Biosynthesis of defensive coccinellidae alkaloids: Incorporation of fatty acids in adaline, coccinelline, and harmonine

Article abstract of DOI:10.1002/ejoc.201101563

In this study, we report on in vitro incorporation experiments of several labelled fatty acids in the ladybird alkaloids coccinelline (Coccinella 7-punctata), adaline (Adalia 2-punctata), and harmonine (Harmonia axyridis). The obtained results clearly indicate that stearic acid is the precursor of coccinelline and harmonine, whereas myristic acid is at the origin of the carbon skeleton of adaline. Possible pathways for the biosynthesis of these alkaloids are presented. In vitro incorporation experiments of labelled fatty acids in the ladybird alkaloids coccinelline (Coccinella 7-punctata), adaline (Adalia 2-punctata) and harmonine (Harmonia axyridis) indicate that stearicacid is the best precursor for the biosynthesis of coccinelline and harmonine, whereas myristic acid is more efficient for the formation of the carbon skeleton of adaline. Copyright

Full text of DOI:10.1002/ejoc.201101563

FULL PAPER
DOI: 10.1002/ejoc.201101563
Biosynthesis of Defensive Coccinellidae Alkaloids: Incorporation of Fatty Acids
in Adaline, Coccinelline, and Harmonine
Eveline Haulotte,[a] Pascal Laurent,[a][‡] and Jean-Claude Braekman*[a]
Keywords: Insects / Alkaloids / Biosynthesis / Fatty acids / Isotopic labeling
In this study, we report on in vitro incorporation experiments
of several labelled fatty acids in the ladybird alkaloids cocci-
nelline (Coccinella 7-punctata), adaline (Adalia 2-punctata),
and harmonine (Harmonia axyridis). The obtained results
clearly indicate that stearic acid is the precursor of coccinel-
line and harmonine, whereas myristic acid is at the origin
of the carbon skeleton of adaline. Possible pathways for the
biosynthesis of these alkaloids are presented.
ferred source of nitrogen, and that the alkaloid biosynthesis
takes place in the insect fat body.[10] In addition, the incor-
poration of specifically deuterated adaline into A. 2-
punctata clearly indicates a close relationship between ad-
aline (2) and adalinine,[9] the latter yielded from the former.
Finally, Camarano et al.[11] reported good incorporations
into the homotropane euphococcinine and two piperidine
alkaloids when feeding adults of Epilachna paenulata on a
diet enriched with [1-13C] and [2-13C]sodium acetate.
In our continued search for a better understanding of
the biosynthetic origin of Coccinellidae alkaloids, we herein
report the incorporation results into coccinelline (1), ad-
aline (2), harmonine (3; see Figure 1), specifically tritiated
fatty acids, namely [14,14,14-3H3]myristic acid (4),
[16,16,16-3H3]palmitic acid (5), and [18,18,18-3H3]stearic
acid (6).
Introduction
Much research has been carried out on the chemical de-
fense systems of Coccinellidae, and it is now well estab-
lished that many coccinellids owe their protection to the
presence of alkaloids.[1–4] More than 50 ladybird alkaloids
have been isolated and characterized, including perhy-
droazaphenalenes, homotropanes, piperidines, pyrrolidines,
azamacrolides, linear amines, and so forth.[3] Despite the
structural diversity of the coccinellid alkaloids, most of
their skeletons are a chain of carbon atoms joined at one
or more sites to a nitrogen atom, suggesting a common bio-
genetic origin associated with a polyacetate pathway. Until
now, only a few incorporation studies supporting this hy-
pothesis have been reported.[5] For example, incorporating
labelled oleic acid and l-serine into Epilachna varivestis lar-
vae and conducting subsequent GC–MS analysis of the col-
lected pupal defensive secretion established the fatty acid
origin of epilachnene.[6] Furthermore, other feeding experi-
ments with specifically deuterated octadec-9-enoic acid
indicated that only the C-15 methylene group of oleic acid
was involved in the process leading to the carbon–nitrogen
bond formation in epilachnene.[7]
In addition, we have shown that the adults of Coccinella
7-punctata[8] and Adalia 2-punctata[9] fed with [1-14C] and
[2-14C]sodium acetate incorporated these precursors into
coccinelline (1) and adaline (2), respectively, supporting a
polyacetate origin for these alkaloids. Moreover, in vitro in-
cubation assays using ladybird tissues demonstrated that 1
and 2 are most likely biosynthesized through a fatty acid
rather than a polyketide pathway, that glutamine is the pre-
Figure 1. Structures of compounds 1, 2, 3, and 10.
Results and Discussion
The labelled fatty acids 4–6 were synthesized as shown
in Scheme 1 by starting from diacids 7–9, respectively. The
starting diacids 7 and 8 are commercially available. This is
not the case for heptadecanedioic acid (9), which had to
be synthesized by a three-step sequence (Scheme 2) from
tridecanedioic acid (7) according to the methodology de-
scribed by Obaza and Smith.[12] The key step in the synthe-
sis of the labelled fatty acids was the alkylation of mono-
[a] Department of Organic Chemistry CP 160/6, Free University
of Brussels, Faculty of Sciences,
50 Av. F. D. Roosevelt, 1050 Brussels, Belgium
Fax: +32-2-6502184
E-mail: braekman@ulb.ac.be
[‡] Current address: Unit of General and Organic Chemistry,
University of Liege-Gembloux Agro-Bio Tech,
2 Passage des déportés, 5030 Gembloux, Belgium
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E. Haulotte, P. Laurent, J.-C. Braekman
FULL PAPER
Scheme 1. Syntheses of [14,14,14-3H3]myristic acid (4), [16,16,16-3H3]palmitic acid (5), and [18,18,18-3H3]stearic acid (6). Reagents and
conditions: (i) CH3OH, H2SO4, reflux; (ii) Ba(OH)2, CH3OH for 4 and CH3OH/THF (6:1) for 5 and 6, then HCl; (iii) (COCl)2, DMF
(N,N-dimethylformamide), hexane, then [3H3]CCdCl, toluene, 0 °C; (iv) p-toluenesulfonylhydrazine, p-TsOH, DMF–sulfolane, then
NaBH3CN, 110 °C; (v) KOH, 18-crown-6, toluene, then HCl.
Scheme 2. Synthesis of heptadecanedioic acid (9). Reagents and conditions: (i) (COCl)2, DMF, hexane, then Meldrum’s acid, DMAP [4-
(dimethylamino)pyridine], THF (tetrahydrofuran); (ii) NaBH3CN, AcOH–THF, 65 °C; (iii) HCl, H2O.
esters 14–16 through a coupling reaction between the corre- the first group of beetles. In both series, [18,18,18-3H3]-
sponding acyl chlorides and [3H3]CCdCl, prepared by the stearic acid had the highest SIR in coccinelline. Finally, and
action of CdCl2 on [3H3]CMgI.[13–16] Ketones 17–19, ob- not surprisingly, for H. axyridis, [18,18,18-3H3]stearic acid
tained in this way, were reduced to form methyl esters 20– was incorporated with a much higher rate into harmonine
22, which were then hydrolyzed to yield the desired labelled than [14,14,14-3H3]myristic acid (Entries 10 and 11).
fatty acids 4–6.[14]
In our previous work dealing with the biosynthesis of
In vitro incubation experiments were carried out by using coccinelline (1) and adaline (2), we postulated that stearic
a protocol identical to the one described in our previous acid could be the precursor of both alkaloids.[10] However,
paper.[10] Thus, each of the three tritiated potential precur- taking into account the data presented in Table 1, we have
sors was immersed with C. 7-punctata, A. 2-punctata, or revised our hypothesis to take into consideration the fact
Harmonia axyridis tissues (20 beetles each) in 1.5 mL of the that although stearic acid was about 20 times more ef-
saline solution for 24 h as described by Ivarsson et al.[17] ficiently incorporated into coccinelline than myristic and
This solution also contained the protease inhibitor PMSF palmitic acid, this was not the case for adaline. Indeed, for
(phenylmethylsulfonyl fluoride), NADPH (reduced nicotin- this alkaloid, the incorporation of myristic acid was about
amide adenine dinucleotide phosphate), and glutamine to 15 times more efficient than the incorporation of palmitic
boost the alkaloid biosynthesis. Then, the respective alka- or stearic acid. Consequently, although the pathway pos-
loids, namely coccinelline (1), adaline (2), and harmonine tulated for the biosynthesis of coccinelline remains correct
(3), were isolated and repeatedly purified to a constant spe- (Scheme 3), the one for adaline is revised as presented in
cific activity (SA), and the specific incorporation rates Scheme 4. The fatty acid incorporation results obtained for
(SIR) were calculated. It should be noted that harmonine A. 2-punctata are better explained if we propose that, in this
was isolated and purified as its diacetyl derivative 10.
species, stearic acid does not undergo two successive β-oxi-
The results obtained are summarized in Table 1. This dations to form myristic acid, but that instead myristic acid
table shows that for A. 2-punctata (Entries 7–9), [14,14,14- is detached from the fatty acid synthetase complex when
3H3]myristic acid was about 15 times more efficiently incor- the chain has grown up to 14 carbon atoms.
porated in adaline than [16,16,16-3H3]palmitic acid and
To refine the biosynthetic pathway of harmonine and to
[18,18,18-3H3]stearic acid. For C. 7-punctata, two series of obtain information about the process leading to the second-
in vitro incorporation experiments were carried out during ary amine formed in this alkaloid, an in vitro incorporation
two successive springs. Whatever the incorporated precur- experiment using [11,11,12,12,13,13,14,14,15,15,16,16,17,
sor, the SIR measured was always higher for the first series 17,18,18,18-2H17]stearic acid (25) was performed with tis-
(Entries 1, 3, and 5) than for the second one (Entries 2, 4, sues of 100 beetles and 5.1 mg of deuterated precursor. This
and 6), probably because of a better metabolic activity for specifically deuterated acid was synthesized by starting
1908
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Biosynthesis of Defensive Coccinellidae Alkaloids
Table 1. Results of the in vitro incorporation experiments of [14,14,14-3H3]myristic acid (4), [16,16,16-3H3]palmitic acid (5), and [18,18,18-
3H3]stearic acid (6) in coccinelline (1), adaline (2), and harmonine (3).[a]
Entry
Alkaloid
(Species)
Incorporated
fatty acid
RI
Alkaloid
amount isolated
[mg]
TR
SA
SIR
[10–3 mCi]
[10–6 mCi]
[mCi/mmol]
[%]
1
2
3
4
5
6
Coccinelline (1)
(C. 7-punctata)
4
4
5
5
6
6
1.45
1.63
1.34
1.82
0.81
1.15
0.87
0.73
1.06
0.57
2.07
0.39
0.13
0.06
0.17
0.06
4.63
0.74
3.05 10–5
1.80 10–5
3.36 10–5
2.20 10–5
4.68 10–4
3.90 10–4
0.010
0.005
0.011
0.006
0.220
0.136
7
8
9
Adaline (2)
(A. 2-punctata)
4
5
6
1.55
1.35
1.37
2.96
2.30
1.33
10.74
0.42
0.42
1.59 10–3
1.19 10–4
7.40 10–5
0.495
0.035
0.021
10
11
Harmonine (3)[b]
(H. axyridis)
4
6
1.37
0.76
2.62
2.36
1.50
28.49
2.09 10–4
0.069
2.090
4.42 10–3
[a] RI = amount of radioactivity incorporated in the sample; TR = total amount of radioactivity recovered in the isolated alkaloid; SA
= specific activity; SIR = specific incorporation rate. [b] Isolated as its diacetyl derivative 10.
Scheme 5. Synthesis of 25. Reagents and conditions: (i) nBuLi,
THF, then [1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-2H17]n-octyl bromide,
DMPU (N,NЈ-dimethyl-N,NЈ-propyleneurea), 0 °C; (ii) H2, Pd/C,
Scheme 3. Biosynthetic pathway proposed for coccinelline (1) in C.
EtOH, then PPTS (pyridinium p-toluenesulfonate), EtOH, 55 °C;
7-punctata.
(iii) CrO3, H2SO4, acetone.
quently, three deuterium atoms had been lost during the
incorporation experiment. A possible explanation for this
observation is that two deuterium atoms were lost during
the oxidation of the C-17 methylene group, and a third one
was lost at the adjacent carbon atom (C-16) through an
intermediary imine undergoing an imine–enamine tauto-
merism. Moreover, as natural harmonine occurs as a single
enantiomer with the (R) configuration at its stereogenic
center,[19] the reduction of the intermediate imine (or en-
amine) produced during the amination step must be stereo-
Scheme 4. Biosynthetic pathway proposed for adaline (2) in A. 2-
punctata.
from protected alkynol 26[18] and [1,1,2,2,3,3,4,4,5,5,6,6,
7,7,8,8,8-2H17]n-octyl bromide using the synthetic pathway
reported by Attygalle et al.[7] and is described in Scheme 5.
The HRMS (EI) spectrum of the sample of diacetylhar-
monine isolated after incorporation of precursor 25 showed
that besides peaks found at 367.3338 (calcd. for
C22H43N2O2 [M + H]+ 367.3325) and 323.3053 (calcd. for
C20H39N2O [M – CH3CO]+· 323.3065) characteristic of un-
labelled diacetylharmonine (10), peaks were found at
381.4182 (calcd. for C22H29[2H14]N2O2 381.4203) and
337.3914 (calcd. for C20H25[2H14]N2O 337.3941), demon-
strating the incorporation of 14 deuterium atoms. Conse- axyridis.
Scheme 6. Biosynthetic pathway proposed for harmonine (3) in H.
Eur. J. Org. Chem. 2012, 1907–1912
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1909

E. Haulotte, P. Laurent, J.-C. Braekman
FULL PAPER
sponding unlabelled compounds, and the spectral properties re-
ported are those of the unlabelled derivatives.
specific. Based on all these data, the broad outlines of the
harmonine biosynthesis in H. axyridis can be established as
depicted in Scheme 6.
Dimethyl Tridecanedioate (11): Concentrated sulfuric acid (0.8 mL)
was added to a solution of 7 (2.002 g, 8.19 mmol) in methanol
(8 mL). The mixture was heated to reflux for 2 h, cooled to room
temp., and diluted with water (25 mL). The resulting mixture was
exhaustively extracted with toluene. The organic layer was washed
successively with water and sodium carbonate (5% aqueous solu-
tion). The toluene layer was then concentrated to dryness under
reduced pressure to afford 11 (2.031 g, 7.46 mmol, 91%) as a white
Conclusions
Based on all these results, the broad outlines of the bio-
syntheses of coccinelline (1), adaline (2), and harmonine (3)
from fatty acids precursors in C. 7-punctata, A. 2-punctata,
and H. axyridis, respectively, are now clearly established. It
is highly probable that the other perhydroazaphenalenes
(e.g., convergine, myrrhine, etc.), the “dimeric” alkaloids
(e.g., exochomine, etc.), and the structurally related ladybird
alkaloids (e.g., calvine, signatipennine, hyperaspine, etc.)[1–4]
are biosynthesized along an analogous pathway that starts
from a fatty acid precursor undergoing straightforward oxi-
dation and amination reactions. Interestingly enough, the
carbon skeleton of ant alkaloids such as the solenopsins[20]
and the tetraponerines[21–22] have been shown to be derived
from polyacetate chains, also most likely of fatty acid ori-
gin. This would indicate that, in insects, such a pathway has
evolved preferentially to the amino acid pathway, which is
more ubiquitous for the biosyntheses of the piperidine and
pyrrolidine alkaloids of plants[23] with the noticeable excep-
tions of coniine in Conium maculatum[24] and pinidine in
Pinus spp.[25]
solid. IR (KBr): ν
= 2930–2851, 1739, 1464, 1438, 1365, 1323,
˜
max
1264, 1208, 1170, 1001, 887 cm–1. H NMR (300 MHz, CDCl3): δ
= 1.27 (m, 14 H), 1.61 (m, 4 H), 2.30 (t, J = 7.5 Hz, 4 H), 3.67 (s,
6 H) ppm. 13C NMR (CDCl3): δ = 25.1, 29.3–29.6 (7 C), 34.2, 51.6,
174.5 ppm. MS (CI, NH3): m/z (%) = 290 (87) [M + NH4]+, 273
(100) [M + H]+, 258 (29), 243 (5), 226 (6). MS (EI): m/z (%) = 272
(Ͻ1) [M]+·, 241 (21), 208 (6), 199 (32), 167 (16), 149 (11), 126 (12),
112 (31), 98 (100), 87 (28), 84 (47), 74 (52), 55 (18).
1
12-(Methoxycarbonyl)dodecanoic Acid (14): A solution of barium
hydroxide (0.5 m in anhydrous methanol, 7 mL) was added to a
solution of diester 11 (1.729 g, 6.35 mmol) in anhydrous methanol
(7 mL) in a sealed tube. The medium was vigorously stirred at room
temp. for 18 h. The barium salt was then removed by filtration,
washed with methanol, and dissolved in HCl (4 n solution). The
resulting solution was extracted with diethyl ether. The organic lay-
ers were combined, and the solvents were evaporated to dryness
under reduced pressure. The solid residue was purified by flash
chromatography on silica gel (hexane/AcOEt, 9:1 then 8:2) to af-
ford 14 (1.066 g, 4.13 mmol, 65%) as a white solid. IR (NaCl disk):
ν
= 3500–2500, 2917–2849, 1733, 1709, 1177 cm–1. 1H NMR
˜
max
Experimental Section
(300 MHz, CDCl3): δ = 1.27 (m, 14 H), 1.62 (m, 4 H), 2.31 (t, J =
7.5 Hz, 2 H), 2.35 (t, J = 7.4 Hz, 2 H), 3.68 (s, 3 H) ppm. 13C
NMR (CDCl3): δ = 24.8, 25.1, 29.1–29.6 (7 C), 34.2, 34.3, 51.7,
174.9, 180.3 ppm. MS (EI): m/z (%) = 259 (Ͻ1) [M + H]+, 227
(18), 208 (11), 199 (10), 167 (8), 149 (8), 126 (11), 112 (31), 98 (100),
87 (29), 84 (52), 74 (82), 69 (30), 60 (14), 55 (50).
General Methods: Thin layer chromatography analyses were per-
formed with 0.25 mm Polygram silica gel SILG/UV254 precoated
plates (Macherey–Nagel) or with neutral alumina 60F254 pre-
coated plates (Merck). Column chromatography was performed
with silica gel columns (MN Kieselgel 60, 0.04–0.063 mm, Mach-
erey–Nagel) by using the flash technique, and alumina filtration
Methyl [14,14,14-3H3]13-Oxotetradecanoate (17): [3H3]CI (112 μL,
1.8 mmol) was added to a suspension of magnesium (45.2 mg,
1.8 mmol) in anhydrous ether (1 mL) in a 5 mL sealed tube. This
mixture was stirred at room temp. for 30 min and then cooled to
0 °C. CdCl2 (331 mg, 1.8 mmol) was added, and the solution was
stirred at room temp. for 1 h. The solvent was carefully removed
under a nitrogen stream to afford the organocadmium compound
[3H3]CCdCl. Simultaneously, ester 14 (101 mg, 0.39 mmol) was dis-
solved in anhydrous hexane (3 mL) in a 5 mL sealed tube. Then,
DMF (30 μL, 0.39 mmol) and oxalyl chloride (200 μL, 2.32 mmol)
were added. After 90 min at room temp., the hexane layer was care-
fully drawn off, and the solvents were evaporated to dryness. The
residue was then dissolved in dry toluene, and the obtained solution
was gently added at 0 °C to the organocadmium compound [3H3]-
CCdCl. The mixture was then stirred under nitrogen at room temp.
for 6 h. The reaction was quenched with sulfuric acid (2% aqueous
solution), and the resulting mixture was extracted with toluene
(3ϫ). The organic layers were combined, and the solvents were
evaporated to dryness under reduced pressure to give a residue,
which was purified by flash chromatography on silica gel (hexane/
AcOEt, 95:5) to afford 17 (69 mg, 0.27 mmol, 69%) as a white so-
1
was realized with basic or neutral alumina (Macherey–Nagel). H
NMR spectroscopic data were recorded in CDCl3 at 300 MHz with
a Bruker Avance TM 300 and are reported in ppm on the δ scale
by using TMS as an internal standard. 13C NMR spectroscopic
data were recorded in CDCl3 at 75.4 MHz with a Bruker Avance
TM 300 instrument. Data are reported in the format of chemical
shift {multiplicity [singlet (s), broad singlet (br. s), triplet (t), mul-
tiplet (m)], coupling constants in Hz, integration}. HRMS (EI),
MS (EI), and MS [CI (chemical ionization)] analyses were per-
formed with a Fison VG Micromass 7070F Autospec instrument
(70 eV). For the MS (CI) analyses, the reagent gas used was ammo-
nia. In all cases, the peak intensities are expressed as percent rela-
tive to the base peak. The IR spectra were recorded with a Bruker
IFS 25 instrument with samples as KBr pellets or as films on an
NaCl disk. Radioactive compounds were assayed in a Packard Tri-
Carb 1600 TR liquid scintillation analyzer. The samples were dis-
solved in methanol (10 mL), and 100 μL of the resulting solution
was added to Packard Insta-Gel Plus liquid scintillation cocktail
(10 mL). Triplicate samples of each compound were counted under
comparable conditions of quenching. All chemicals were obtained
from Aldrich or Acros Organics and used without further purifica-
tion. [3H3]CI, (10 mCi, 85 mCi/mmol, 10 mCi/mL) and [1,1,2,2,3,3,
4,4,5,5,6,6,7,7,8,8,8-2H17]n-octanol, were purchased from Amer-
sham (UK) and Aldrich, respectively. All synthetic steps involving
tritiated compounds were developed beforehand by using the corre-
lid. IR (KBr): ν
= 2917–2849, 1732, 1714, 1472, 1463, 1441,
˜
max
1364, 1320, 1261, 1234, 1207, 1178, 888 cm–1. 1H NMR (300 MHz,
CDCl3): δ = 1.27 (m, 14 H), 1.59 (m, 4 H), 2.13 (s, 3 H), 2.30 (t, J
= 7.5 Hz, 2 H), 2.41 (t, J = 7.4 Hz, 2 H), 3.66 (s, 3 H) ppm. 13C
NMR (CDCl3): δ = 23.9, 25.0, 29.2–29.6 (7 C), 29.9, 34.2, 43.9,
1910
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Eur. J. Org. Chem. 2012, 1907–1912

Biosynthesis of Defensive Coccinellidae Alkaloids
51.5, 174.4, 209.4 ppm. MS (EI): m/z (%) = 256 (9) [M]+·, 241 (3), 1,13-Bis(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)tridecane
(24):
225 (35), 199 (72), 167 (62), 149 (41), 143 (8), 126 (18), 112 (22),
98 (58), 87 (49), 83 (59), 74 (47), 69 (54), 58 (100).
NaBH3CN (1.9 g, 28.42 mmol) was added to a solution of 23
(3.528 g, 7.10 mmol) in acetic acid (28 mL) and THF (42 mL). The
mixture was stirred at room temp. for 5 min and then warmed at
60 °C for 2 h. The crude mixture was then diluted with water
(160 mL), and the resulting mixture was acidified with HCl. The
obtained precipitate was removed by filtration and purified by flash
chromatography on silica gel (CH2Cl2) to afford 24 (1.602 g,
Methyl [14,14,14-3H3]Tetradecanoate (20): p-Toluenesulfonic acid
(1 mg) and p-toluenesulfonylhydrazine (56 mg, 0.58 mmol) were
added to a solution of 17 (66 mg, 0.26 mmol) dissolved in a 1:1
mixture of DMF and sulfolane (1 mL). The mixture was stirred
at room temp. for 24 h before the addition of NaBH3CN (25 mg,
0.37 mmol). After stirring at 110 °C for 5 h, the crude mixture was
diluted with water, and the resulting mixture was extracted with
hexane (3ϫ). The organic layers were combined and concentrated
under reduced pressure to give a residue, which was purified by
flash chromatography on silica gel (hexane/AcOEt, 99:1) to afford
20 (31 mg, 0.13 mmol, 49%) as a colorless oil. 1H NMR (300 MHz,
CDCl3): δ = 0.88 (t, J = 6.7 Hz, 3 H), 1.25 (m, 20 H), 1.61 (m, 2
H), 2.30 (t, J = 7.5 Hz, 2 H), 3.67 (s, 3 H) ppm. 13C NMR (CDCl3):
δ = 14.3, 22.8, 25.1, 29.3–29.8 (8 C), 32.1, 34.3, 51.6, 174.5 ppm.
MS (EI): m/z (%) = 242 (17) [M]+·, 211 (5), 143 (6), 87 (47), 74
(100), 69 (18), 59 (22), 55 (39).
3.42 mmol, 48%). IR (NaCl disk): ν
= 2918–2849, 1743, 1339,
˜
max
1208, 1061, 984 cm–1. H NMR (300 MHz, CDCl3): δ = 1.25 (m,
18 H), 1.44 (m, 4 H), 1.75 (s, 6 H), 1.79 (s, 6 H), 2.08 (m, 4 H),
3.52 (t, J = 5.0 Hz, 2 H) ppm. 13C NMR (CDCl3): δ = 26.6, 26.8,
27.0, 28.5, 29.3–29.6, 46.2, 104.9, 165.8 ppm. MS (CI, NH3): m/z
(%) = 486 (1) [M + NH4]+, 326 (12), 309 (15), 299 (7), 282 (100),
265 (22), 112 (13). MS (EI): m/z (%) = 468 (Ͻ1) [M]+·, 142 (8), 128
(6), 112 (18), 98 (100), 84 (23), 67 (23), 58 (64), 55 (72).
1
Heptadecanedioic Acid (9): Compound 24 (101 mg, 0.22 mmol) was
dispersed in HCl (6 n aqueous solution, 2 mL), and the reaction
mixture was heated to reflux overnight. After cooling, the solid
obtained was removed by filtration, washed with water, and dried
to afford diacid 9 (48 mg, 0.16 mmol, 74%) as a white solid. IR
[14,14,14-3H3]Myristic Acid (4): Ester 20 (29 mg, 0.12 mmol), po-
tassium hydroxide (25 mg, 0.44 mmol), and 18-crown-6 (9 mg,
0.03 mmol) were placed in a 1 mL sealed flask containing anhy-
drous toluene (0.5 mL). The mixture was stirred at room temp.
overnight and then acidified with HCl (4 n solution), and the re-
sulting mixture was extracted with toluene (3ϫ). The combined
organic layers were concentrated under reduced pressure, and the
obtained residue was purified by flash chromatography on silica gel
(hexane/AcOEt, 9:1) to afford 4 (20 mg, 0.09 mmol, 72%) as a
(NaCl disk): ν
= 2919–2849, 1699, 1471, 1411, 1290, 927,
˜
max
720 cm–1. H NMR (300 MHz, CDCl3): δ = 1.24 (m, 22 H), 1.48
(m, 4 H), 2.18 (t, J = 7.3 Hz, 4 H), 11.92 (br. s, 2 H) ppm. 13C
NMR (CDCl3): δ = 24.5, 28.5–29.0 (11 C), 33.7, 174.5 ppm. MS
(CI, NH3): m/z (%) = 318 (64) [M + NH4]+, 300 (92), 282 (100),
264 (10), 223 (5), 126 (7), 112 (22). MS (EI): m/z (%) = 300 (Ͻ1)
[M]+·, 256 (9), 126 (9), 112 (34), 98 (100), 84 (66), 73 (26), 69 (37),
60 (31), 55 (60).
1
white solid. IR (KBr): ν
= 3500–2500, 2918–2849, 1699, 1471,
˜
max
1
1411, 1290, 927, 720 cm–1. H NMR (300 MHz, CDCl3): δ = 0.88
(t, J = 6.7 Hz, 3 H), 1.27 (m, 20 H), 1.61 (m, 2 H), 2.35 (t, J =
7.5 Hz, 2 H) ppm. 13C NMR (CDCl3): δ = 14.3, 22.8, 24.8, 29.2–
29.8 (8 C), 32.1, 34.2, 180.0 ppm. MS (EI): m/z (%) = 228 (58)
[M]+·, 211 (11), 199 (5), 185 (32), 171 (10), 143 (11), 129 (49), 115
(15), 101 (9), 87 (19), 85 (30), 83 (18), 73 (100), 71 (40), 69 (31), 60
(78), 57 (55), 43 (63). SA = 0.3034 mCi/mmol.
[18,18,18-3H3]Stearic Acid (6): The labelled acid 6 was prepared
from diacid 9 according to the same procedure as described for
acids 4 and 5. The solvent for the selective hydrolysis was CH3OH/
THF (6:1). SA of 9 = 0.2107 mCi/mmol. The yields for each step
in the synthesis are reported in Scheme 1. The spectral properties
of all the intermediates are compatible with those reported in the
literature.
[16,16,16-3H3]Palmitic Acid (5): The same synthetic scheme was ap-
plied to prepare acid 5, but in this case, pentadecanedioic acid (8)
was used as the starting material, and the solvent for the selective
hydrolysis was CH3OH/THF (6:1) instead of CH3OH. SA of 5 =
0.3193 mCi/mmol. The yields for each step in the synthesis are re-
ported in Scheme 1. The spectral properties of all the intermediates
are compatible with those reported in the literature.
[11,11,12,12,13,13,14,14,15,15,16,16,17,17,18,18,18-2H17]Stearic
Acid (25): The labelled acid 25 was prepared according to the pro-
cedure described by Attygalle et al.[7] The yields for each step in
the synthesis are reported in Scheme 5.
In Vitro Incubation Assays: Adults of A. 2-punctata were purchased
from Horpi Systems (Verlaine, Belgium). Adults of C. 7-punctata
and of H. axyridis were field-collected in Belgium. The experimen-
tal setup for the in vitro incubation assays has already been de-
scribed.[10] The purifications of each alkaloid were carried out until
a constant radioactivity was measured. For adaline (2), the crude
extract was purified by successive flash chromatography procedures
on silica gel by using AcOEt then AcOEt/MeOH/NH4OH (95:5:1)
as eluents, and on neutral alumina by using hexane/CH2Cl2 (5:5)
then pure CH2Cl2 as eluents. For coccinelline (1), the crude extract
was purified by successive flash chromatography procedures on sil-
ica gel by using AcOEt then AcOEt/MeOH/NH4OH (5:5:0.1), and
CH2Cl2 then CH2Cl2/MeOH/NH4OH (9:1:0.1) as eluents. For har-
monine (3), the crude extract was filtered through neutral alumina
by using CH2Cl2 then CH2Cl2/MeOH/NH4OH (8:2:0.2) as eluents.
The sample of harmonine thus obtained was then treated with a
1:1 mixture of pyridine/acetic anhydride (1 mL) at room temp. for
2 h. After the addition of MeOH, the mixture was concentrated
1,13-Bis(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)tridecane-1,13-di-
one (23): Diacid 7 (100 mg, 0.41 mmol) was dissolved in anhydrous
hexane (3 mL) in a 5 mL sealed tube, and DMF (64 μL, 0.82 mmol)
and oxalyl chloride (422 μL, 4.91 mmol) were added. The mixture
was kept at room temp. under nitrogen for 90 min. Then, the hex-
ane layer was carefully drawn off, and the solvents were evaporated
to dryness. The diacyl dichloride thus obtained was dissolved in
dry THF, and the obtained solution was added dropwise at room
temp. to a solution containing Meldrum’s acid (261 mg, 1.81 mmol)
and DMAP (150 mg, 1.22 mmol) in anhydrous THF (0.5 mL). The
mixture was stirred at room temp. under nitrogen overnight and
quenched with water (6 mL). The pH was adjusted to 1 with HCl,
and the organic solvent was removed under reduced pressure.
Through filtration of the resulting aqueous layer, the desired prod-
uct 23 (178 mg, 0.36 mmol, 88%) was obtained as a light yellow
1
solid. H NMR (300 MHz, CDCl3): δ = 1.28 (m, 14 H), 1.69 (m,
4 H), 1.73 (s, 12 H), 3.06 (t, J = 7.6 Hz, 4 H), 15.29 (br. s, 2 H) under reduced pressure, and the residue was purified by flash
ppm. 13C NMR (CDCl3): δ = 26.3, 26.9, 29.3–29.4 (7 C), 35.9, chromatography on silica gel (AcOEt then AcOEt/MeOH, 95:5) to
91.4, 104.9, 160.3, 170.7, 198.5 ppm.
afford pure N,N-diacetylharmonine (10).
Eur. J. Org. Chem. 2012, 1907–1912
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1911

E. Haulotte, P. Laurent, J.-C. Braekman
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Received: October 26, 2011
Published Online: January 4, 2012
1912
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 1907–1912