and X) is somewhat surprising since, in contrast to these other alkaloids, a significant amount of anabasine formation has been demonstrated to take place in the aerial portions of the Nicotiana plants (36-38). The results of the biosyntheses from 14C02 therefore indicate that the glutinosa root is the most active site of biosynthesis of each of the four nicotine alkaloids examined. Since these experiments have also shown that about 15 % of the nicotine of N. glutinosa is formed independently in the aerial portions of the plant (26) and as cell culture experiments (42) have recently shown that N. tabacum cells from the root and the leaf have about equal capacity to form nicotine, it appears that in different Nicotiana species the various nicotine alkaloids are synthesized independently in the aerial portions of the plant at differing rates. Thus, the observation that 14C from 14C02 is incorporated into root anabasine more rapidly than into the aerial
BIOSYNTHESIS
OF NICOTINE
anabasine by N. glutinosa does not conflict in any way with a fairly rapid synthesis of anabasine by the aerial portions of N. glauca. When the specific activities of anabasine and anatabine listed in Table II are compared with the activities of the corresponding nicotine samples, it is apparent that the determined anabasine and anatabine activities are similar to the nicotine activities. In view of the larger pools of nicotine present in both root and aerial portions of N. glutinosa, this suggests that if any precursorproduct relationship exists between the pyrrolidine and piperidine ring alkaloids, the relationship must be nicotine 4 anabasine or anatabine, or both, in the direction proposed by Iljin (17, 39, 40). Obviously, the results reported here can only indicate that the conversion of nicotine into anabasine or anatabine or both is potentially possible. Furthermore, the possibility that very slow interconversions among the various nicotine alkaloids, of the type indicated by the experiments involving 15N-enriched alkaloids (5), take place within N. glutinosa cannot be eliminated on the basis of short term biosyntheses from 14C02. REFERENCES 1. DAWSON, R. F., Am. SC. 48, 337 (1960). 2. BATTERSBY, A. R., Quart. Rev. XV, 259 (1961). 3. MOTHES, K., AND SCHUTTE, H. R., Angew. Chem., 76, 265 (1963). of Natural Prod4. LEETE, E., in “Biogenesis ucts” (P. Bernfeld, ed.), p. 746. MacMillan, New York (1963). 5. Tso, T. C., AND JEFFREY, It. N., Arch. Biothem. Biophys. 80, 46 (1959). 6. Tso, T. C., Arch. Biochem. Biophys. 92, 248 (1961). 7. Tso, T. C., AND JEFFREY, R. N., Arch. Biothem. Biophys. 97, 4 (1962). 8. Tso, T. C., AND JEFFREY, R. N., Plant Physiol. 31, 433 (1956). lab, 334 (1957). 9. SCHRBTER, H., 2. Naturjorsch. 10. DAWSON, R. F., J. Am. Chem. Sot. 73, 4218 (1951). 11. DEWEY, L. J., BYERRUM, R. U., AND BALL, C. D., J. Am. Chem. Sot. 78, 3997 (1954). 12. BROWN, S. A., AND BYERRUM, R. U., J. Am. Chem. Sot. 74, 1523 (1952). 13. BYERRUM, R. U., RINGLER, R. L., HAMILL, R. L., AND BALL, C. D., J. Biol. Chem. 218, 371 (1955).
ALKALOIDS
53
14. KISAKI, T., AND TAMAKI, E., Arch. Biochem. Biophys. 92, 351 (1961). 15. KISAKI, T., AND TAMSKI, E., Arch. Biochem. Biophys. 94, 252 (1961). 23, 16. KISAKI, T., AND TAMAKI, E., Naturwiss. 540 (1960). 14, 553 (1949). 17. ILJIN, G. S., Biochimia 18. MOTHES, K., AND SCHR~TER, H. B., Arch. Pharmazie 294, 99 (1961). 19. Wa~a, E., Arch. Biochem. Biophys. 82, 471 (1956). R. B., V~LLEAU, W. D., AND 20. GRIFFITH, STOKES, G. W., Science 121, 343 (1955). 21. Tso, T. C., AND JEFFREY, R. N., Plant Physiol. 32, 86 (1957). 22. Waoa, E., KISAKI, T., AND IHIDA, M., Arch. Biochem. Biophys. 80, 258 (1959). 23. DAWSON, R. F., Am. J. Botany 32, 416 (1945). 24. LAWSON, R. F., Am. J. Botany 29, 66 (1942). 25. RAPOPORT, H., STERMITZ, F. R., AND BBKER, D. R., J. Am: Chem. Sot. 82, 2765 (1966). 26. AL~ORTH, W. L., DESELMS, R. C., AND RAPOPORT, H., J. Am. Chem. Sot. 86, 1608 (1964). 27. ALWORTH, W.L., LIEBMAN, A.A., AND RAPOPORT, J. Am. Chem. Sot. 86, 3375 (1964). 28. HOAGLhND, D. R., AND ARNON, D. I., California Agri. Exptl. Station Circular 347, revised 1950. College of Agriculture, Univ. of Calif., Berkeley. 29. LEETE, E., AND BELL, V. M., J. Am. Chem. Sot. 81, 4358 (1959). Natural Products 30. Tso, T. C., Abstracts IUPAC Symposium, Kyoto, Japan (1964), p. 125. 31. BYEI~RUM, R. U., AND WING, R. E., J. Biol. Chem. 206, 637 (1953). 32. BYERRUM, R. U., S~TO, C. S., AND BILL, C. D., Plant Physiol. 31, 374 (1956). 33. BYERRUM, R. U., DEWEY, L. J., HAMILL, R. L., AND BILL, C. D., J. Biol. Chem. 219, 345 (1956). R. L., AND BALL, 34. BYERRUM, R. U., &MILL, C. D., J. Biol. Chem. 210, 645 (1954). 35. LEETE, E., GROS, E. G., AND GILBERTSON, T. J., Tetrahedron Letters 587 (1964). 36. DAWSON, R. F., Am. J. Botany 31, 351 (1944). 37. SCHMUCK, A. A., SMIRNOV, A., AND ILJIN, G. S., Dokl. Akad. Nauk SSSR 32, 365 (1941). 31, 355 (1957). 38. ARONOFF, S., Plant Physiol. 39. ILJIN, G. S., Dokl. Akad. Nauk SSSR 69, 99 (1948) 40. ILJIN, G. S., Probl. Biochem. in der Mitshurinschen Biol. Sammlg. I, 169 (1949). 41. SOLT, M. L., DAWSON, R. F., AND CHRISTMAN, D. R.., Plant Physiol. 36, 887 (1960). 42. SPEAEE, T., MCCLOSKEY, P., SMITH, W. K., SCOTT, T. A., AND HUSSEY, H., Nature 201, 614 (1964).
OF
BIOCHEMISTRY
ANII
Interrelationships
among Nicotine,
WILLIAM of Chemistry
45-53 (19%)
112,
of the Nicotine
Biosynthesis
Department
BIOPHYSICS
Alkaloids Nornicotine,
L. ALWORTH
AND
in Nicofiana Anabasine,
HENRY
and Lawrence Radiation Laboratory, Berkeley, California Received
April
glufinosa and Anatabine’
RAPOPORT University
of California,
15, 1965
Biosyntheses from WO2 carried out with both intact Nicotiana glutinosa plants and seedlings have demonstrated that nornicotine is not the precursor of nicotine in either the root or the aerial portions of the plant. This conclusion contrasts with that reached on the basis of 3H incorporations into the alkaloids of Nicotiana rustica. The most probable relationship, based on WOZ biosyntheses and supporting some previous conclusions, is that nornicotine arises by demethylation of nicotine in both the root and aerial portions. The relative specific activities of anabasine and anatabine after the 14COt biosyntheses indicated that anabasine is not the precursor of anatabine. In the case of nicotine, nornicotine, anatabine, and anabasine, higher specific activities were noted in the samples isolated from the root than in those isolated from the aerial portions. This shows that these four alkaloids all can be formed in the Nicotiana root, and furthermore, since the l4C in these experiments first enters into the metabolism of the aerial portion of the plants, these results indicate that the N. glutinosa root is the most active site of formation of each of these alkaloids. Finally, a comparison of the specific activities of anabasine, anatabine, and nicotine suggests that if a precursor-product relationship exists among these alkaloids it must. be nicotine -+ anatabine or anabasine, or both.
The biosynthesis of the Nicotiana, alkaloids has been more extensively studied than that of any other family of alkaloids, and the results and conclusions of these studies have been summarized in several comprehensive reviews (l-4). In addition to the work described in these reviews, which has resulted in some degree of understanding of the biosynthetic pathways to nicotine and anabasine, a large number of experiments have been carried out which bear upon possible biosynthetic interrelationships among the various Nicotiana alkaloids. A clear understanding of these interrelationships has not been gained, however, since interpretations of the per-
tinent investigations have been complicated by certain details of the experimental procedures. Several different Nicotiana species have been used in these investigations, and certain of the observations, therefore, may be due to species differences. Furthermore, several of the pertinent studies (5-7) were carried out with Nicotiana. plants which had been decapitated by the removal of the flower buds and some of the leaves (topping and suckering). This modification of the plant was carried out because it is known to stimulate nicotine alkaloid synthesis (8). Since it has been found that the relative amounts of nicotine and nornicotine in the N. glutinosa and N. tabacum plant are altered by this decapitation procedure (8), and since there is no guarantee that the synthesis of the nicotine alkaloids is not altered qualitatively as well as quantitatively by this procedure, it would seem to
1 Sponsored in part by the United States Atomic Energy Commission. 2 Public Health Service Predoctoral Research Fellow of the National Institute of General Medicsl Sciences. 45
46
ALWORTH
AND
be potentially dangerous in experiments where interrelationships among the various nicotine alkaloids are being investigated. Finally, in several of the experiments (6, 9-18) the alkaloids were extracted only after the plant material had been dried. Several instances of an apparent conversion of nicotine into nornicotine during the drying of Nicotiana plant material have been noted (8, 19-al), and experiments involving use of 15N have shown that the conversion of nicotine into nornicotine is only one of several transformations occurring during the drying process (21). In consideration of these observations, data pertaining to the relationship between nicotine and nornicotine which were obtained by examining the alkaloid content of dried plants must be viewed with extreme skepticism. Our studies of the biosyntheses of the nicotine alkaloids from 14C02 were undertaken, in part, to investigate various possible interrelationships among nicotine, nornicotine, anabasine, and anata.bine. Experimental procedures were therefore designed to eliminate those features discussed above which had weakened the interpretations of previous studies. Our experiments were performed with a single species, Nicotiana glutinosa, which has a suitable distribution of the various Nicotiana alkaloids (8, 22). The plants used were not topped, grafted, or modified in any manner and were frozen in liquid nitrogen immediately after the biosynthesis experiments. The alkaloids were then isolated from the still frozen fresh plant material. Root and aerial fractions were treated separately since other studies, in particular grafting experiments, had indicated that the leaf and root carried out independent and different processes of alkaloid biosynthesis. (23, 24). The kinetics of 14C02 incorporation were instrumental in establishing the sequence thebaine + codeine + morphine in Papaver somniferum (25). It was felt that a similar examination of the rate of incorporation of 14C02 into the alkaloids of N. glutinosa might indicate at least the possible interrelationships among these alkaloids. The rate of labeling of various portions of the nicotine molecule led to several interesting observations which have been reported pre-
RAPOPORT
viously (26, 27). In this paper, the rates of incorporation of 14C02 into nornicotine (II), anabasine (III), and anatabine (IV) are reported and compared with those found previously for nicotine (I) isolated from the same biosyntheses. These relative rates of labeling are discussed in terms of possible interrelationships among the alkaloids of Nicotiana
glutinosa. --
I, R =CH, II, R = H
/A
I’ -‘1
III
IV
METHODS The method of growing the N. glutinosa plants used in these biosynthetic experiments, exposing them to 14C02 for the desired period of time, and isolating and purifying the various alkaloids separately from the root and aerial portions, has been described in detail previously (26, 27). The N. glutinosa seedlings used in separate biosynthetic experiments reported here were grown in a Petri dish on filter paper (Whatman No. 1) which was dampened with half-strength nutrient solution (28). The seeds were germinated and the seedlings were grown under regular laboratory lighting with a slow stream of air passing over the germinating seeds to maintain aerobic conditions. The seedlings were exposed to %OZ under the conditions described for expt. XI, i.e., the CO* concentration was maintained at the normal air level throughout the exposure period (27). The alkaloids were extracted from 8 gm of fresh N. glutinosa seedlings; 250 ml of 5ooj, aqueous acetone was used in the same manner as described for the root and aerial portions of the mature plants. The relative concentrations of the various Nicotiana alkaloids in the root and aerial portions of the N. glutinosa plants were determined by gas-liquid chromatography (g.1.c.) as previously described (26). For those alkaloids below the limit of mass detection during the fractionation of the crude alkalid samples, i.e., anabasine in both the root and aerial samples and nornicotine and anatabine in the majority of the root samples, the original g.1.c. collections were made by retention times. Aliquots of the alkaloids collected in this manner were reinjected into a more sensitive g.1.c. apparatus, and peaks corresponding to the respective alkaloid were observed. The integrated areas of the peaks were compared with a plot of peak
BIOSYNTHESIS
OF NICOTINE
area versus mass that had been prepared by injecting known amounts of a standard solution of the alkaloid in question and the amount of alkaloid collected by g.1.c. was calculated. These values, corrected for the collection efficiency, gave the relative amounts of those alkaloids which did not show a visible peak during the initial g.1.c. fractionation. The g.l.c.-proportional counting technique that had been utilized to determine the specific activity of the nicotine samples and described in detail previously (26) was also used to determine the specific activities of the other alkaloids. However, the apparatus was not that described previously, but was modified in design so as to be more suitable for the investigation of microgram samples,3 and 0.25 pg of nicotine could be easily detected. The minimum specific activity that could be detected in the case of the nicotine alkaloids was OJ pC per millimole. The g.1.c. column used was a polybutylene glyco1 column of the type previously described (5foot X $-inch column, 10% polybutylene glycol on 60/80 KOH treated firebrick) (26). When operated at 180-200”, with an argon flow of 60 ml per minute, nicotine, nornicotine, anabasine, and anatabine were satisfactorily resolved by this column, and the identical retention times of the isolated radioactive samples and of standard samples of these alkaloids were used as the criteria of identification. In addition certain representative samples of each alkaloid were chromatographed on paper (phosphate buffered paper developed with water saturated t-amyl alcohol) (26) versus the standard alkaloid samples and in each case alkaloid spots with R,‘s identical to those of the standards were noted. The iv-methyl analysis was performed on the radioactive nicotine sample obtained from the 5day-old N. glzhtinosa seedlings after the alkaloid material was diluted with 50 ~1 of nicotine and purified by g.1.c. The Herzig-Meyer determination was carried out and the methyltriethylammonium iodide was isolated and purified as previously described (27). Liquid scintillation counting of the ammonium iodide salt indicated that less than 1% of the total nicotine activity was located in the N-methyl group. RESULTS
The 2-3-month-old N. glutinosa plants used for the biosyntheses were each found to contain from 1.5 to 7.5 mg of nicotine in 3 This apparatus was designed and assembled by Dr. R. 0. Martin of this laboratory, and will be described in a forthcoming publication.
47
ALKALOIDS TABLE
I
RELATIVE ALKALOID CONTENT OF NICOTIANA QLUTINOSA Alkaloid
Nicotine Nornicotine Anabasine Anatabine
Determination by g.1.c. Root
Aerial
14 2 1 6
15 9 1 6
Previous analysis (8) Root Aerial 28 1 1
5 16 1
the roots and 4.7-43 mg of nicotine in the aerial portions. The relative alkaloid content of N. gZutinosa is presented in Table I along with an analysis of a mature plant reported by Tso and Jeffrey (8). Although these workers failed to identify anatabine as a component, others (22) have reported that the concentration of anatabine in the roots of this species is greater than that of nornicotine. Because of dilution effects, these relative pool sizes are important in determining the relative specific activities found within each plant portion after short term biosyntheses from 14C02. The specific activities of nornicotine, anabasine, and anatabine isolated from N. glutinosa after biosyntheses from 14C0, are listed in Table II along with those previously determined for nicotine. In those cases where no activity could be detected in the alkaloid samples, the upper limits of the specific activities, as estimated from the amount of sample examined, are listed. DISCUSSION
Nicotine-Nornicotine
Relationships
The majority of the investigations concerning the interrelationships among the Nicotiana alkaloids have dealt with the possible relationships between nicotine and nornicotine. The results of these investigations are summarized below. (a) In mature N. glutinosa and glauca the major amount of nornicotine seems to be formed in the aerial portions by demethylation of nicotine translocated from the roots. This was concluded from studies of the alkaloid content of grafted tomato and N. glutinosa plants (23). The results obtained from
48
ALWORTH
AND TABLE
SPECIFIC ACTIVITIES OF NICOTIANA Exposure (hours)
Expt.”
ft
4
IX ;
6
XI ;
6
II ;
6
VIII
12 xii III
;
12
ALKALOIDS
RAPOPORT II ISOLATED
AFTER
Specific Activities
BI~SYNTHESES
FROM ‘GO,
(pC/mmole)
Nicotine
Nornicotine
0.14 4.9 0.9 5.9 0.4 7.4 3.2 54.6 2.6 41.4 40.7 152
0.1* 1.2b O.lb 2.9 O.lb 3.7 lb 25 -
None None None
lib 4 10
None
Anabasine
46 8.26 2b detected detected detected 8b 74 2.6 3.9 detected 26.4
Anatabine
4b 8.2 1
2.3 73 2.4 5 36 63
a The biosynthesis experiments listed here refer to those previously described (26, 27); Roman numeral designates the chronological sequence, and the capital letters refer to the plant portions: A to the aerial portions (leaves and stems); R to the roots. b No activity peak was noted. The values reported are upper limits estimated from the amounts of sayple injected.
stddies of these grafted plants were consistent with the previous observations that nicotine was formed in the root of the n’icotiana plant (24) and further indicated that nornicotine was produced only in the Nicotiana leaf at the expense of translocated nicotine. Experiments involving the use of isotopically labeled nicotine have also shown a conversion of nicotine to nornicotine by the Nicotiana plant. Radioactive nornicotine, as well as radioactive nicotine, was isolated 4 days after N. glauca shoots were supplied with nicotine-14C (9). In other experiments (5), nicotine enriched equally in both heterocyclic rings with 15N was fed hydroponically to an intact N. glauca plant. After 3 weeks’ metabolism, 80% of the 15K found in the alkaloid fraction was recovered as nornicotine. Since the nornicotine isolated was found to be equally labeled with 15N in both rings, this suggested that the nicotine was converted directly to nornicotine. While these experiments with isotopically labeled nicotine indicate a conversion of nicotine to n?rnicotine in N. glauca, it should be emphasized that in N. rustica, a species which contains nicotine as the major alkaloid, the results were totally different (5). When 15N nicotine was supplied to N. rustica in an identical manner to that used in the experi-
ments with N. glauca, no conversion to nornicotine was observed. On the other hand, when 15N-enriched nornicotine was supplied to N. rustica, nearly half of the 15N excess present in the isolated alkaloids was found in the form of nicotine. Other experiments using excised N. glutinosa leaves that had been obtained from grafted plants with tomato roots and therefore originally contained negligible amounts of nicotine and nornicotine, demonstrated that 22 % of the l-nicotine supplied to the leaf could be recovered as nornicotine after 2 days’ metabolism (10). (b) Studies involving the use of 15N-enriched nutrients indicate that nomicotine can also arise independently of nicotine in Nicotiana plants. When grafted plants consisting of tomato scions on N. tabacum root stocks were exposed to nutrient solutions containing 20 atom % excess 15N for 4 weeks, the excess 15N in the root nornicotine (9.38%) was higher than the l5S excess in the shoot nornicotine (7.49%), and both were higher than the excess in the root nicotine (6.43 %) or the shoot nicotine (.i.30%) (21). When intact N. glutinosa plants were exposed to 15N-enriched nutrients t’hroughout their entire growth period, the nornicotine isolated also was found to have a greater 15N
BIOSYNTHESIS
OF NICOTINE
content than did the nicotine, although plants exposed to 15N-enriched nutrients only during the final stages of growth showed a greater 15N content in the nicotine (6). Since the transformation of nicotine into nornicotine would not involve a change in the amount of nitrogen in the molecule, the higher 15N content in the nornicotine isolated in these insta,nces (6, 21) indicates that the nornicotine was derived independently of the nice tine. (c) The rate of incorporation of 3H from tritiuted water into nornicotine b?~N. rustica i-s more rapid than the rate of 3H incorporation into nicotine. Using topped N. rustica plants, a species in which nicotine accounts for about 95 % of the total alkaloids, Tso and Jeffrey (7) found that 30 minutes after exposure times of 0.5, 2.5, and 70 hours, the highest 3H specific activity was not found in nicotine but in nornicotine. The ratios of the specific activities of nicotine to nornicotine were 0.48, 0.75, and 0.88, respectively. The authors concluded the 3H was being incorporated into nornicotine and the nornicotine-3H was then being slowly converted into nicotine, that is, that the nornicotine was an actual precursor of the nicotine. (d) Nicotine and nornicotine take part in transmethylation reactions within the Nicotiana pla.nt with various methyl donors. The Nmethyl-14C group of nicotine can be transferred to choline (29) ; also the S-methyl group of methionine can be transferred intact to become the N-methyl group of nicotine (11). Most recently it has been reported that when nornicotine-15N and L-methionine--methyl-14C were supplied to tobacco plants, nicotine labeled with both 15N and 14Cwas isolated (30). In connection with these observations, however, it should be noted that a myriad of nicotine methyl precursors have been established including formic acid (12), formaldehyde (13), serine (13), methionine (11, 12), choline (31), betaine (32), glycolic acid (33), and glycine (34), and that under comparable conditions formaldehyde-14C (13) was found to be a somewhat better precursor of the N-methyl group of nicotine than was methioninemethyl-14C (12). (e) During the air drying of Nicotiana,
ALKALOIDS
49
changes in alkaloid content and relative concentrations occur. As noted in the introduction one such change is a decrease in the nicotine content and a corresponding increase in the nornicotine content (8, 21). In previous papers dealing with the rate of labeling of the nicotine molecule from 14COz (26, 27), we reported that the rate of labeling of the N-methyl group of the nicotine molecule was surprisingly slow. In the majority of cases the percent,age of the activity located in the N-methyl group was between 1 and 5 % (whereas the statistical value would be 10%) and the maximum incorporation noted was only 8.2 % after twelve hours exposure to 14C02. This means that when the specific activities of nicotine and nornicotine listed in Table I are compared, one is essentially comparing amounts of incorporation into the two heterocyclic rings. With this fact in mind, a comparison of the values in Table II allows us to eliminate several of the theoretically possible nicotine-nornicotine interrelationships. Contrary to the conclusions reached from the rate of incorporation of 3H into nicotine and nornicotine (7) the 14Cspecific activities of nicotine and nornicotine listed in Table I indicate that nornicotine is not the precursor of nicotine in either the root or the aerial portions of N. glutinosa. Even after 12 hours’ exposure to an atmosphere of 14C02 (expt. III), the specific activity of the nornicotine in both the aerial and root portions of the plant were found to be several times less than the specific activity of the corresponding nicotine samples. Similar results were observed in the nicotine and nornicotine isolated from t’he root after biosyntheses runs in which “CO2 was added to maintain a normal CO2 concentration (expt. X). When 12C02was added, however, the lower resulting specific activities prevented meaningful comparisons of samples from the aerial portions. In the case of the nornicotine samples obtained from 14C02 exposures of less than 12 hours, the g.l.c.proportional counting determinations also showed that the nornicotine specific activities were less than the corresponding nicotine specific activities, although the slow rate of incorporation of 14C02 into nornico-
50
ALWORTH
AND RAPOPORT
tine prevented as definitive a comparison as was possible in the case of the twelve hour biosyntheses. Since an insignificant amount of 14C is incorporated into the N-methyl of nicotine under the conditions of our experiments, the observations that activity from 14C02 is incorporated into nicotine more rapidly than into nornicotine clearly show that nornicotine is not the precursor of nicotine in N. glutinosa. The opposite conclusion reached after studying the relative rates of 3H incorporation by N. rustica (7) may be explained by several factors. First, the N. rustica plants used had been topped and suckered, a modification which may be affecting the relative rates of alkaloid synthesis. Second, in view of the fact that about 95 % of the alkaloid content of N. rustica is nicotine, the failure to separate the root and aerial portions of the plants may have resulted in the active root nicotine being diluted by a large pool of nicotine from the aerial portions. This could have led to a large amount of nicotine of lower specific activity than was found in the smaller amount of nornicotine. Third, as was pointed out previously, the experiments involving the metabolism of 15N-enriched alkaloids by Nicotiana (5) indicated that the nicotine-nornicotine relationships of N. rustica might not be typical. Thus, although nicotine was converted readily to nornicotine by N. gluuca, in N. rustica only the opposite transformation was observed. Fourth, the contrasting results may truly represent different relative rates of incorporation of 3H and l4C into nicotine and nornicotine. While the nicotine isolated from Nicotiana has been found to be pure levorotatory, the nornicotine isolated has always been found to be a mixture of enantiomers (14). Some experimental evidence indicates that the conversion of nicotine to nornicotine by Nicotiana may involve a change in stereochemistry at C-2’ (14-16). Such a change could indicate that the conversion takes place via a dehydro compound such as myosmine or N-methylmyosmine. If this were the case, under the experimental conditions used, 3H would have been incorporated into nornicotine from tritiated water after the formation of the nicotine-nornicotine carbon
skeleton. Therefore the 3H incorporations, the sites of which were not determined, need not be consistent with the relative rate of formation of the carbon skeletons of these two alkaloids as indicated by the rates of 14C02 incorporation. It has been observed that while nornicotine seemed to be derived independently of nicotine at the earlier stages of growth of N. glutinosa, at later stages the relative labeling from 15N indicated that the nornicotine could be derived largely from nicotine (6). The N. glutinosa used for the experiments listed in Table I were from 2 to 3 months old. The observation that the plants were just beginning to bud and the fact that they contained comparable am0unt.s of nicotine and nornicotine in their aerial portions indicated that they were at a relatively early growth stage (8). Nevertheless, to test if our conclusions concerning the nicotine-nornicotine relationship had been unduly influenced by the age of the N. glutinosa plants used, an additional biosynthesis was carried out with five day old seedlings. About 8 gm (fresh weight) of N. glutinosa plants, 5 days from seeding, was exposed for 2 hours to 14C02. When the alkaloid fraction was isolated from these seedlings in the usual manner and examined in the g.l.c.-proportional counting apparatus, activity was detected in nicotine but not in nornicotine. Activity was readily detected in 20 pg of nicotine, but no activity could be detected in about 2.8 pg of nornicotine. As an upper limit, t,herefore, the nornicotine could have had a specific activity seven times that of nicotine. However, the amount of nicotine isolated from the 5-day seedlings was 30 times the amount of nornicotine isolated. Considering the relative pool sizes, therefore, in order for nornicotine to be the precursor of nicotine in these 5-day-old N. glutinosa seedlings, the specific activity of the nornicotine would have had to be at least 30 times that of nicotine after a short exposure to 14C02.This specific activity would have been over four times the estimated upper limit of the relative nornicotine activity. When the radioactive nicotine isolated from these 5-day-old seedlings was degraded, less than 1% of the incorporated label was found to be located in
BIOSYNTHESIS
OF NICOTINE
the N-methyl group. This labeling pattern was consistent with that observed after 14C02 biosynt,heses performed with mature N. glutinosa (26, 27) and further indicated that the direct comparison of nicotine and nornicotine specific activities made in the above discussion is valid. The relative rate of 14C incorporation from 14C02 into the alkaloids of 5-day-old N. glutinosa seedlings therefore leads to the same conclusion reached from 14Cincorporation into nicotine and nornicotine in 2-3-month-old plants; namely, that nornicotine is not the precursor of nicotine. Although the proposed schemes for the biosynthesis of the pyrrolidine ring of nicotine (27, 35) lead to a Alpyrroline intermediate which, when coupled with the pyridine ring precursor would lead to nornicotine, these schemes would not be altered to a significant extent by adding the N-methyl function at an ea.rlier stage. In view of the fact that the grafting experiments (23) indicated that nicotine was the precursor of the majority of the nornicotine in the aerial portions of N. glutinosa, it is tempting to propose that the root and aerial nornicotine in our N. glutinosa are arising by demethylation of the root and aerial nicotine, respectively. The relative specific activities listed in Table II would be consistent with this nicotine-nornicotine relationship. Experiments involving the use of 15N enriched nutrients, however, have indicated that the nornicotine of N. glutinosa can be derived independently of the nicotine (6, 21). Since the determined specific activities of Table II would also be consistent with the formation of nicotine and nornicotine independently from different precursors, or from a common precursor if the rate of nicotine formation from the precursor is many times that of nornicotine formation, additional experiments will be necessary before the relationship can be finally resolved and the results of both the 14C and the 15N incorporations reconciled. Whatever the nicotine-nornicotine relationship might be, the values listed in Table II clearly indicate that the nornicotine found in the roots of our N. glutinosa was not derived from translocation of nornicotine from the aerial portions of the plants. Thus, the relative specific activities of the
51
ALKALOIDS
nornicotine derived from the root and aerial portions of the plants after 6 (expts. IX, XI, and II) and 12 hours (expt. III) exposure to 14C02 clearly indicate that the nornicotine in the root is‘ ‘d&rived independently of the nornicotine in the aerial portions of the plant, since the root nornicotine has a higher specific activity than the aerial nornicotine. This observation is consistent with experiments mentioned previously that were carried out with grafted N. tabacum plants (21), where the relative rate of 15N incorporation also indicated that nornicotine in the Nicotiana root was not derived from translocation of the nornicotine found in the aerial portions of the grafted plants. Anabasine-Anatabine
Relationships
Only a few experiments have been reported in which the possible interrelationships between anabasine and anatabine and their relationship to nicotine have been investigated. The conclusions that can be derived from these experiments are summarized below. (a) Considerable amounts of anabasine are formed independently in the aerial portions of Nicotiana plants. Investigations involving grafted N. glauca plants showed that anabasine was formed both in the root and aerial portions of the Nicotiana plants (36, 37). The fact that excised N. glauca leaves were capable of anabasine synthesis was also demonstrated (38) by the isolation of uniformly labeled anabasine 51 hours after the leaf was exposed to 14C02.While these results suggested that the biosynthesis of anabasine and nicotine are separate processes, it has been proposed that in addition, a transformation of nicotine into anabasine was possible in the aerial portions of N. glauca (17, 39, 40). (b) Experiments have indicated an interrelationship may exist between nicotine and nornicotine, and anabasine in Nicotiana. When 15N was supplied to N. rustica in the form of anabasine, it was largely recovered in the form of nicotine (5). The 15N supplied in the form of nornicotine was recovered in both nicotine and anabasine, while the 15N supplied to these plants in the form of nicotine was recovered as such. In the case of N. glauca, however, 15N supplied to the
52
ALWORTH
AND RAPOPORT
plants in the form of nicotine was recovered in anabasine and in nornicotine. In contrast to the 15N experiments, no conversion of nicotine-W into anabasine was observed in the N. glauca shoot after 4 days (9). Fina,lly, in one case, N. glauca, when supplied with a radioactive pyrrolidine ring precursor, began forming radioactive nicotine, nornicotine, and anabasine (18). These results could be obtained only during months when the plants were in a dormant state; during months when the plants were growing vigorously only negative results were obtained and other experiments (41) carried out with excised N. glauca root cultures which were producing nicotine and anabasine concurrently indicated that it was impossible to divert the alkaloid biosynthesis from one to the other. These observations, therefore, although indicating that some relationship between nicotine and anabasine may exist in the Nicotiana plant cannot be satisfactorily explained. (c) Experimental results indicate that anabasine is not serving as the precursor of anatabine. Thus in the experiments involving the re-feeding of W-enriched alkaloids to N. rustica, no conversion of anabasine to anatabine was noted (5). Furthermore, in the studies involving the incorporation of 3H into the alkaloids of N. rustica (7), the specific activity of anata,bine after exposure times of 0.5 or 2.5 hours was greater than that of anabasine, indicating again that anabasine is not t’he precursor of anatabine. In view of the confusing experimental results that have appeared in the literature dealing with the possible relationships of anabasine and anatabine to nicotine, it is unfortunate that the anabasine and anatabine specific activities determined after biosyntheses from 14C02 could not have led to definite conclusions as was possible in the case of nornicotine and nicotine. However, the relative specific activities determined for anabasine and anatabine and listed in Table II are such that only tenta,tive conclusions are justified. In every case where the specific activities of both the anabasine and anatabine samples could be determined, the specific activity of anatabine was either equal to, or greater than, the specific activity of t,he corresponding anabasine samples. This was true both in the root (III,, Xx,
and IIR) and in the aerial (X, and IX,) samples. If anabasine were the precursor of anatabine, the relative pool sizes (1 anabasine: 6 anatabine) are such that the specific activity of anabasine should have been at least six times that of anatabine after short term exposures to 14C02. The values listed in Table I show that this was not the case and therefore the results of biosyntheses from 14C02 are consistent with other experiments which have indicated that anabasine is not the precursor of anatabine. The specific activities listed in Table II are consistent either with the independent syntheses of anabasine and anatabine from 14C02 at rat,es comparable to their relative pool sizes, or with the formation of anabasine from anatabine at a fairly rapid rate. Although i4C first enters the aerial portions of the Nicotiana plants during the biosyntheses from 14C02,the values in Table I show that the specific activity of the root anatabine is higher than the specific activity of the corresponding aerial anatabine sa.mples. This observation indicates that the Nicotiana root is the site of the most rapid formation of anatabine, just as has been observed in the case of nornicotine (Table II) and previously in the case of nicotine (26). The observation, however, that this same relationship appears to be true also in the case of anabasine (expts. II and X) is somewhat surprising since, in contrast to these other alkaloids, a significant amount of anabasine formation has been demonstrated to take place in the aerial portions of the Nicotiana plants (36-38). The results of the biosyntheses from 14C02 therefore indicate that the glutinosa root is the most active site of biosynthesis of each of the four nicotine alkaloids examined. Since these experiments have also shown that about 15 % of the nicotine of N. glutinosa is formed independently in the aerial portions of the plant (26) and as cell culture experiments (42) have recently shown that N. tabacum cells from the root and the leaf have about equal capacity to form nicotine, it appears that in different Nicotiana species the various nicotine alkaloids are synthesized independently in the aerial portions of the plant at differing rates. Thus, the observation that 14C from 14C02 is incorporated into root anabasine more rapidly than into the aerial
BIOSYNTHESIS
OF NICOTINE
anabasine by N. glutinosa does not conflict in any way with a fairly rapid synthesis of anabasine by the aerial portions of N. glauca. When the specific activities of anabasine and anatabine listed in Table II are compared with the activities of the corresponding nicotine samples, it is apparent that the determined anabasine and anatabine activities are similar to the nicotine activities. In view of the larger pools of nicotine present in both root and aerial portions of N. glutinosa, this suggests that if any precursorproduct relationship exists between the pyrrolidine and piperidine ring alkaloids, the relationship must be nicotine 4 anabasine or anatabine, or both, in the direction proposed by Iljin (17, 39, 40). Obviously, the results reported here can only indicate that the conversion of nicotine into anabasine or anatabine or both is potentially possible. Furthermore, the possibility that very slow interconversions among the various nicotine alkaloids, of the type indicated by the experiments involving 15N-enriched alkaloids (5), take place within N. glutinosa cannot be eliminated on the basis of short term biosyntheses from 14C02. REFERENCES 1. DAWSON, R. F., Am. SC. 48, 337 (1960). 2. BATTERSBY, A. R., Quart. Rev. XV, 259 (1961). 3. MOTHES, K., AND SCHUTTE, H. R., Angew. Chem., 76, 265 (1963). of Natural Prod4. LEETE, E., in “Biogenesis ucts” (P. Bernfeld, ed.), p. 746. MacMillan, New York (1963). 5. Tso, T. C., AND JEFFREY, It. N., Arch. Biothem. Biophys. 80, 46 (1959). 6. Tso, T. C., Arch. Biochem. Biophys. 92, 248 (1961). 7. Tso, T. C., AND JEFFREY, R. N., Arch. Biothem. Biophys. 97, 4 (1962). 8. Tso, T. C., AND JEFFREY, R. N., Plant Physiol. 31, 433 (1956). lab, 334 (1957). 9. SCHRBTER, H., 2. Naturjorsch. 10. DAWSON, R. F., J. Am. Chem. Sot. 73, 4218 (1951). 11. DEWEY, L. J., BYERRUM, R. U., AND BALL, C. D., J. Am. Chem. Sot. 78, 3997 (1954). 12. BROWN, S. A., AND BYERRUM, R. U., J. Am. Chem. Sot. 74, 1523 (1952). 13. BYERRUM, R. U., RINGLER, R. L., HAMILL, R. L., AND BALL, C. D., J. Biol. Chem. 218, 371 (1955).
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