Absolute configuration of anabasine from Messor and Aphaenogaster ants

Article abstract of DOI:10.1023/A:1010335003297

A method has been developed to assign the absolute configuration and enantiomeric excess of anabasine based on small amounts of material (in the microgram range), by derivatization with (+)-menthylchloroformate followed by capillary GC analysis of the resulting carbamate(s). This method was applied to three samples of anabasine isolated from Messor and Aphaenogaster ants. In Messor sanctus, only (2′S)-anabasine was present, whereas in Aphaenogaster subterranea and A. miamiana (2′S)-anabasine was determined to have an ee of 78 and 24%, respectively.

Full text of DOI:10.1023/A:1010335003297

Journal of Chemical Ecology, Vol. 27, No. 5, 2001
ABSOLUTE CONFIGURATION OF ANABASINE FROM
Messor AND Aphaenogaster ANTS
SABINE LECLERCQ,1 SEBASTIEN CHARLES,1 DESIRE DALOZE,1
JEAN-CLAUDE BRAEKMAN,1, SERGE ARON,2 and JACQUES M.
PASTEELS2
1Laboratory of Bio-organic Chemistry
Department of Organic Chemistry, CP 160/07
2Laboratory of Cellular and Animal Biology, CP 160/12
Free University of Brussels
Av. F. D. Roosevelt
50 - 1050 Brussels, Belgium
(Received June 28, 2000; accepted January 3, 2001)
Abstract—A method has been developed to assign the absolute configuration
and enantiomeric excess of anabasine based on small amounts of material (in the
microgram range), by derivatization with (+)-menthylchloroformate followed
by capillary GC analysis of the resulting carbamate(s). This method was applied
to three samples of anabasine isolated from Messor and Aphaenogaster ants. In
Messor sanctus, only (20S)-anabasine was present, whereas in Aphaenogaster
subterranea and A. miamiana (20S)-anabasine was determined to have an ee of
78 and 24%, respectively.
Key Words—Alkaloids, anabasine, ants, Messor, Aphaenogaster, absolute con-
figuration.
INTRODUCTION
The alkaloid anabasine (1) has been isolated from several plant genera, such
as Anabasis, Nicotiana, Duboisia, Malacocarpus, and Marsdenia (Leete, 1983).
20
( )-Anabasine ([ ]D
82.2, neat) from Anabasis aphylla L. was shown to have
the (20S) absolute configuration (Lukes et al., 1962) (Figure 1), but values of
the optical rotation of anabasine from different sources vary considerably (from
82.2 to 0) (Leete, 1983). More recently, anabasine has also been reported from
animal sources, e.g., in two of the 19 species of hoplonemertine worms surveyed
To whom correspondence should be addressed. e-mail: braekman@ulb.ac.be
945
C
0098-0331/01/0500-0945$19.50/1 2001 Plenum Publishing Corporation

946
LECLERCQ ET AL.
FIG. 1. Structures of (20S)-( )-anabasine [(20S)-1] and anabaseine (2).
(Kem, 1988), in the poison gland of the ants Messor ebeninus (Coll et al., 1987) and
M. bouvieri (Jackson et al., 1989), and as a minor component in M. capensis (Brand
and Mpuru, 1993) and Aphaenogaster rudis, where it coexists with anabaseine (2)
(Figure 1) (Attygalle et al., 1998).
From a biosynthetic point of view, it would be interesting to compare the abso-
lute configuration of anabasine (1) isolated from plants and from animals (worms
and ants). To this end, an effective and practical procedure to assign the absolute
configuration of this alkaloid and applicable to quantities in the microgram range
was needed. Although the separation of the enantiomers of anabasine as their tri-
fluoroacetyl derivatives on a dipentyl -cyclodextrin GC column has been reported
(Armstrong et al., 1990), this method is not very convenient since this column is
not commercially available. Our attempts to separate the anabasine enantiomers
on two commercial chiral columns (CP-Chirasil Val and CP-Chirasil-Dex CB)
were unsuccessful as the alkaloids did not elute from these columns at the tem-
perature limit. In this paper, we have developed a method based on the reaction
of ( )-anabasine with (+)-menthylchloroformate, affording the corresponding
menthoxycarbonyl derivatives, followed by comparison of their retention time in
capillary gas chromatography with that of a standard of known absolute configura-
tion. This procedure allowed us to assign the absolute configuration and to measure
the ee of anabasine (1), which was detected in three ant species: Messor sanctus,
Aphaenogaster subterranea, and A. miamiana (Myrmicinae: Myrmicini).
METHODS AND MATERIALS
Sample Collection. Colonies of Messor barbarus and M. sanctus were collec-
ted at Port Leucate (southern France). Colonies of Messor structor were collected
in the area of Toulouse (southern France). Colonies of Aphaenogaster subterranea
were collected in Bouconne forest (Midi-Pyre´ne´es), whereas Aphaenogaster se-
nilis were collected at Consprons (Banyuls-sur-Mer, southern France). Colonies
of Aphaenogaster miamiana were collected near Gainesville, Florida.
Analyses of Ants for Alkaloids. Ants were separated manually from the soil
and dipped into MeOH. Five hundred ants of each species were then exhaustively

ANABASINE CONFIGURATION
947
extracted with CHCl3–MeOH (1 : 1). The extract was evaporated under vac-
uum and submitted to filtration on an alumina column, first eluting with CH2Cl2
(10 ml) to remove nonpolar compounds, then with CH2Cl2–MeOH (1 : 1; 20 ml)
to yield a polar fraction which was analyzed by TLC and GC-MS for the presence
of alkaloids.
Chemical Analyses. UV spectra were taken on a Philips PU 8700 UV-VIS
spectrophotometer in MeOH. IR spectra were recorded on a Bruker IFS 25 instru-
ment as a film on a NaCl disk. NMR spectra were recorded in CDCl3 at 600 and
150.87 MHz (Varian Unity 600 instrument) or at 250 MHz (Bruker WM 250).
Chemical shifts ( ) are reported in parts per million from internal TMS, and the
coupling constants are given in hertz. Mass spectra were performed on a Fisons
VG Autospec mass spectrometer in the electron impact (70 eV) mode. Optical
rotations were measured on a Perkin Elmer 141 polarimeter (Na vapor lamp) in a
10-cm cell at room temperature. Flash liquid chromatography was performed over
Macherey-Nagel Si gel (0.04–0.063 mm) and thin-layer chromatography analy-
ses (TLC) on Polygram SilG/UV254 precoated plates (0.25 mm). The compounds
were visualized under UV254 light, and/or by spraying with Dragendorff reagent.
GC-MS analyses were carried out on a Fisons VG Autospec mass spectrometer in
the electron impact (70 eV) mode, coupled to a Carlo Erba GC 8065 gas chromato-
graph equipped with a split injector, and with a 25-m 0.25-mm CP-Sil 5CB fused
silica column (Chrompack). The conditions were: 1 min at 150 C, then increased
10 C/min to 320 C, then maintained 2 min at 320 C. Carrier gas was helium, and
injector and detector temperatures were 250 C. GC analyses were performed on a
Varian gas chromatograph equipped with a 25-m 0.53-mm CP-Sil 19CB fused
silica column (Chrompack). The carrier gas was nitrogen, and oven and injector
temperatures were maintained at 250 C and the detector temperature at 270 C.
Derivatization of ( )-Anabasine with (+)-(1S,2R,5S)-Menthylchloroformate
[(+) 3]. To ( )-anabasine (1.0 mg, 0.0062 mmol) in a 300- l Reacti-vial was
added anhydrous THF (150 l), NEt3 (5 l, 0.036 mmol) and finally (+)-3 (4.0 l,
0.018 mmol). After stirring at 60 C under argon for 2 hr, the reaction mixture
was poured into 10% aqueous NaOH (2 ml), and the aqueous phase extracted
with diethyl ether (3 1 ml). Drying and concentration of the combined organic
extracts in vacuo afforded an oily residue, which was purified by flash chromatog-
raphy on silica gel (CH2Cl2, then CH2Cl2–MeOH 9 : 1) to quantitatively afford
a mixture of (20R)-4 and (20S)-4 (2.1 mg, 0.0061 mmol). They were separated
by reverse-phase HPLC (Chromspher C18, 5 m, 10 250 mm, CH3CN–H2O,
65 : 35, flow: 5 ml/min).
20
(20 R)- [(1S,2R,5S)-Menthoxycarbonyl]anabasine [(20R) 4]. [ ]D +110
+.
(c 0.135, CH2Cl2); EI-MS (70 eV) m/z 344 (M , C21H32N2O2, 6), 216 (10), 207
(100), 206 (66), 189 (22), 162 (38), 138 (7), 133 (14), 128 (7), 92 (21), 83 (53), 69
1
(26), 55 (38); IR (film) 2928–2866, 1694, 1422, 1265, 1169, 1037, 810-714 cm
;
1
UV (MeOH)
(ε) 203 (6800), 262 (1900); GC: tr = 13.0 min; H NMR
max

948
LECLERCQ ET AL.
(600 MHz, CDCl3) 8.52 (m, 2 H, H-2, H-6), 7.57 (d, 8.0 Hz, 1 H, H-4), 7.32
(m, 1 H, H-5), 5.51 (bs, 1H, H-20), 4.65 (td, J = 11.0, 4.0, 1 H, H-80), 4.12 (bd,
J = 13.0, 1 H, H-60eq), 2.79 (td, J = 14.0, 5.0, 1 H, H-6 0ax), 2.28 (bd, J = 14.0,
1 H, H-30eq), 2.12 (bd, J = 11.0, 1 H, H-130eq), 1.96 (tt, J = 13.8, 4.8, 1 H,
H-30ax), 1.67 (m, 4 H, H-40eq, H-10 0eq, H-110eq, H150), 1.62 (m, 1 H, H-50eq),
1.52 (m, 2 H, H-50ax and H-120ax), 1.43 (bq, J = 13.0, 1 H, H-40ax), 1.30 (m,
1 H, H-90ax), 1.04 (m, 1 H, H-100ax), 0.98 (q, J = 11.8, 1 H, H-130ax), 0.91 (d,
J = 6.7, 3 H, CH3-140), 0.86 (m, 1H, H-110ax), 0.78 (d, J = 5.0, 3 H, CH3-160
or 170), 0.73 (d, J = 6.3, 3 H, CH3-160 or 170).
20
(20S)-[(1S,2R,5S)-Menthoxycarbonyl]anabasine [(20S)-4]. [ ]D
100 (c
0.485, CH2Cl2); EI-MS (70 eV) m/z 344 (M+., C21H32N2O2, 6), 216 (10), 207
(100), 206 (66), 189 (22), 162 (38), 138 (7), 133 (14), 128 (7), 92 (21), 83 (53), 69
1
(26), 55 (38); IR (film) 2956–2872, 1699, 1410, 1261, 1038, 963, 815-712 cm
;
1
UV (MeOH)
(ε) 203 (4600), 262 (1800); GC: tr = 13.5 min; H NMR
max
(250 MHz, CDCl3) 8.54 (m, 2 H, H-2, H-6), 7.62 (d, J = 8.0, 1 H, H-4), 7.36
(dd, J = 8.0, 4.0, H-5), 5.53 (bs, J = bd, 1 H, H-20), 4.61 (td, J = 11.0, 4.0,
1 H, H-80), 4.13 (bd, J = 14.0, 1 H, H-60eq), 2.79 (ddd, J = 14.0, 11.0, 4.0, 1 H,
H-60ax), 0.92 (d, J = 6.7, 3 H, CH3-140), 0.79 (d, J = 6.9, 3 H, CH3-16 0 or -170),
0.73 (d, J = 6.9, 3 H, CH3-160 or 170). The derivatization procedure described
above was applied to the alkaloid fractions coming from M. sanctus, A. subter-
ranea, and A. miamiana. The menthoxycarbamates thus obtained were analyzed
by capillary GC and by GC-MS.
Hydrolysis of (20S)-Anabasine-p-NO2-Benzamide [(20S)-5] into (20S)-Anab-
asine. (20S)-5 (Pfrengle and Kunz, 1989) was kindly provided by Prof. H. Kunz
20
(University of Mainz). (20S)-5, [ ]D
130 (c 3, MeOH) (19.6 mg, 0.063 mmol)
was dissolved in a mixture of 33% HBr–AcOH (0.6 ml) in a 1 ml Reacti-vial.
The mixture was stirred at 80 C for 45 hr and was then poured into NH4OH
(8 M) and the aqueous phase quickly extracted with CH2Cl2. The combined organic
extracts were concentrated in vacuo to afford an oily residue that was purified by
flash chromatography on a silica gel column (CH2Cl2–MeOH 9 : 1) to afford
(8.5 mg) (20S)-anabasine in an 84% yield.
20
1
(20S)-Anabasine [(20S)-1]. [ ]D
21 (c 0.5, MeOH); H NMR data of
(20S)-1 were identical to those of our synthetic material and to literature data
(Yang and Tanner, 1997). Treatment of this sample with (+)-3 as described above
furnished (20 S)-[(1S,2R,5S)-menthoxycarbonyl]anabasine [(20S)-4], which gave a
single peak at tr = 13.5 min in capillary GC.
RESULTS
Development of a Procedure to Assign the Absolute Configuration of Anaba-
sine. We first applied to a sample of ( )-anabasine [( )-1], synthesized according

ANABASINE CONFIGURATION
949
SCHEME 1. Derivatization of ( )-1 with (+)-(1S,2R,5S)-menthylchloroformate, and syn-
thesis of (20S)-4. Reagents and conditions: (a) NEt3, THF, 60 C, 2 hr; (b) 33% HBr–AcOH,
80 C, 45 hr; (c) (+)-(1S,2R,5S)-3, NEt3, THF, 60 C, 2 hr.
to Deo and Crooks (1996), the procedure that was developed for the solenopsins
(Leclercq et al., 1994), but we could not separate the resulting diastereomeric
MTPA amides by capillary GC or by HPLC. In contrast, treatment of ( )-1 with
an excess of (+)-menthylchloroformate [(+)-3] quantitatively furnished the di-
astereomeric carbamates (20R)-4 and (20S)-4 (Scheme 1), which could be sepa-
rated by reverse-phase HPLC on a C18 column. Moreover, their retention times
in capillary GC on a CP-Sil 19CB column were also different (Figure 2). The
two diastereomers were identified by GC comparison with a reference sample
of (20S)-4, which was prepared from (20S)-anabasine-p-nitrobenzamide [(20S)-5]
(Pfrengle and Kunz, 1989) (Scheme 1). Under our GC conditions, the retention
time of (20R)-4 was 13.0 min and that of (20S)-4, 13.5 min (Figure 2). We have also
assigned all the proton signals of (20R)-4 by a 2D NMR study at 600 MHz.
Detection of Anabasine and Anabaseine in Messor and Aphaenogaster Ants.
Five hundred ants of each species were exhaustively extracted, and each extract
was checked for the presence of anabasine (1) by TLC and GC-EI-MS. Compound
1 was present as the sole alkaloid in two of the six species, Messor sanctus and
Aphaenogaster miamiana. In TLC, the alkaloidic fraction of A. senilis exhibited
one major spot less polar than anabasine. GC-EI-MS analysis revealed the presence
of one major peak identified as anabaseine (2) (Wheeler et al., 1981). No trace
of anabasine was detected in this species. In contrast, the two compounds were
present in the venom of A. subterranea, in an approximately 60 : 40 ratio. Finally,
neither anabasine nor anabaseine could be detected in the venom of M. barbarus
and M. structor. The relative amounts of anabasine (1) and anabaseine (2) identified
in the venom of Messor and Aphaenogaster ants are reported in Table 1.

950
LECLERCQ ET AL.
FIG. 2. GC (FID) separation of (20S)-4 and (20R)-4.
Absolute Configuration and Enantiomeric Excess of Anabasine from Messor
and Aphaenogaster Ants. The procedure described above was applied to determine
the absolute configuration of anabasine isolated from the venom of M. sanctus,
A. subterranea, and A. miamiana. In M. sanctus, only one diastereoisomer was
present after derivatization, whose retention time corresponded to that of synthetic
TABLE 1. RELATIVE AMOUNTS OF 1 AND 2 IN VENOMS OF Messor AND Aphaenogaster
ANTS
Anabasine (1)
Anabaseine (2)
M. sanctus
M. barbarus
M. structor
A. subterranea
A. senilis
100
0
0
60 a
0
0
0
0
40 a
100
0
A. miamiana
100
aApproximate figures, due to peak superimposition.

ANABASINE CONFIGURATION
951
(20S)-4. Thus, anabasineispresentinthevenomofM. sanctusasasingleenantiomer
having the 20S configuration.
In A. subterranea and A. miamiana, anabasine was present as a mixture
of R and S enantiomers, with the S enantiomer predominating. Since we have
checked that no kinetic resolution occurs during conversion of anabasine into
diastereoisomers 4, the enantiomeric excess of (S)-anabasine from A. subterranea
and A. miamiana could be determined to be 78% and 24%, respectively, from the
(20S)-4/(20R)-4 ratio.
DISCUSSION
The data reported here on the ee of anabasine from ants are too limited to
make any generalization. However, it should be pointed out that our results are
similar to those obtained with plants, where anabasine either exists as a single
enantiomer with the S configuration or as S/R mixtures, with the S enantiomer
being generally predominant (Leete, 1983). Several hypotheses can be put forward
to explain the wide range of enantiomeric purity observed for anabasine samples
isolated from both plants and ants: (1) poor stereochemical control of the enzymatic
reaction leading to the formation of the (20S)-stereogenic carbon, (2) co-occurrence
of two different biosynthetic routes for the (+) and ( ) enantiomers, and (3)
chemical or enzymatic isomerization of enantiomerically pure anabasine. The
relative contribution of these different possibilities is not known yet. In any case,
the detection of a single anabasine enantiomer in M. sanctus shows that our method
of analysis does not lead to any racemization.
The presence of anabasine and anabaseine in the genus Aphaenogaster could
be of taxonomic significance, since it supports the current classification of these
ants with both genera being placed within the same tribe Pheidolini (Bolton, 1995);
thegenusMessorwasevenonceconsideredasasubgenusofAphaenogaster(Forel,
1890). However, it is striking that two of the three Messor species analyzed in this
study do not contain alkaloids, at least in amounts sufficient to be detected by TLC
and CG-MS.
Acknowledgments—We are grateful to Prof. Luc Passera (University of Toulouse) for collect-
ing M. structor ants and to Dr. R. K. Vander Meer (USDA, Gainesville, Florida) for collecting A.
miamiana ants. We also thank Mr. S. Rossay for his participation in the preliminary aspects of this work,
Dr. M. Luhmer for the NMR spectra and Mr. C. Moulard for the mass spectra. S. A. and S. L. thank
the Fonds National de la Recherche Scientifique of Belgium for financial support.
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