Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures

Article abstract of DOI:10.1016/j.phytochem.2005.07.012

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Furthermore, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.

Full text of DOI:10.1016/j.phytochem.2005.07.012

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 1890–1897
www.elsevier.com/locate/phytochem
Stereoselectivity of the demethylation of nicotine piperidine
homologues by Nicotiana plumbaginifolia cell suspension cultures
a,b,1
b
´
Trixie Ann Bartholomeusz
, Roland Molinie c,1, Albrecht Roscher ,
d
a
d
Franc¸ois-Xavier Felpin , Franc¸oise Gillet , Jacques Lebreton ,
a,
c,
*
Franc¸ois Mesnard *, Richard J. Robins
a
Laboratoire de Phytotechnologie, EA 3900, Universite´ de Picardie Jules Verne, Faculte´ de Pharmacie, 1 rue des Louvels, 80037 Amiens, France
b
Ge´nie Enzymatique et Cellulaire, CNRS UMR6022, Universite´ de Picardie Jules Verne, 33 rue St. Leu, 80039 Amiens, France
c
Laboratoire dÕAnalyse Isotopique et Electrochimique de Me´tabolisme, CNRS UMR6006, Universite´ de Nantes,
Faculte´ des Sciences et Techniques, 2 rue de la Houssinie`re, 44322 Nantes, France
`
´ ´
Laboratoire de Synthese Organique, CNRS UMR6513, Universite de Nantes, Faculte des Sciences et Techniques,
d
`
2, rue de la Houssiniere, 44322 Nantes, France
Received 20 May 2005; received in revised form 11 July 2005; accepted 11 July 2005
Abstract
The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of
Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the
medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-
methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product
occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Fur-
thermore, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the
metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine
homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different
metabolic profiles in relation to the demethylation activity are discussed.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Nicotiana plumbaginifolia; (R,S)-N-Methylanabasine; (R,S)-N-Methylanatabine; (S)-Nicotine; Demethylation; Cell suspension culture;
Chiral selectivity
1. Introduction
established during the condensation of N-methylpyrro-
linium ion with ÔactivatedÕ nicotinic acid. It is usually
accompanied by smaller amounts of other alkaloids
(Leete, 1983; Saitoh et al., 1985). While some of these
– such as nornicotine, cotinine and myosmine – are bio-
synthetically derived from nicotine, the extent to which
the configuration at the C20 is conserved is variable
(Armstrong et al., 1999; Leete, 1983; Mesnard et al.,
2001). In a few species, such as Nicotiana alata and N.
otophora, nornicotine can accumulate in roots to levels
as high as, or even higher than, nicotine.
The principal alkaloid found in most Nicotiana spe-
cies is (S)-nicotine (Fig. 1), the chiral centre at C20 being
*
Corresponding authors. Tel.: +33 3 2282 7494; fax: +33 3 2282
7469 (F. Mesnard); Tel.: +33 2 5112 5701; fax: +33 2 5112 5712
(R. Robins).
E-mail addresses: francois.mesnard@u-picardie.fr (F. Mesnard),
richard.robins@univ-nantes.fr (R.J. Robins).
1
These two authors participated equally in the experimental work
reported in this presentation.
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.07.012

T.A. Bartholomeusz et al. / Phytochemistry 66 (2005) 1890–1897
1891
N
R
N
R
N
R
N
N
N
R = CH3 Nicotine
R = CH3 N-Methylanabasine
R = H Anabasine
R = CH3 N-Methylanatabine
R = H Anatabine
R = H
Nornicotine
Fig. 1. Structures of the alkaloids.
These alkaloids are frequently accompanied by vari-
able amounts of other minor components – notably
anabasine and anatabine – which are produced by
related biosynthetic pathways (Bush et al., 1999;
Robins, 1998). In some species, such as N. glauca, anab-
asine is the major alkaloid found in all tissues (Saitoh
et al., 1985). While superficially similar to nicotine, these
alkaloids differ in three aspects: the presence of a piper-
idine ring in place of the pyrrolidine ring; the total ab-
sence of any N-methyl derivatives; and their tendency
to be found as mixtures of the (S)- and (R)-isomers
(Armstrong et al., 1999).
Both biochemical and chemical evidence have shown
that nornicotine is produced by the demethylation of
nicotine (Bush et al., 1999; Leete, 1983), not by a parallel
pathway from putrescine via the pyrrolinium ion. In
contrast, all the evidence is that anabasine and anata-
bine are not synthesised via an N-methylated intermedi-
ate (Fecker et al., 1993; Robins, 1998). The reactions
responsible for the N-demethylation of nicotine to nor-
nicotine have attracted interest both for their biochemi-
cal elucidation and for their potential economic
exploitation. It is an important reaction, and it is pri-
marily due to the efficient demethylation of nicotine by
hepatic detoxifying systems that the extreme toxicity of
this compound to man is not more evident (Gorrod
and Schepers, 1999). Nevertheless, nicotine derivatives
are showing an increasing potential in treating neurolog-
ical disorders (Brennan, 2000). Mammalian detoxifica-
tion involves the intervention of a P-450 oxidase
to activate the pyrrolinium ring at the C50 position
(Carlson et al., 1995).
In contrast, the mechanism of this process in plants is
still not well understood. It has been established that the
C50 position is not involved in nicotine oxidation in
plants (Botte et al., 1997), indicating that the mechanism
of degradation is not equivalent. It is also apparent that
demethylation can be effectively carried out on a wide
range of compounds modified either in the heterocycle
or in the N-substituent (Boswell et al., 1993, 1999;
Dawson, 1951; Leete, 1983; Mesnard et al., 2001).
As part of a programme aimed at establishing the
mechanism of nicotine demethylation in plants, the che-
mio- and stereo-specificity of the reaction in terms of the
substrates that can be demethylated is being examined in
cell suspension cultures of N. plumbaginifolia, which
have previously proved to be efficient at converting
exogenous nicotine to nornicotine (Manceau et al.,
1989). In order to evaluate the involvement of each cycle
in the reaction and the degree of selectivity of the reac-
tion, three types of nicotine analogue have been synthes-
ised. In the first type, modification was made to the
N-substituent. This showed that the demethylation
activity was limited to only small increases in the size
of the substituent group present (Boswell et al., 1999).
In the second type, modification was made to the pyri-
dine ring, notably the replacement of the heterocycle
with a phenyl group. These analogues were efficiently
metabolised and competed with nicotine for the demeth-
ylation activity (Mesnard et al., 2001). It was shown that
both isomers in (R,S)-nicotine or pyridine analogues
were demethylated, but at markedly different rates (Mes-
nard et al., 2001) indicating a degree of chiral selectivity
in the process.
In this paper, we report the demethylation of the
third type of modification, in which the pyrrolidine
cycle has been replaced by a piperidine ring. (R,S)-N-
Methylanatabine and (R,S)-N-methylanabasine, the
non-natural N-methyl derivatives of anatabine and
anabasine, have been synthesised (Felpin et al., 2001)
and fed to cell suspension cultures of N. plumbaginifolia.
This paper reports the metabolism of these compounds.
2. Results and discussion
2.1. Metabolism of (R,S)-N-methylanabasine or(R,S)-
N-methylanatabine
The metabolism in cell cultures of N. plumbaginifolia of
(R,S)-N-methylanabasine and (R,S)-N-methylanatabine
was followed by HPLC in an initial experiment over 10
days. In order to confirm the stability of these products
under biotransformation conditions, either 7.1 lmol of
(R,S)N-methylanabasine or 6.2 lmol of (R,S)N-methyla-
natabine was incubated in FMS medium without cells. No
degradation of either substrate occurred (data not
shown).
In contrast, incubation of 7.1 lmol of (R,S)N-
methylanabasine with 2.0 g FW of N. plumbaginifolia

1892
T.A. Bartholomeusz et al. / Phytochemistry 66 (2005) 1890–1897
cells in non-growth-stimulating conditions led to the
demethylation of approximately 50% of the (R,S)-N-
methylanabasine within 4 days (Fig. 2(a)). Within 2
days, both N-methylanabasine and anabasine were
detected in the cells at about 0.2 lmol (Fig. 2(b)). There-
after, the intracellular concentration of N-methylanaba-
sine remained constant, while the level of the anabasine
steadily increased, reaching 0.5 lmol by day 10. Cells re-
mained healthy throughout this time course. This is a
similar profile to that observed with nicotine (Mesnard
et al., 2001), indicating that demethylation was almost
certainly intracellular, N-methylanabasine being taken
up from the medium and anabasine being released. Such
a proposal is supported by the steady accumulation of
anabasine in the medium (Fig. 2(a)), reaching 3.3 lmol
(40%) at 4 days and slowly increasing up to 3.5 lmol
at 8 days. At this point, 49% of the substrate was unme-
tabolised and 49% was present as the product of
demethylation. No other product of metabolism was
detected.
Similarly, when 6.2 lmol of (R,S)-N-methylanatabine
was incubated with 2.0 g FW of N. plumbaginifolia cells
in non-growth-stimulating conditions, 62% of the
(R,S)N-methylanatabine was degraded within 4 days
(Fig. 2(c)). Metabolism again appeared to be intracellu-
lar, as shown in Fig. 2(d), and the product of demethyl-
ation, anatabine, was the only compound seen to
accumulate. Within 2 days, the intracellular substrate
concentration reached a maximum (0.6 lmol), while
the product maximal (0.5 lmol) was approximately 2
days later (Fig. 2(d)). Thereafter, both substrate and
product could be detected in the cells in steadily decreas-
ing amounts, reaching zero around 10 days. Cells re-
mained healthy throughout this time course. This
profile differs slightly from those of (S)-nicotine and
(R,S)-N-methylanabasine, which both remained ele-
vated in the cells at day 10. In parallel, anatabine accu-
mulated in the medium, but only about 0.6 lmol
(approximately 11%) had accumulated at 4 days
(Fig. 2(c)). Thus, in contrast to the degradation of
(R,S)-N-methylanabasine, about 40% of the compound
was not detected at 4 days in either substrate or product
of demethylation, a profile closer to that for nicotine
metabolism than that for (R,S)-N-methylanabasine
metabolism (Fig. 2(a) and (b)). Thereafter, however,
the amount of anatabine accumulated steadily, such that
by 8 days, 2.0 lmol (39%) of demethylation product and
1.9 lmol (37%) of substrate remained, a total recovery
of about 76%.
Thus, efficient demethylation of both (R,S)-N-meth-
ylanatabine and (R,S)-N-methylanabasine occurs, as
seen for nicotine and analogues altered in the pyridine
moiety (Mesnard et al., 2001). The profiles of degrada-
tion differ slightly, in that (R,S)-N-methylanatabine does
not persist in the cells and, by 8 days, about 25% of the
initial compound present in substrate is not recovered in
product. This profile is closer to that of nicotine degra-
dation than that of (R,S)-N-methylanabasine in that net
loss of alkaloid has occurred. However, no minor prod-
ucts that could represent either further metabolism of
8
6
4
2
0
6
4
2
0
0
2
4
6
8
10
12
0
2
4
6
8
10
12
(a)
(c)
Time (days)
Time (days)
0.8
0.6
0.6
0.4
0.2
0.0
0.4
0.2
0.0
0
2
4
6
8
10
12
0
2
4
6
8
10
12
(b)
(d)
Time (days)
Time (days)
Fig. 2. Metabolism of (a and b) (R,S)-N-methylanabasine or (c and d) (R,S)-N-methylanatabine by cell cultures of N. plumbaginifolia. (a) and (c)
show the alkaloid content of the medium, (b) and (d) the alkaloid content of the cells. Each point represents the mean s.d. for 2 cultures and at least
3 determinations by HPLC. (h) (R,S)-N-methylanabasine, ( ) (R,S)-anabasine, (n) (R,S)-N-methylanatabine, (m) (R,S)-anatabine, (d) total
n
alkaloid (substratecells + substratemedium + productcells + productmedium).

T.A. Bartholomeusz et al. / Phytochemistry 66 (2005) 1890–1897
1893
anatabine or alternative metabolites of N-methylanata-
bine were identified. For the demethylation of N-methy-
lanabasine, only anabasine was detected as product and
no decrease was observed in the total (substrate + prod-
uct) during the middle period of the culture.
analysed, it was seen to contain both isomers of (R,S)-
N-methylanabasine, with the (S)-isomer in excess of
the (R) (Fig. 4). This is compatible with both isomers
being taken up by the cells but the (R)-isomer being
more rapidly demethylated, the (R)-anabasine being ex-
creted and accumulating in the medium. It was not pos-
sible to obtain a reliable determination of the isomeric
composition of the low intracellular levels of (R)- and
(S)-anabasine.
2.2. Chiral selectivity of demethylation of (R,S)-N-
methylanabasine and (R,S)-N-methylanatabine
A simple explanation of the demethylation profiles
observed by HPLC is that the demethylation activity is
not equivalent for the (S)- and (R)-isomers. The chirality
of the residual substrates and the demethylation prod-
ucts was therefore followed in duplicate experiments
by chiral GC.
A very similar profile was seen for the selectivity of
demethylation of the two isomers of (R,S)-N-methyla-
natabine (Fig. 3(c) and (d)). Again, it was the (R)-isomer
of (R,S)-N-methylanatabine that rapidly disappeared
from the medium and (R)-anatabine that accumulated.
The (S)-isomer also underwent demethylation, but more
slowly, and (S)-anatabine accumulated in the medium to
only ca. 35% of the level of the (R)-isomer. The medium
content of (R)-N-methylanatabine was essentially
reduced to zero within 4 days. While the overall profile
of the chirality of demethylation is very close to that of
(R,S)-N-methylanabasine, there was ca. 25% loss of
alkaloid from the system (Fig. 2(c) and (d)). However,
it cannot be ascertained whether this loss is of only
one isomer or whether both isomers of anatabine are
subject to further metabolism. What is apparent is that,
as with (R,S)-nicotine, the rate of demethylation for the
two isomers was not equivalent and that the (R)-isomer
was degraded preferentially (Mesnard et al., 2001).
Indeed, for both (R,S)-N-methylanabasine and (R,S)-
N-methylanatabine, it cannot be definitively concluded
whether the (S)-isomer of the product is derived from
The profiles of the residual (R)- and (S)-N-methylan-
abasine and of the (R)- and (S)-anabasine accumulated
in the medium are presented in Fig. 3(a) and (b). It is
seen that, as with (R,S)-nicotine, the (R)-isomer was
very much more rapidly demethylated than the (S)-
isomer (Fig. 3(a)), leading to a rapid accumulation of
(R)-anabasine, which was accompanied by a slower
accumulation of (S)-anabasine, to about 15% of the
(R)-isomer (Fig. 3(b)). When compared with (R,S)-
nicotine (Mesnard et al., 2001), (R)-N-methylanabasine
was more rapidly lost than (R)-nicotine, essentially no
(R)-N-methylanabasine remaining in the medium after
4 days. This observation is open to two interpretations:
either only the (R)-isomer is transported into the cells or
the (R)-isomer is a very much better substrate for the N-
demethylating activity. When the intracellular pool was
100
80
60
40
20
0
100
80
60
40
20
0
0
2
4
6
8
10
12
0
2
4
6
8
10
12
(a)
(c)
100
Time (days)
Time (days)
50
40
30
20
10
0
80
60
40
20
0
0
2
4
6
8
10
12
0
2
4
6
8
10
12
Time (days)
(b)
(d)
Time (days)
Fig. 3. Metabolism of (a and b) (R,S)-N-methylanabasine or (c and d) (R,S)-N-methylanatabine by cell cultures of N. plumbaginifolia indicating the
amount of (R)- and (S)-isomer present in the medium for each compound. (a) and (c) show the substrate content, (b) and (d) the product content.
Each point represents the mean s.d. for 2 cultures and at least 2 determinations by GC. The total quantity of alkaloid was determined by GC and
the proportion of each isomer present by chiral GC. For substrates (a and c), ( ) total, (r) (R)-isomer, (m) (S)-isomer; for products (b and d), (h)
n
total, (e) (R)-isomer, (n) (S)-isomer.

1894
T.A. Bartholomeusz et al. / Phytochemistry 66 (2005) 1890–1897
10
8
120
100
80
60
40
20
0
6
4
0
2
4
6
8
10
2
(a)
Time (days)
0
120
100
80
0
2
4
6
8
10
12
Time (days)
Fig. 4. Metabolism of (R,S)-N-methylanabasine by cell cultures of N.
plumbaginifolia indicating the amount of (R)- and (S)-isomer present in
60
40
20
the cells. Each point represents the mean s.d. for at least
2
determinations by GC. The total quantity of alkaloid was determined
0
0
2
4
6
8
10
by GC and the proportion of each isomer by chiral GC. ( ) total, (r)
n
(b)
Time (days)
(R)-isomer, (m) (S)-isomer.
80
60
40
20
0
the (S)-isomer of the substrate or whether some racemi-
sation has occurred. From the data presented in
Fig. 3(a) and (c), it would appear that, from day 4 on-
wards, the residual alkaloids in the medium are com-
posed of approximately 100% of the (S)-isomer.
However, the (S)-isomers of both products accumulate
steadily over days 4–10 (Fig. 3(b) and (d)). Hence, it
would appear that either the cells contain a racemase,
slowly converting the (R)- to the (S)-isomer, or the
demethylase does not retain enantiomeric purity at
100%. In view of the fact that both anatabine and anab-
asine naturally accumulate as enantiomeric mixtures
(Armstrong et al., 1999), the presence of a racemase is
likely. Nicotine biosynthesis produces exclusively the
(S)-isomer (Leete, 1983), although whether such rigid
stereoselectivity also occurs for anatabine and anabasine
biosynthesis is not known. However, both isomers of
nornicotine have been reported, consistent with race-
mase activity on the N-demethylated product.
0
2
4
6
8
10
(c)
Time (days)
Fig. 5. Metabolism of (S)-nicotine and (R,S)-N-methylanabasine
simultaneously by cell cultures of N. plumbaginifolia. (a) The (S)-
nicotine, nornicotine and total alkaloid content in the medium in the
presence (r, e, m), respectively) or absence ( , h, d, respectively) of
(R,S)-N-methylanabasine; (b) and (c) the N-methylanabasine (filled
symbols) and anabasine (open symbols) content in the medium in the
n
presence of (S)-nicotine. (r, e) (R)-isomer, (m, n) (S)-isomer, ( , h)
n
total. Each point represents the mean s.d. for 2 cultures and at least
3 determinations by HPLC or GC.
present at day 8, consistent with previous data indicat-
ing that nornicotine shows substantial further
catabolism.
When exhibited simultaneously, both (S)-nicotine
and (R,S)-N-methylanabasine were taken into the cells
and demethylated (Fig. 5). The intracellular concentra-
tions of nicotine and N-methylanabasine and their
demethylation products were similar to when either
alkaloid was supplied alone (data not shown). Both
products of demethylation, nornicotine and anabasine,
were seen to accumulate steadily in the medium. The
presence of (R,S)-N-methylanabasine had no apparent
effect on the rate of metabolism of (S)-nicotine,
although the extent to which nornicotine accumulated
was enhanced. Whether this is an influence on the
further metabolism of nornicotine, leading to higher
accumulation, needs further investigation. However,
the overall loss of the nicotine alkaloid series from
the system occurred to an equivalent level both in the
presence and absence of (R,S)-N-methylanabasine
(Fig. 5(a)).
2.3. Co-feeding with (S)-nicotine and (R,S)-N-
methylanabasine
It was previously demonstrated that demethylation
by N. plumbaginifolia of nicotine and analogues in the
pyridine moiety showed competition (Mesnard et al.,
2001). Experiments were therefore conducted with
(R,S)-N-methylanabasine, homologue in the pyrrolin-
ium moiety, to see whether this modification had a sim-
ilar effect.
Cell cultures supplied with nicotine alone showed
essentially the same profile of nicotine catabolism as
seen previously, in that nicotine was consumed continu-
ously and nornicotine accumulated (Mesnard et al.,
2001). Within 8 days, for 6.2 lmol of nicotine con-
sumed, ca. 4 lmol of nornicotine accumulated
(Fig. 5(a)). Thus, only 71% of the initial alkaloid was

T.A. Bartholomeusz et al. / Phytochemistry 66 (2005) 1890–1897
1895
Furthermore, the presence of (S)-nicotine had no
apparent effect on the demethylation of (R,S)-N-methy-
lanabasine (compare Figs. 3 and 5): neither the rate of
demethylation nor the stereoselectivity of the reaction
was affected. In addition, the total alkaloid for the anab-
asine series remained essentially unaltered, as for (R,S)-
N-methylanabasine alone.
Thus, neither the rate of anabasine accumulation nor
the stereoselectivity of the demethylation of (R,S)-N-
methylanabasine was influenced by the presence of nic-
otine, nor was the metabolism of nicotine in the presence
of N-methylanabasine markedly different to that for nic-
otine alone.
Hence, the co-presence of the two substrates does not
appear to have mutually influenced their demethylation,
the kinetics of degradation not being modified. The only
notable effect is that of enhanced nornicotine accumula-
tion. This indicates that, in contrast to the situation of
substrates modified on the pyridine ring of nicotine
(Mesnard et al., 2001), demethylation activity is sensitive
to the size and/or configuration of the ring containing
the N-methyl group against which the activity is
directed.
compounds was any other metabolite identified. In its
profile, the demethylation of (R,S)-N-methylanatabine
is thus closer to that of nicotine than that of (R,S)-N-
methylanabasine.
However, both substrates show the same stereose-
lectivity of demethylation as (R,S)-nicotine, in that
the (R)-isomer is strongly preferred to the (S)-isomer.
Thus, over the time-course of the experiment, the
culture medium is relatively enriched in (S)-N-methy-
lanabasine or (S)-N-methylanatabine, while (R)-anaba-
sine and (R)-anatabine, respectively, accumulate. In
this accumulation of the products of demethylation,
the metabolism of these substrates differs very mark-
edly from that of nicotine, in that (R)-nornicotine is
rapidly further degraded. While it has been suggested
that the degradation probably is initiated with a
ring-opening reaction, as described for the degrada-
tion of nicotine by various micro-organisms (Kaiser
et al., 1996), this has not yet been experimentally
confirmed.
With (S)-nicotine, it is increasingly evident that
demethylation proceeds via oxidation of the N-methyl
group, without involving the ring (Botte et al., 1997;
´
Molinie, 2005), in contrast to the proposed mechanism
for the oxidation of (S)-nicotine by liver P450 oxidase
(Carlson et al., 1995). It is now shown that the demeth-
ylation activity present in N. plumbaginifolia can also
act on non-natural N-methylpiperidine homologues of
nicotine. The stereoselectivity of demethylation of
(R,S)-N-methylanatabine and (R,S)-N-methylanabasine
is the same as found for nicotine (Mesnard et al.,
2001), in that the (R)-isomer is preferentially degraded,
which could imply that the same enzyme system is
responsible for the demethylation of all these alkaloids.
However, N-methylanabasine does not apparently
compete with nicotine for demethylation activity, sug-
gesting the possibility that more than one demethyla-
tion activity might be present. Further study of these
demethylation processes is required to resolve these
discrepancies.
3. Conclusions
It has previously been shown that Nicotiana spp. cell
cultures are active in demethylating a range of substrates
(Boswell et al., 1999; Dawson, 1951; Leete, 1983; Man-
ceau et al., 1989; Mesnard et al., 2001), including those
with N-methylation or N-ethylation on the piperidine
ring of C6 homologues of nicotine (Dawson, 1951). It
is now confirmed that this demethylation is intracellular,
although, as frequently observed with in vitro cultures,
the majority of the product accumulates in the medium.
This is to be expected in that what little evidence is avail-
able indicates that demethylation in Nicotiana probably
involves a P-450 oxidase in the microsomal fraction
(Chelvarajan et al., 1993) or a mixed function oxidase
(Hao and Yeoman, 1998). Either case will have a
requirement for reduced pyridine cofactor. Further-
more, it is apparent that the methyl group can be trans-
ferred to the tetrahydrofolate pool (Mesnard et al.,
2002).
4. Experimental
4.1. Materials
The two homologues tested show slightly different
profiles of demethylation, in that the total (sub-
strate + product) remained close to 100% for (R,S)-N-
methylanabasine throughout the analytical period,
whereas for (R,S)-N-methylanatabine 30–40% of the
alkaloid was undetected, depending on the extent of
advancement of the reaction. It would appear that,
while efficient at demethylating both these compounds,
the cell cultures do not possess the capacity to degrade
further anabasine, but that anatabine may be sub-
ject to metabolism. However, for neither series of
(R,S)-N-Methylanabasine, (R,S)-N-methylanatabine
(R,S)-anabasine and (R,S)-anatabine were synthesised
as described previously (Felpin et al., 2001). An authen-
tic sample of synthetic (S)-(ꢀ)-anabasine (Amat et al.,
2002) was obtained from Pr. M. Amat (University of
Barcelona, Barcelona, Spain). A non-racemic mixture
of (R,S)-anabasine ((R):(S) approximately 4:1) was ob-
tained by extracting Burley tobacco leaf, as described
previously (Armstrong et al., 1999).

1896
T.A. Bartholomeusz et al. / Phytochemistry 66 (2005) 1890–1897
4.2. Cell suspension cultures
4.5. HPLC analysis (quantitative)
A stable culture of green N. plumbaginifolia was
cultured on medium ÔFMDÕ as described previously
(Mesnard et al., 2001). Essentially, this medium is a
modified ÔLSÕ medium (Linsmaier and Skoog, 1965)
and contains sucrose (87.6 mM), 2,4-dichlorophenoxy-
acetic acid (0.9 lM) and 6-benzylaminopurine (0.4 lM)
to promote growth.
Following optimisation of the conditions, alkaloids
were separated on an HPLC system composed of a
Merck 6200 pump with a Merck 4250 UV detector in
the following conditions: column, C18-Nucleosyl
(250 · 4.6 mm, Macherey Nagel, France); eluant, per li-
tre: 5 ml CH3CN, 4 ml THF, 0.5 ml H3PO4 (85%), 1 g
KH2PO4, 10 mg NaN3, 0.5 ml triethylamine (pH 3.5
with KOH); detector, 260 nm; flow, isocratic at 0.8 ml/
min. Each sample was analysed three times. In these
conditions, satisfactory resolution of all target alkaloids
was obtained. Detection limits were 10 ng for nicotine
and nornicotine, 25 ng for N-methylanabasine and
anabasine and 25 and 50 ng for N-methylanatabine
and anatabine, respectively. For all alkaloids, calibra-
tion was linear to at least 100 ng.
4.3. Protocol for bioconversion assays
Cells grown in culture medium ÔFMDÕ were harvested
aseptically during the exponential growth phase (day 8
of the culture cycle) and transferred into a non-
growth-stimulating medium ÔFMSÕ (Mesnard et al.,
2001). This minimal medium contains the macro-ele-
ments of medium ÔFMDÕ but lacks micro-elements, vita-
mins, sucrose and phytohormones. Very limited growth
occurs under these conditions. Biotransformation was
conducted as described previously (Mesnard et al.,
2001) in ÔFMSÕ medium supplemented with nicotine
(15.4 lmol), N-methylanabasine (7.1 lmol) or N-meth-
ylanatabine (6.2 lmol). Competition experiments were
conducted under the same conditions using nicotine
(7.7 lmol) with N-methylanabasine (3.6 lmol). All
experiments were carried out at least twice, with dupli-
cate analyses made in each experiment.
4.6. GC analysis (quantitative)
Quantification of alkaloids was performed by GC on
a Hewlett Packard 6890N (www.hp.com) chromato-
graph fitted with a Hewlett Packard 6783 injector; car-
rier gas, helium at 1.2 ml/min (constant flow); split
ratio, 1:20; column, fused poly(5% diphenyl/95% dim-
ethylsiloxane) deactivated for non-specific interaction
with basic compounds (PTA-5, Supelco, 30 m, i.d.
0.32 mm, film thickness, 0.52 lm), injector temperature,
220 ꢁC, detection by FID at 280 ꢁC; injected volume
(manual), 1 ll. Elution was by thermal gradient: 80 ꢁC
for 1 min; 10 ꢁC/min to 175 ꢁC, 6 min at 175 ꢁC,
40 ꢁC/min to 260 ꢁC, 1 min at 260 ꢁC. Calibration was
by reference to external standards (range 0.05–0.5 mg/
ml; nicotine r2 = 0.9979, nornicotine r2 = 0.9994, N-
methylanabasine r2 = 0.9981, anabasine r2 = 0.9990 N-
methylanatabine r2 = 0.9991, anatabine r2 = 0.9995)
and 2,6-di-tert-butyl-4-methylphenol (BHT) (0.5 mg/
ml) was used as internal correction reference. The iden-
tity of the eluted products was confirmed by co-elution
with authentic standards and by GC-MS.
4.4. Extraction procedures
Cells were separated from medium by filtration. Cells
were freeze-dried and medium stored at ꢀ20 ꢁC until re-
quired. Alkaloids were recovered from freeze-dried cells
by extraction with 25 ml CHCl3–NH4OH 30% (24/1 v/v)
twice under reflux for 2 h. After filtration, the pooled ex-
tract was dried under reduced pressure (33 ꢁC).
For HPLC, cellular extracts were dissolved in mobile
phase (1–5 ml) and filtered. Culture media were diluted
as required in mobile phase, filtered and directly injected
onto the HPLC system.
For chiral GC, a portion of the cellular extract in
mobile phase was rigorously extracted with CHCl3, the
organic phases pooled and dried, and the residue taken
into MeOH. Alkaloids were recovered from culture
media by solid phase extraction chromatography. Fol-
lowing basification to pH > 10 (NHþ4 solution), this
solution was applied to a preconditioned (washed with
5 ml MeOH and 5 ml 1% NHþ4 solution) solid phase
extraction cartridge (500 mg Discovery C18, Supelco,
www.sigmaaldrich.com) fitted to a Visiprep vacuum
manifold (Supelco). Sample was introduced by low suc-
tion, the cartridge rinsed with 1% NHþ4 solution (5 ml)
and dried with a flow of N2 gas for 60 min. Compounds
were recovered in methanol (10 ml) and the volume re-
duced to approximately 0.5 ml.
4.7. GC analysis (chiral)
Alkaloid stereoisomers were separated by GC on a
Hewlett Packard 6510 (www.hp.com) chromatograph;
carrier gas, hydrogen at 1.0 ml/min (constant flow); split
ratio, 1:10; column, BETA CHIRODEX (Supelco,
30 m, i.d. 0.32 mm, film thickness, 0.52 lm), injector
temperature, 220 ꢁC, detection by FID at 250 ꢁC;
injected volume (manual), 1 ll. Elution was by thermal
gradient. For N-methylanatabine and anatabine:
100 ꢁC for 1 min; 20 ꢁC/min to 150 ꢁC, 6 min at
150 ꢁC, 20 ꢁC/min to 170 ꢁC, 6 min at 170 ꢁC, 20 ꢁC/min
to 210 ꢁC, 1 min at 210 ꢁC. For N-methylanabasine:
100 ꢁC for 1 min; 10 ꢁC/min to 180 ꢁC, 20 ꢁC/min
to 210 ꢁC, 2 min at 210 ꢁC. Under these conditions,

T.A. Bartholomeusz et al. / Phytochemistry 66 (2005) 1890–1897
1897
Carlson, T., Jones, J., Peterson, L.A., Castagnoli, N.J., Iyer, K.,
Trager, W., 1995. Stereoselectivity and isotopic effects associated
with cytochrome P450-catalyzed oxidation of (S)-nicotine. Drug
Met. Disp. 2, 749–756.
Chelvarajan, R., Fannin, F., Bush, L.P., 1993. Study of nicotine
demethylation in Nicotiana otophora. J. Agric. Food Chem. 41,
858–862.
Dawson, R.F., 1951. Alkaloid biogenesis. III. Specificity of the
nicotine–nornicotine conversion. J. Am. Chem. Soc. 73, 4218–4221.
Fecker, L.F., Rugenhagen, C., Berlin, J., 1993. Increased production
¨
(R)- and (S)-anabasine were not resolved. (R)- and (S)-
anabasine were therefore determined as their trifluoro-
acetyl derivatives (Armstrong et al., 1999) under the
same chromatographic conditions with the following
thermal gradient: 100 ꢁC for 1 min; 5 ꢁC/min to
210 ꢁC, 1 min at 210 ꢁC.
Peak assignment was by reference to external stan-
dards: (R)- and (S)-anatabine on the ratio present in
an extract of Burley tobacco leaf, previously shown to
contain (R):(S)-anatabine in a ratio of 4:1 (Armstrong
et al., 1999); (R)- and (S)-anabasine by reference to an
authentic synthetic sample of (S)-(ꢀ)-anabasine (Amat
et al., 2002).
of cadaverine and anabasine in hairy root cultures of Nicotiana
tabacum expressing a bacterial lysine decarboxylase gene. Plant
Mol. Biol. 23, 11–21.
Felpin, F.X., Girard, S., Vo-Thanh, G., Robins, R.J., Villieras, J.,
Lebreton, J., 2001. Efficient enantiomeric synthesis of pyrrolidine
and piperidine alkaloids from tobacco. J. Org. Chem. 66, 6305–
6312.
Gorrod, J.W., Schepers, G., 1999. Biotransformation of nicotine in
mammalian systems. In: Gorrod, J.W., Jacob, P. (Eds.), Analytical
Determination of Nicotine and Related Compounds and their
Metabolites. Elsevier, Amsterdam, pp. 45–67.
Hao, D.Y., Yeoman, M.M., 1998. Evidence in favour of an oxidative
N-demethylation of nicotine to nornicotine in tobacco cell cultures.
J. Plant Physiol. 152, 420–426.
Kaiser, J.-P., Feng, Y., Bollag, J.-M., 1996. Microbial metabolism of
pyridine, quinoline, acridine, and their derivatives under aerobic
and anaerobic conditions. Microbiol. Rev. 60, 483–498.
Leete, E., 1983. Biosynthesis and metabolism of the tobacco alkaloids.
In: Pelletier, S. (Ed.), Alkaloids: Chemical and Biological Perspec-
tives. Wiley, Chichester, pp. 85–152.
Acknowledgements
We are most grateful to Professor Mercedes Amat
(University of Barcelona, Barcelona, Spain) for an
authentic sample of synthetic (S)-(ꢀ)-anabasine.
References
Amat, M., Canto, M., Llor, N., Bosch, J., 2002. Enantioselective
synthesis of 2-arylpiperidines from chiral lactam.
A concise
synthesis of (-)-anabasine. J. Chem. Soc. Chem. Commun., 526–
527.
Armstrong, D.W., Wang, X., Lee, J.-T., Liu, Y.-S., 1999. Enantio-
meric composition of nornicotine, anatabine and anabasine in
tobacco. Chirality 11, 82–84.
Linsmaier, E.M., Skoog, F., 1965. Organic growth factor requirements
of tobacco tissue cultures. Physiol. Plant. 18, 100–127.
Manceau, F., Fliniaux, M.-A., Jacquin-Dubreuil, A., 1989. Ability of a
Nicotiana plumbaginifolia cell suspension to demethylate nicotine
into nornicotine. Phytochemistry 28, 2671–2674.
Boswell, H.D., Watson, A.B., Walton, N.J., Robins, D.J., 1993.
Formation of N0-ethyl-S-nornicotine by transformed root cultures
of Nicotiana rustica. Phytochemistry 34, 153–155.
Mesnard, F., Girard, S., Fliniaux, O., Bhogal, R.K., Gillet, F.,
Lebreton, J., Fliniaux, M.-A., Robins, R.J., 2001. Chiral specificity
of the degradation of nicotine by Nicotiana plumbaginifolia cell
suspension cultures. Plant Sci. 161, 1011–1018.
Mesnard, F., Roscher, A., Garlick, A., Girard, S., Baguet, E., Arroo,
R., Lebreton, J., Robins, R.J., Ratcliffe, R.G., 2002. Evidence for
the involvement of tetrahydrofolate in the demethylation of
nicotine by Nicotiana plumbaginifolia cell-suspension cultures.
Planta 214, 911–919.
Boswell, H.D., Drager, B., Eagles, J., McClintock, C., Parr, A.,
Portsteffen, A., Robins, D.J., Robins, R.J., Walton, N.J., Wong,
C., 1999. Biosynthesis of novel alkaloids part 1 – Metabolism of N-
alkyldiamines and N-alkylnortropinones by transformed root
cultures of Nicotiana and Brugmansia. Phytochemistry 52, 855–869.
Botte, M., Mabon, F., Le Mouillour, M., Robins, R.J., 1997.
Biosynthesis of nornicotine in root cultures of Nicotiana alata
does not involve oxidation at C-50 of nicotine. Phytochemistry 46,
117–122.
Molinie´, R., 2005. Effet isotopique cine´tique de lÕazote au cours de la
´
´
´
demethylation des alcalo¨ıdes par les plantes et les bacteries. Ph.D.
thesis, University of Nantes, Nantes.
Brennan, M., 2000. The good side of nicotine. Chem. Eng. News
March 27, 23–26.
Bush, L.P., Hempfling, W.P., Burton, H.R., 1999. Biosynthesis of
nicotine and related alkaloids. In: Gorrod, J.W., Jacob, P. (Eds.),
Analytical Determination of Nicotine and Related Compounds
and their Metabolites. Elsevier, Amsterdam, pp. 13–44.
Robins, R.J., 1998. The biosynthesis of alkaloids in root cultures. In:
Roberts, M.F., Wink, M. (Eds.), Alkaloids: Biochemistry, Ecology,
and Medicinal Applications. Plenum Press, New York, pp. 199–
218.
Saitoh, F., Noma, M., Kawashima, N., 1985. The alkaloid contents of
sixty Nicotiana species. Phytochemistry 24, 477–480.