Semiochemicals of the scarabaeinae. VII: Identification and synthesis of ead-active constituents of abdominal sex attracting secretion of the male dung beetle, Kheper subaeneus

Article abstract of DOI:10.1023/A:1021440220329

Using gas chromatography with flame ionization detection (FID) and electroantennographic detection (EAD) in parallel, butanoic acid, skatole, and (E)-2,6-dimethyl-6-octen-2-ol were identified as constituents of the abdominal sex-attracting secretion of the male dung beetle, Kheper subaeneus, which reproducibly elicited EAD responses in male and female antennae. This is the first report of the occurrence of (E)-2,6-dimethyl-6-octen-2-ol as a natural product, for which the name (E)-subaeneol is proposed. In some experiments, a few other constituents of the secretion also gave reproducible responses in specific male and female antennae but did not elicit responses when the analyses were repeated with other antennae. The major volatile constituent of the secretion, identified as (S)-(+)-2,6-dimethyl-5-heptenoic acid, is one of these EAD-active compounds. Both this compound and (E)-2,6-dimethyl-6-octen-2-ol were synthesized from authentic starting materials for comparison with the natural products.

Full text of DOI:10.1023/A:1021440220329

Journal of Chemical Ecology, Vol. 28, No. 12, December 2002 (�C 2002)
SEMIOCHEMICALS OF THE SCARABAEINAE. VII:
IDENTIFICATION AND SYNTHESIS OF EAD-ACTIVE
CONSTITUENTS OF ABDOMINAL SEX ATTRACTING
SECRETION OF THE MALE DUNG BEETLE, Kheper subaeneus
�
B. V. BURGER, W. G. B. PETERSEN, W. G. WEBER, and Z. M. MUNRO
Laboratory for Ecological Chemistry
Department of Chemistry
University of Stellenbosch
Stellenbosch 7600, South Africa
(Received January 15, 2002; accepted August 7, 2002)
Abstract—Usinggaschromatographywithflameionizationdetection(FID)and
electroantennographic detection (EAD) in parallel, butanoic acid, skatole, and
(
E)-2,6-dimethyl-6-octen-2-ol were identified as constituents of the abdominal
sex-attracting secretion of the male dung beetle, Kheper subaeneus, which re-
producibly elicited EAD responses in male and female antennae. This is the first
report of the occurrence of (E)-2,6-dimethyl-6-octen-2-ol as a natural product,
for which the name (E)-subaeneol is proposed. In some experiments, a few other
constituents of the secretion also gave reproducible responses in specific male
and female antennae but did not elicit responses when the analyses were repeated
with other antennae. The major volatile constituent of the secretion, identified as
(
S)-(+)-2,6-dimethyl-5-heptenoic acid, is one of these EAD-active compounds.
Both this compound and (E)-2,6-dimethyl-6-octen-2-ol were synthesized from
authentic starting materials for comparison with the natural products.
Key Words—Scarabaeinae, Kheper subaeneus, dung beetle, sex attractant, in-
sect semiochemicals, electroantennography, terpenes, semiochemical synthesis.
INTRODUCTION
Male ball-rolling dung beetles of the genus Kheper produce a white, flocculant
proteinaceous carrier material containing minute amounts of complex mixtures of
volatile organic compounds acting as sex attractants for females. The secretion is
extruded from several hundred tiny openings in a depression on either side of the
�
To whom correspondence should be addressed. E-mail: lecus@sun.ac.za
2
527
0
098-0331/02/1200-2527/0 �C 2002 Plenum Publishing Corporation

2
528
BURGER, PETERSEN, WEBER, AND MUNRO
first abdominal sternite. It is then broken up into fine particles and released into the
air by brushes on the tibia of the male. The ecological importance of dung beetles in
general and biological aspects of the production and function of the abdominal sex
attractant secreted by male Kheper dung beetles has been discussed in previous
communications on this subject (Burger et al., 1983, 1990, 1995; Burger and
Petersen, 1991, 2002). Hexadecanoic acid, 2,6-dimethyl-5-heptenoic acid, (E)-
nerolidol, and skatole, were previously identified as the major volatile organic
constituents of the abdominal secretion of K. lamarcki. (Burger et al., 1983). The
abdominalsecretionofK. nigroaeneuscontainstheEAD-activeconstituents(R)-3-
methylheptanoic acid and skatole. A third EAD-active constituent of this secretion
was not identified, as it is present in such a low concentration that it could barely
be detected by gas chromatography (GC) using flame ionization detection (FID)
(
Burger and Petersen, 2002). A third Kheper species, K. subaeneus, occurs in
parts of the Hluhluwe Game Reserve, about 50 km from the Mkuzi Game Reserve
both in South Africa) where K. nigroaeneus and K. lamarcki are found. In this
(
communication, we report the identification of the EAD-active constituents of the
abdominal sex-attracting secretion of male K. subaeneus.
METHODS AND MATERIALS
Collection of Material. We initially attempted to catch K. subaeneus dung
beetles in pitfall traps baited with rhinoceros or horse dung in Hluhluwe Game
Reserve, amethodthatwasusedeffectivelytocatch K. nigroaeneusinMkuziGame
Reserve. Probably due to the availability of large quantities of rhinoceros dung in
the parts of the game reserve where these beetles are found, this method proved
ineffective in catching K. subaeneus in Hluhluwe. K. subaeneus were, therefore,
collected annually during the first week of November from 1988 to 1995 from
rhinoceros dung middens along dirt roads near the main entrance gate of Hluhluwe
Game Reserve. The dung beetles were transported to Stellenbosch, South Africa,
and were kept in greenhouses. The secretion was collected as described in the
previous paper in this series on semiochemicals of the Scarabaeinae (Burger and
Petersen, 2002).
Analytical Methods. Gas chromatographic determinations were carried out
with a Carlo Erba 4160 gas chromatograph with parallel flame ionization and
electroantennographic detection (FID/EAD). The instrument was equipped with a
4
0-m � 0.3-mm glass capillary column coated with OV-1701-OH at a film thick-
ness of 0.375 �m. Helium was used as carrier gas at a linear velocity of 28.5 cm/sec
at 40 C, and a temperature program of 2 C/min from 40 C to 250 C (hold) was
employed. Chiral gas chromatographic analyses were done on a glass column
�
�
�
�
(
25 m � 0.3 mm) coated with 10% heptakis(2,3,6-tri-O-methyl)-�-cyclodextrin
in OV-1701-OH at a film thickness of 0.25 �m, using H2 as carrier gas at a linear
velocity of 50 cm/sec. The column was programmed at 2 C/min from 40 C to
�
�

SCARABAEINAE SEMIOCHEMICALS VII
2529
�
1
90 C (hold). The instrumentation for GC analysis of dung beetle secretions with
FID and EAD recording in parallel using dung beetle antennae as sensing elements
has been described by Burger and Petersen (1991). In short, an antenna was inserted
lengthwise into the air duct, as this arrangement produced a less noisy baseline
than positioning the antenna sideways in the airstream, a position in which the club
of the antenna is subjected to the effect of the turbulent mixing of the effluent and
ambient air. The antenna was protected against heat radiation from the gas chro-
�
matograph by a heat shield cooled with water at 18 C. Electrical responses in the
antenna were recorded via Ag–AgCl electrodes placed in pipets filled with saline
solution containing NaCl (7.5 g/liter), CaCl2 (0.21 g/liter), KCl (0.35 g/liter), and
NaHCO3 (0.2 g/liter), as well as polyvinylpyrrolidone K90 (Fluka, 49 g/liter) to
increase its viscosity (Van der Pers, 1980). Antennal responses were amplified with
a Murphy Developments AMS-025 amplifier and were recorded on an Omniscribe
recorder. Over a period of about 5 years, EAD/FID analyses were done with at
least 80 male and as many female insects. Only one antenna of each dung beetle
was used so that the beetle could still be used for the collection of secretion.
Mass spectra were recorded on a Carlo Erba QMD 1000 quadrupole mass
spectrometer using the column and gas chromatographic conditions specified
1
13
above. H and C NMR spectra of synthetic compounds and intermediates were
�
recorded at 299.905 and 75.42 MHz, respectively, at 25 C on a Varian VXR
300 NMR spectrometer.
Reference Compounds. Commercially available reference compounds and
starting compounds for the synthesis of other reference compounds were purchased
from Merck (Darmstadt, Germany), Fluka (Buchs, Switzerland), or Sigma-Aldrich
(Cape Town, South Africa).
(
E)-2,6-Dimethyl-6-octen-2-ol (4a) was prepared from geraniol (3,7-
dimethyl-2,6-octadien-1-ol) (1a) according to Scheme 1. Tetrachloromethane
�
(
69.3 g; 450 mmol) was added during 90 min to a stirred and chilled (0 C)
solution of geraniol (1a) (61.6 g; 400 mmol) and triphenylphosphine (115.3 g;
4
40 mmol) in dry acetonitrile (350 ml). The temperature of the reaction mixture
�
was allowed to rise gradually to 22 C at which temperature it was stirred for a fur-
ther 19 hr. Pentane (300 ml) was added to the reaction mixture, which was stirred at
�
room temperature for 15 min and left overnight at � 15 C to allow precipitation of
triphenylphosphineoxide. Theprecipitatewasremovedbyfiltrationofthecoldsus-
pension. Pentane and acetonitrile were removed on a rotary evaporator, the remain-
�
ing material diluted with pentane, the solution cooled to � 15 C, and the precipi-
tate filtered off. This procedure was repeated until no further triphenylphosphine
oxide precipitated. Removal of the solvent and repeated fractionation gave a color-
�
less oil (27.4 g; 39.7%), bp 106–107 C/22 mm Hg, containing (E)-8-chloro-2,6-
dimethyl-2,6-octadiene (2a) in 97.6% purity (GC-MS). MS: m/z (%) = 159(0.3),
1
6
57(1), 136(10), 129(4), 121(9), 105(5), 93(57), 91(26), 79(25), 77(23), 69(100),
8(23), 67(23), 53(14), 41(53), 39(30). 13C NMR (CDCl3): � = 41.17 (t, C-1);

2
530
BURGER, PETERSEN, WEBER, AND MUNRO
SCHEME 1.
1
20.31 (d, C-2); 142.78 (s, C-3); 39.47 (t, C-4); 26.25 (t, C-5); 123.61 (d, C-6);
31.99 (s, C-7); 25.68 (q, C-8); 16.12 (q, CH3C-3); 17.71 (q, CH3C-7).
E)-8-Chloro-2,6-dimethyl-2,6-octadiene(2a)(27.4g;159mmol)wasslowly
added to a stirred solution of lithium aluminum hydride in tetrahydrofuran (THF)
380 ml; 0.02 g/ml; 200 mmol). The reaction mixture was refluxed for 4 hr, allowed
1
(
(
to cool to room temperature, and diluted with pentane (150 ml). The precipitated in-
organic material was filtered off. Conventional work-up procedures and distillation
�
of the product gave a colorless oil (16.9 g; 77.2%); bp 77 C/ 40 mm Hg, containing
9
1
4
8.0% (GC-MS) of (E)-2,6-dimethyl-2,6-octadiene (3a); MS: m/z (%) = 138(3),
23(15), 109(1), 107(2), 95(38), 81(3), 79(3), 77(2), 69(100), 67(11), 55(5), 53(7),
1(53), 39(8). 13C NMR (CDCl3): � = 26.38 (q, C-1); 131.98 (s, C-2); 125.11
(d, C-3); 27.45 (t, C-4); 40.42 (t, C-5); 136.49 (s, C-6); 118.93 (d, C-7); 14.02
q, C-8); 18.34 (q, CH3C-2); 16.34 (q, CH3C-6).
(
Solid mercury(II) acetate (3.2 g; 10 mmol) was added in one batch to a
stirred solution of the diene (3a) (1.38 g; 10 mmol) in THF (40 ml) and water
�
(
10 ml) at 22 C to produce a yellow reaction mixture (Brown et al., 1972). The
reaction mixture became colorless after 6 min, and it was stirred for a further
hr. A solution of sodium hydroxide (1.2 g NaOH in 10 ml H2O) was added
2
in one batch to the reaction mixture, followed immediately by the rapid addition
of a solution of sodium borohydride and sodium hydroxide (0.19 g NaBH4 and
�
1
.2 g NaOH in 10 ml H2O). The temperature of the reaction mixture rose to 38 C,
and mercury started to precipitate. The reaction mixture was stirred for 30 min,

SCARABAEINAE SEMIOCHEMICALS VII
2531
after which the supernatant solution was decanted from the mercury and saturated
with K2CO3, resulting in the separation of the THF layer containing the organic
material from the saturated K2CO3 solution. Careful concentration of the THF
phase on a rotary evaporator gave a mixture of the organic products (1.41 g)
containing the starting material (3a), an unidentified product that could possibly
be a cyclic ether formed from the starting material (Brown et al., 1972), the two
terpenols(4a)and(5), and2,6-dimethylocta-2,6-diol(6)inaratioof24:33:29:3:11.
This procedure was repeated several times and the products (10.4 g) combined
for column chromatographic fractionation on silica gel (450 g, Merck 60, 230–
400 mesh). Elution with 10% ether in hexane gave a fraction (1.16 g) containing the
desired terpenol (4a) and the terpenol (5) in a ratio of 97:3 (GC-MS). This fraction
�
was subjected to bulb-to-bulb distillation (135 C/25 mm Hg) for spectrometric
characterization. The major constituent was 2,6-dimethyl-6-octen-2-ol (4a); m/z
(
5
4
%) = (138)7, 123(20), 109(3), 95(6), 83(19), 82(100), 69(23), 67(80), 59(57),
5(23), 43(30), 41(3). 13C NMR (CDCl3): � = 29.87 (q, C-1); 71.67 (s, C-2);
4.17 (t, C-3); 23.31 (t, C-4); 40.67 (t, C-5); 136.31 (s, C-6); 119.15 (d, C-7); 13.95
(q, C-8); 29.87 (q, CH3C-2); 16.14 (q, CH3C-6). The minor constituent was 3,7-
dimethyl-6-octen-3-ol (5); m/z (%) = 156(2), 141(17), 138(9), 127(8), 123(40),
1
5
13(13), 109(18), 95(13), 85(19), 81(18), 71(100), 69(25), 67(23), 58(19), 55(30),
3(12), 43(68), 41(45), 39(20).
(
R)-(� )-2,6-Dimethyl-5-heptenoic acid (10a), was prepared from (R)-(� )-
3
,7-dimethyl-1,6-octadiene [(� )-�-citronellene] (7a) according to Scheme 2. A
solution of 3-chloroperbenzoic acid (containing 6% 3-chlorobenzoic acid) (8.23 g;
SCHEME 2.

2
532
BURGER, PETERSEN, WEBER, AND MUNRO
4
4.85 mmol) in chloroform (100 ml) was added over 1.25 hr to (R)-(� )-3,7-
dimethyl-1,6-octadiene (7a) in chloroform (40 ml) at such a rate that the tem-
�
perature of the reaction mixture did not exceed � 10 C. The progress of the par-
tial epoxidation of the �-citronellene was monitored by GC analysis and was
�
completed after stirring the reaction mixture an additional 2 hr at � 10 C. The 3-
chlorobenzoicacidwasfilteredoff, andthetemperatureofthefiltrateallowedtorise
to room temperature. The filtrate was washed with NaOH solution (0.45 M, 40 ml),
Na2CO3 solution (5%), saturated brine and dried. Removal of the solvent under
reduced pressure gave a colorless oil (6.87 g; 99.3%) containing 95.9% (GC-MS)
of (R)-(� )-6,7-epoxy-3,7-dimethyl-1-octene (8a). MS: m/z (%) = 139(4), 125(2),
1
6
21(3), 111(6), 95(14), 85(13), 82(16), 81(100), 79(18), 71(30), 69(39), 68(50),
7(60), 59(64), 55(53), 54(40), 53(20), 43(44), 41(64), 39(37).
Ozone was passed through a solution of the epoxide (8a) (6.22 g, 40.32 mmol)
�
in dry dichloromethane (350 ml) at � 70 C until, after about 35 min, the reaction
mixture developed a light purple color. The excess of ozone and oxygen was
removed as quickly as possible by passing nitrogen through the reaction mixture for
�
4
0 min. This solution was carefully concentrated to 40 ml, cooled to 0 C, and added
to a cold suspension of zinc (15.0 g; 229.4 mmol) in acetic acid (39 ml) containing
sodium acetate (3.41 g; 41.52 mmol) and sodium iodide (10.72 g; 68.64 mmol)
(Kn o¨ ll and Tamm, 1975). The bulk of the dichloromethane was removed under
reducedpressure, andtheresultingsolutionwasstirredfor2hratroomtemperature.
The zinc was filtered off, and the solution of the reaction products in acetic acid was
extracted repeatedly with dichloromethane. The combined extracts were washed
with saturated brine and water, dried, and the solvent removed to yield a liquid
product (5.52 g; 97.7%) containing 71.5% (GC-MS) of the target product, (R)-
(
9
4
� )-2,6-dimethyl-5-heptenal (9a). MS: m/z (%) = 140(3), 107(2), 97(1), 93(1),
1(2), 83(12), 82(100), 81(7), 70(5), 69(24), 67(46), 57(5), 56(16), 55(21), 53(10),
3(8), 42(7), 41(51), 39(16).
(
R)-(� )-2,6-Dimethyl-5-heptenal (9a) was oxidized to the corresponding
acid with chromic acid. Chromic acid solution (24 ml, containing 4.79 g of
CrO3 in 3 M H2SO4) was slowly added to a stirred solution of (R)-(� )-2,6-
dimethyl-5-heptenal (9a) (3.85 g; 27.46 mmol) in acetone (40 ml). The color
of the reaction mixture changed from orange to a dirty green, and a precipi-
tate of inorganic salts was formed. The acetone solution of the organic prod-
ucts was decanted from the inorganic material, the acetone evaporated under
reduced pressure, and the residual material dissolved in dichloromethane. The
inorganic precipitate was dissolved in water (100 ml) and repeatedly extracted
with dichloromethane. All of the dichloromethane extracts were combined and
extracted with NaOH solution (3 M; 30 ml). This basic solution was extracted
five times with dichloromethane to remove all of the unsaponified material, after
which it was acidified with dilute sulfuric acid (3 M; 5.5 ml) and the organic acid
extracted with dichloromethane. Conventional work-up methods and distillation

SCARABAEINAE SEMIOCHEMICALS VII
2533
gave the target compound, (R)-(� )-2,6-dimethyl-5-heptenoic acid (10a) (2.07 g) in
a yield of 48.3%, bp 112–114/5 mm Hg, [�]2 = � 18.27 (neat), 95.7% ee (GC).
5
�
D
MS: m/z (%) = 156(9), 138(13), 110(4), 95(7), 83(88), 82(49), 74(58), 69(46),
6
3
2
7(38), 55(48), 53(12), 41(100), 39(30). 13C NMR (CDCl3): � = 183.04 (s, C-1);
8.84 (d, C-2); 33.56 (t, C-3); 25.62 (t, C-4); 123.48 (d, C-5); 132.42 (s, C-6);
5.68 (q, C-7); 16.86 (q, CH3C-2); 17.65 (q, CH3C-6).
(
S)-(+)-2,6-Dimethyl-5-heptenoic acid (10b) was prepared from (+)-�-
citronellene (7b) according to Scheme 2 and the procedures described for its
R enantiomer. (S)-(+)-6,7-Epoxy-3,7-dimethyl-1-octene (8b) was obtained as a
colorless oil (6.73 g; 97.3%) in a purity of 96.8% and converted to (S)-(+)-2,6-
dimethyl-5-heptenal (9b), which was used without purification for the prepara-
tion of (S)-(+)-2,6-dimethyl-5-heptenoic acid (10b) (2.12 g; 48.1%). bp 117–
5
�
1
1
4
18/5 mm Hg; [�]2 = +24.6 (neat), 96.3% ee (GC). MS: m/z (%) = 156(10),
D
38(14), 110(4), 95(7), 83(97), 82(52), 74(65), 69(48), 67(43), 55(50), 53(11),
1(100), 39(28). 13C NMR (CDCl3): � = 183.02 (s, C-1); 38.82 (d, C-2); 33.55
(
t, C-3); 25.61 (t, C-4); 123.45 (d, C-5); 132.44 (s, C-6); 25.68 (q, C-7); 16.86 (q,
CH3C-2); 17.65 (q, CH3C-6).
RESULTS AND DISCUSSION
In their natural habitat, male K. subaeneus can often be observed exhibiting
the typical secretion-producing behavior, which can be summarized as follows.
Oriented with the head upwind and the front two pairs of legs on the dung surface,
themaleraiseshishindlegsandlowershisheadintothedung. Thelegsareretracted
simultaneously inwards towards the sides of the body and are then simultaneously
extended. This movement results in the tibial brushes brushing the controlled-
release proteinaceous material (Burger et al., 1990), extruded from the abdomen
asfinefibersandcontainingthesexattractant, againsttherowsofcurvedabdominal
bristles. This causes puffs of attractant to rise into the air from both sides of the
insect. After a short time of approximately 20–30 sec, the retraction and extension
of the hind legs are repeated. This procedure is kept up until a female arrives or the
male is disturbed by other insects in or on the dung (Burger et al., 1983). Although
their population density was rather low in Hluhluwe Game Reserve during the
late 1980s, five or more males were often found calling on one large rhinoceros
dung midden. In contrast, calling behavior has not been observed with the same
frequency in K. lamarcki in Mkuzi Game Reserve. In captivity, however, this
situation was reversed, and the secretion of male K. subaeneus was collected only
in small quantities on only a few occasions. A total of only a few milligrams were
collected over a period of 5 years from about 700 males. A gas chromatogram of
the volatile organic fraction of the abdominal secretion of K. subaeneus is shown
in Figure 1. The general appearance of this gas chromatogram is similar to that of

2
534
BURGER, PETERSEN, WEBER, AND MUNRO
FIG. 1. Total ion chromatogram (GC-MS) of an extract of the male abdominal secretion of
the dung beetle Kheper subaeneus. Gas chromatographic conditions are given in the text.
K. nigraeneus (Burger and Petersen, 2002) in that both secretions contain mostly
heavy constituents. However, only a relatively small percentage of the compounds
present are common to both secretions.
A typical GC analysis with FID and EAD recording in parallel using a female
antenna as sensing element is shown in Figure 2. The constituent eluting at a
retention time of 4.98 min was identified as butanoic acid (n-butyric acid) from
its mass spectrum and coelution with the synthetic compound. Although the EAD
response is weak, it was present in all EAD chromatograms. The constituent eluting
at 28.27 min was identified as 3-methylindole (skatole) and is also present in the
abdominal secretions of K. lamarcki (Burger et al., 1983) and K. nigroaeneus
(Burger and Petersen, 2002). Although barely detectable by FID, the constituent
eluting at 17.75 min gave a strong and reproducible EAD response. This constituent
elutes with a shorter retention time than the EAD-active constituent that remained
unidentified in the male abdominal secretion of Kheper nigroaeneus (Burger and
Petersen, 2002). Male antennae produced similar EAD/FID analyses.
After the gas chromatographic analyses had been completed, only about ten
times the quantity used for the analysis shown in Figure 2 was available for further
analytical work. All of this material, collected during the previous five summer
seasons, was used in a final attempt at obtaining some mass spectral information
on the structure of the constituent eluting at 17.75 min. Using a very high elec-
tron multiplier setting, the mass spectrum shown in Figure 3 was obtained. The
spectrum contains several ions typically found in the spectra of the monoterpenes.
This and the presence of compounds with terpenoid character in the abdominal
secretions of other Kheper species prompted us to compare the retention time
and mass spectrum of the unknown compound with those of selected model ter-
penoid compounds. It became clear that the unknown compound was not a cyclic

SCARABAEINAE SEMIOCHEMICALS VII
2535
FIG. 2. First part of a gas chromatographic analysis of an extract of the male abdominal
secretion of Kheper subaeneus with EAD and FID recording in parallel using a female
antenna as sensing element. Chart speed was 0.5 cm/min. Gas chromatographic conditions
are given in the text. The poor separation of later eluting peaks is due to overloading of the
column. Assignment of EAD-active constituents: 1 = butanoic acid; 2 = (E)-2,6-dimethyl-
6
-octen-2-ol; 3 = skatole.
or acyclic monoterpene or a primary monoterpene alcohol and that it could prob-
ably be a tertiary monoterpene alcohol, albeit not one of the tertiary terpenoid
alcohols that we had available for comparison. Partial hydroxylation of (E)- and
(
Z)-3,7-dimethyl-1,6-octadiene with mercury(II) acetate gave a mixture of alco-
hols containing, among others, 2,6-dimethyl-7-octen-2-ol, which had a retention
time similar to that of the EAD-active constituent of the secretion but a slightly
different mass spectrum. It was, therefore, concluded that the unknown constituent
could possibly be a similar tertiary alcohol but with its double bond in a different
position. Conversion of a mixture of geraniol (1a) and nerol (1b) [(E)- and (Z)-
3,7-dimethyl-2,6-octadien-1-ol, respectively], viathecorrespondingchlorides(2a)

2
536
BURGER, PETERSEN, WEBER, AND MUNRO
FIG. 3. Mass spectrum of the EAD-active constituent [(E)-2,6-dimethyl-6-octen-2-ol] of
the male abdominal secretion of Kheper subaeneus eluting at 17.75 min in the gas chro-
matogram shown in Figure 2.
and (2b), to (E)- and (Z)-2,6-dimethyl-2,6-octadiene, (3a) and (3b), respectively
(Scheme 1), and subjecting this mixture to partial hydroxylation, gave a mixture
containing the two unchanged terpenes (3a and 3b), the two terpenols (4a and 4b),
the terpenol (5), and the terpenediol (6). One of the terpenols was EAD-active and
had the retention time and mass spectrum of the unknown compound. From the
relative retention times of the three terpenols, it was concluded that the EAD-active
one could possibly be (E)-2,6-dimethyl-6-octen-2-ol (4a).
The configuration of the double bond in the EAD-active compound was con-
firmed by repeating the synthesis with geraniol according to Scheme 1. Geraniol
[(E)-3,7-dimethyl-2,6-dien-1-ol] (1a) was, thus, converted to (E)-8-chloro-2,6-
dimethyl-2,6-octadiene (2a). Reduction of this chloride with lithium aluminum
hydride gave the diene (3a). Partial hydroxylation of the diene (3a) by mercuration-
demercuration according to Brown and Geoghegan, (1967) i.e., by the addition of
the diene to a quantity of mercury(II) acetate equivalent to the hydroxylation
of one of the double bonds, gave a mixture of the unchanged diene (2a), the
target compound, (E)-2,6-dimethyl-6-octen-2-ol (4a), 3,7-dimethyl-6-octen-3-ol
(5), and 2,6-dimethyloctane-2,6-diol (6) in which the diols 4a and 5 were present
in an approximate ratio of 1:1. Although the two trisubstituted double bonds in the
diene 3a are so similar that totally regioselective hydroxylation was not expected
to be feasible, reverse addition, i.e., addition of the mercury(II) acetate to the diene
according to Brown et al. (1972), gave the terpenols 4a and 5 in a ratio of 91:9. A
fraction containing the desired terpenol, 4a, and the terpenol 5 in a ratio of 97:3
was isolated from the crude product by column chromatography. As far as could
be ascertained, (E)-2,6-dimethyl-6-octen-2-ol (4a) is a new natural product for
which the name (E)-subaeneol is proposed.
In addition to butanoic acid, (E)-2,6-dimethyl-6-octen-2-ol, and skatole,
which reproducibly elicited EAD responses in male and female antennae of K.

SCARABAEINAE SEMIOCHEMICALS VII
2537
subaeneus, some other constituents of the abdominal secretion in a few instances
also gave EAD responses. Because of the nonreproducibility of the EAD results,
the minor EAD-active constituents of the secretion could not be identified. How-
ever, the major volatile constituent of the secretion, eluting at 47.2 and 22.1 min
in the gas chromatograms shown in Figures 1 and 2, respectively, also gave an
EAD response in about 5–10% of the analyses. This compound was identified
as the chiral 2,6-dimethyl-5-heptenoic acid, which had previously been identified
in K. lamarcki (Burger et al., 1983). (R)-(� )-2,6-Dimethyl-5-heptenoic acid (9a)
was synthesized from commercially available (R)-(� )-3,7-dimethyl-1,6-octadiene
[
(� )-�-citronelene] (7a) by epoxidation of the trisubstituted double bond to give
the unsaturated epoxide (8a), ozonolysis of the double bond in this compound, and
simultaneous deprotection of the double bond (Kn o¨ ll and Tamm, 1975) giving the
unsaturated aldehyde (9a) and oxidation of the aldehyde to (R)-(� )-2,6-dimethyl-
5
-heptenoic acid (10a) according to Scheme 2.
Racemic 2,6-dimethyl-5-heptenoic acid could be resolved by gas chromato-
graphy on a capillary column coated with heptakis(2,3,6-tri-O-methyl)-�-
cyclodextrin/OV-1701. It was found that the synthetic acid (10a) and the natural
compound did not coelute from this chiral column. Therefore, the natural com-
pound had to be (S)-2,6-dimethyl-5-heptenoic acid (10b). This enantiomer, which
coeluted with the natural compound was synthesized from (S)-(+)-3,7-dimethyl-
1,6-octadiene [(+)-�-citronellene] (7b) according to a sequence of steps similar
to those used for the synthesis of the R enantiomer (10a), as shown in Scheme 2.
Although the mass spectrum of the synthetic acid is identical to that of the
natural compound and it coeluted with the natural compound from the chiral GC
column, it did not elicit EAD responses in a large number of male and female
antennae that were used in EAD/FID measurements. This is not unexpected in
view of the observation that the natural compound also did not reproducibly elicit
EAD responses. From the research done with dung beetles over many years, it is
clear that semiochemical communication in dung beetles is extraordinarily com-
plex, and as far as the inconsistency of antennal responses in EAD analyses with
the antennae of this and other Kheper species is concerned, there are various fac-
tors that could be taken into consideration. It is, for example, possible that there
could be some natural variation in the population in terms of antennal responses.
Because the research had to be done with captured insects, it was not possible to
determine the age, physiological state, etc of the insects that were used in these
experiments, factors that could have influenced the EAD results. The possibility
that the response of an antenna to the volatile constituents of the secretion could
depend on the concentration of the components flowing over the antenna was in-
vestigated, but this does not seem to explain the inconsistent results obtained with
(
S)-2,6-dimethyl-5-heptenoic acid (10b) and some of the other minor constituents
of the secretion. Another factor that has to be considered is that the best EAD re-
sults are mostly obtained when an EAD-active compound is eluted as a sharp peak

2
538
BURGER, PETERSEN, WEBER, AND MUNRO
from the GC column. Better results are often obtained when higher programming
rates are used. Many of the volatile constituents of the secretion are carboxylic
acids or other polar compounds that elute as more or less pretailing peaks from
apolar columns. If an EAD active constituent is eluted as a pretailing peak, it is
possible that the receptors on the antenna become saturated at a level below the
detection threshold of the system. In the present investigation, a capillary column
coated with OV-1701-OH was used because it is known that underivatized fatty
acids are eluted with better peak shapes from this phase than from other apolar and
even moderately polar stationary phases. A detailed investigation of the effect of
the choice of stationary phase and programming rate on EAD/FID analyses with
dung beetle antennae have, however, not yet been undertaken. Using a modulating
device, which delivers the effluent of the column as sharp pulses into the humidi-
fied airstream of the EAD setup, could possibly solve the problem of pretailing
peaks in EAD analyses.
Unfortunately, as in the research on K. nigroaeneus (Burger and Petersen,
2002), field tests with synthetic materials have so far not produced positive results.
The problems associated with field tests of the synthetic attractant constituents
of Kheper dung beetles were discussed in a previous communication (Burger and
Petersen, 2002).
The results obtained in interspecific EAD analyses revealed that considerable
interspecificsemiochemicalcommunicationcouldtakeplacebetweendungbeetles
in the same area. This aspect will be dealt with in a forthcoming communication.
Acknowledgments—Support of this work by the University of Stellenbosch and the Foundation
for Research Development, Pretoria, is gratefully acknowledged. The authors are indebted to the Natal
Parks, Game, and Fish Preservation Board for permission to study dung beetles in Mkuzi Game Reserve.
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