Lizard epidermal gland secretions. II. Chemical characterization of the generation gland secretion of the sungazer, cordylus giganteus

Article abstract of DOI:10.1021/np1008366

In lizards, the epidermal glands of the femoral and precloacal regions are involved in the production of semiochemicals. In addition to its femoral glands, the giant girdled lizard, or sungazer, Cordylus giganteus, which is endemic to South Africa, has generation glands as an additional potential source of semiochemicals. These epidermal glands are described as glandular scales that overlay the femoral glands and are included in the normal epidermal profile located in the femoral (thigh) and anterior antebrachial (fore-leg) regions of the male sungazer. GC-MS analysis of the generation gland secretions and the trimethylsilyl derivatives of some of the steroidal constituents was employed to identify 59 constituents, including alkenes, carboxylic acids, alcohols, ketones, aldehydes, esters, amides, nitriles, and steroids. The quantitative differences of the volatile constituents of the fore- and hind-leg generation glands were compared between individuals. This is the first report on the chemical composition of generation glandular material of lizards.

Full text of DOI:10.1021/np1008366

ARTICLE
pubs.acs.org/jnp
Lizard Epidermal Gland Secretions. II. Chemical Characterization of the
Generation Gland Secretion of the Sungazer, Cordylus giganteus
Stefan Louw,*,† Ben V. Burger,† Maritha Le Roux,† and Johannes H. Van Wyk‡
Laboratory for Ecological Chemistry, †Department of Chemistry and Polymer Science, and Ecophysiology Laboratory,
‡Department of Botany and Zoology, Stellenbosch University, Stellenbosch, 7600, South Africa
S Supporting Information
b
ABSTRACT: In lizards, the epidermal glands of the femoral and
precloacal regions are involved in the production of semiochemicals.
In addition to its femoral glands, the giant girdled lizard, or sungazer,
Cordylus giganteus, which is endemic to South Africa, has generation
glands as an additional potential source of semiochemicals. These
epidermal glands are described as glandular scales that overlay the
femoral glands and are included in the normal epidermal profile
located in the femoral (thigh) and anterior antebrachial (fore-leg)
regions of the male sungazer. GC-MS analysis of the generation gland secretions and the trimethylsilyl derivatives of some of the
steroidal constituents was employed to identify 59 constituents, including alkenes, carboxylic acids, alcohols, ketones, aldehydes,
esters, amides, nitriles, and steroids. The quantitative differences of the volatile constituents of the fore- and hind-leg generation
glands were compared between individuals. This is the first report on the chemical composition of generation glandular material of
lizards.
everal potential sources of pheromones in lizards have been
described, including epidermal and cloacal glands and the
constituents. Additionally, the quantitative differences of the
volatile constituents of the fore- and hind-leg generation glands
were compared between individuals. The histology of the gen-
eration glands of C. giganteus is also briefly described.
S
bloodꢀskin barrier.1 A comprehensive review of the natural pro-
ducts originating from the integument of reptiles, including a
summary of the chemicals secreted from the femoral (thigh) and
precloacal (preanal) glands of a number of lizard species, was
recently published.2 The chemical characterization of the secretion
of the femoral glands of the giant girdled lizard, or sungazer, Cordylus
giganteus (Squamata, Cordylidae), located in the ventral femoral
region, has also been reported.2,3 Lꢀopez and co-workers4ꢀ7
recently summarized the potential functions of femoral gland
semiochemicals, which could, for example, include mate choice,
maleꢀmale competition, and interspecific recognition. In addition
to femoral glands, cordylid lizards have holocrine secretory cells
located in the beta-layer of the epidermis, termed generation
glands,8,9 which may occur in different body locations, for example,
in the femoral, precloacal, antebrachial (fore-arm), and dorsal
epidermal regions.10 In most cases, the glandular material is stacked
as a series of beta-layers.10
’ RESULTS AND DISCUSSION
Basic Histology. In Figure 1 the generation glands in the
ventral femoral region as well as the fore-leg antebrachial region
can be clearly discerned from the normal epidermal scales. The
histological profiles of both the femoral and generation gland
(Figure 2) correspond to the description given by Van Wyk and
Mouton.10 The stacked beta-layer generations are observed in
the generation glands located in the femoral region (Figure 2).
Typical of the beta-type generation glands is the absence of the
outer oberhautchen layer, indicating that the outer layer of the
generation series is exposed on a continuous basis. Although the
generation glands on the fore-legs appear more swollen than
those in the ventral femoral region, histological appearance is
similar and the bulging is created by the underlying enlarged
epidermal plates.10
Chemical Characterization. GC-MS analyses of the dichlor-
omethane extracts of the glandular material showed that the
samples collected from the fore- and hind-legs were qualitatively
identical. The compounds identified in the material collected
from the generation glands of C. giganteus are listed in Table 1. Of
the 168 chromatographic peaks observed in the GC flame
In the case of C. giganteus, only males have generation glands,
as patches of “glandular scales” anterior to the single row of
femoral gland pores and on the ventral aspect of the fore-arm
region (Figure 1).10 In other cordylid species, females may also
possess generation glands, the number which may vary according
to geographical region.11,12 It has been suggested that the
generation glands could have a semiochemical function in
combination with other sources such as the femoral glands.12,13
The purpose of this study was to identify the volatile con-
stituents secreted from the generation glands by GC-MS analysis
of the secretion and the trimethylsilyl derivatives of some of its
Received: November 16, 2010
Published: May 13, 2011
r
Copyright
2011 American Chemical Society and
1364
dx.doi.org/10.1021/np1008366 J. Nat. Prod. 2011, 74, 1364–1369
American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
Figure 1. Generation glands in C. giganteus lizards. (A) Ventral view of
the hind-leg of a male lizard showing a single row of femoral pores (fp)
and an anterior patch of generation glands (gg). (B) Ventral view of the
fore-leg of a male lizard showing groups of generation glands (gg).
ionization detector (FID) analyses, 59 components, constituting
more than 92% of the total amount of the detected compounds,
could be analyzed with sufficient sensitivity utilizing GC-MS to yield
mass spectra amenable to MS interpretation and comparison with
Wiley, National Bureau of Standards (NBS), National Institute of
Standards and Technology (NIST), and the in-house mass spectra
libraries. A typical GC-FID chromatogram is depicted in Figure 3.
The structures of the majority of these compounds were confirmed
by GC coelution with authentic reference compounds. Quantitative
GC-FID analyses using solventless sample introduction revealed
quantitative differences between fore- and hind-leg samples, as well
as between individuals. The results from the quantitative analyses
are summarized in Table 1.
Figure 2. Histological cross-section of the epidermis in the ventral
femoral region of an adult male C. giganteus. The femoral gland (fg) is
located in the dermis (d), and the femoral gland tube (ft) opens on the
ventral surface of the femoral region of the hind-leg. The stacked
generation layers of the generation gland (gg) overlays the femoral
gland as part of the epidermal profile.
employing published MS data. An interesting application of this
technique is illustrated by the identification of the three cholestan-
3-ol isomers 49, 50, and 51, the mass spectra of which did not allow
unambiguous differentiation between the three isomers. However,
some differences exist between the isomers with respect to the relative
abundance of the ions formed by the elimination of water from the
molecular ion due to the influence of the compounds’ configuration
on the mechanism of this elimination.15,16 In general, the mass spectra
of C17-substituted steroidsþwith cis-A/B ring junctions (5β-H) have
prominent [M ꢀ H2O]3 ions, whereas the corresponding mass
spectra of steroids with trans-A/B ring junctions (5R-H) have [M ꢀ
H2O]•þ ions with low relative abundances.15 From this information it
was deduced that constituent 49 is a 5β-H isomer of cholestan-3-ol
and that constituents 50 and 51 are the 5R-H isomers. Comparison of
the mass spectra of the TMS derivatives of these constituents with
those in mass spectra libraries revealed that constituent 49 is probably
5β-cholestan-3R-ol, while confirming that constituents 50 and 51 are
the two 5R-H isomers of cholestan-3-ol. The C-3 configuration of the
latter two constituents could not be determined. The sterols 54,55,56,
and 58, the identities of which were not confirmed by retention time
comparison with standards, were positively identified on the basis of
thegoodcorrelationoftheirmassspectra,aswellasthemassspectraof
their TMS derivatives, with those of the mass spectra libraries.
Constituents 52, 53, and 57 were tentatively identified as 3-ethox-
ycholestane, cholestan-2/3-one, and cholest-4-en-3-one, respectively,
by MS interpretation and comparison of their EI-mass spectra with
mass spectra libraries, but their identities could not be confirmed.
The aliphatic nitriles hexadecanitrile (21) and octadecanitrile (30)
and the aliphatic amides pentadecanamide (28), (Z)-9-hexadecena-
mide (33), hexadecanamide (35), heptadecanamide (38), octadeca-
namide (41), nonadecanamide (43), and icosanamide (45) have not
been found in the secretions of any lizard species, although long-chain
aliphatic amides have been identified in secretions from the scent
glands of two snake species, Dumeril’s ground boa, Acrantophis
dumerili,17 and the western diamondback rattlesnake, Crotalus atrox.18
The scent glands produce secretions that contain chemicals that are,
for example, used as allomones to repel predators.2 A few short-chain
nitriles have been identified in the sternal gland secretions used by the
male koala, Phascolarctos cinereus, for territorial marking.19
The EI-MS of constituents 5 and 7 displayed promineþnt ions
corresponding to the general formulas [CnH2nꢀ1
]
and
[CnH2n]þ and decreasing in abundance with increasing mass,
characteristic of monounsaturated alkenes and saturated alipha-
tic alcohols.14 Comparison of the mass spectra of these consti-
tuents with the available mass spectral libraries indicated that
they could be either 1-alkenes or 1-alcohols. Although no
molecular ions were present in their mass spectra, they were
identified as 1-tetradecene (5) and 1-pentadecene (7) by co-
injection of the generation gland extract with authentic synthetic
samples of these compounds. Alkenes have been found in the
skin of some lizards and snakes, but not in the secretions of
lizards.2 The occurrence of alkenes in the generation gland
secretions could, however, be due to the fact that the generation
glands are in fact glandular scales, forming part of the lizard’s skin.
A number of additional compound classes, commonly found
in lizard secretions,2 including the femoral gland secretion of C.
giganteus,3 were also identified in its generation gland secretions,
i.e., the C9, C10, and C12ꢀ22 unbranched saturated and unsatu-
rated fatty acids 3, 4, 8, 11, 15, 18, 24, 26, 27, 32, 34, 37, 40, 42,
and 44, the C12, C14, C15, and C16 primary alcohols 6, 12, 16, and
20, the C14, C15, and C17 methyl ketones 9, 13, and 22, the C9,
C10, and C14ꢀ17 saturated and unsaturated aldehydes 1, 2, 10, 14,
17, and 19, the methyl-C16 and dodecyl-C16 saturated and
unsaturated esters 23, 47, and 48, 11 steroids (49ꢀ59), squalene
(46), and a γ-lactone, hexadecan-4-olide (29). It is interesting to
note that cholesterol, one of the major components identified in
the femoral gland secretions of C. giganteus,3 does not occur in
the generation gland material. Hexadecanoic acid (26) is the
major component of all the generation gland secretions analyzed.
The identification of steroids 49ꢀ58 could not be confirmed by
retention time comparison, due to the unavailability of standards.
However, in some cases additional diagnostic information was
obtained by the interpretation of the EI-mass spectra of these steroids
1365
dx.doi.org/10.1021/np1008366 |J. Nat. Prod. 2011, 74, 1364–1369

Journal of Natural Products
ARTICLE
Figure 3. GC-FID chromatogram of an extract of fore-leg generation gland material of a male sungazer, C. giganteus. The analytical parameters are given
in the text, and the identified compounds are numbered in order of elution from the GC column. *Impurity.
Even though GC-MS analysis yielded good mass spectra for
constituents 25, 31, 36, and 39, these constituents could not be
identified by MS library comparison or the interpretation of their
mass spectra, due to the paucity of relevant mass spectrometric
information.
organic material from collected samples. Syringes were cleaned with the
same solvent.
GC analyses were performed on a Carlo Erba 5300 gas chromatograph,
using Borwin Intuitive Chromatography software, version 1.22, for data
acquisition. The determination of the absolute abundance of constituents
was done with an HP 5890 Series II GC, equipped with an autosampler.
Agilent Chemstation software, version A.09.01, was used for instrument
control and data acquisition. These instruments were equipped with FIDs
and split/splitless inlet systems, and their injectors and detectors were
operated at 220 and 280 °C, respectively. Glass capillary columns (40 m ꢁ
0.3 mm i.d.) were coated with PS-089-OH, a silanol-terminated (95%)-
methyl-(5%)-phenylpolysiloxane copolymer, at a film thickness of 0.25 μm.
Helium was used as carrier gas at a linear flow velocity of 28.6 cm/s. Samples
were injected in the split mode using a split ratio of 1:10. The sample
constituents were thermally focused on the column at a temperature of
30 °C, after which the column was heated ballistically to 40 °C and then
programmed at 2 °C/min from 40 to 280 °C (hold 60 min).
EI-MS data were acquired at 70 eV on a Carlo Erba QMD 1000 GC-
MS instrument, using the above-mentioned column and conditions,
with He as carrier gas at an average linear velocity of 28.6 cm/s. Steroid
derivatives were analyzed using a mass range of m/z 25 to 550. Ion
source and interface temperatures of 200 and 250 °C, respectively, were
used for all analyses. Some GC-MS analyses were done on a Fisons MD-
800 instrument using the parameters specified above.
The present investigation showed that the generation glands
produce more diverse chemicals than the femoral glands, includ-
ing groups of compounds (e.g., nitriles and amides) that are
absent from the femoral gland secretions.3 C. giganteus is the only
cordylid that is recognized as a terrestrial grassland species, and
the unique presence of generation glands on the fore-legs10 may
relate to marking of the grass substrate. The fact that the chemical
composition of the hind- and fore-leg generation glands is
qualitatively identical supports the assumption that the glands
on the fore-legs not only constitute a functional increase in the
secretion volume but might also facilitate effective depositing of
glandular material in the grassland environment. Although the
quantities of chemicals produced by the generation glands differ
between individual lizards, no pattern could be found in these
differences (Table 1). The information made available in the
present study should facilitate investigations into the semio-
chemical communication of C. giganteus and other species.
’ EXPERIMENTAL SECTION
NMR spectra of synthetic reference compounds were recorded in
CDCl3 on Varian Unity INOVA 600 NMR, Varian Unity INOVA 400
NMR, and Varian 300 VNMRS instruments (Varian, Palo Alto, USA).
Chemical shifts are given in parts per million (δ) relative to CHCl3 (7.26
and 77.04 ppm for 1H and 13C NMR, respectively).
General Experimental Procedures. All Pyrex glassware used for
the chemical analysis was thoroughly cleaned with H2O and acetone and
heated to 500 °C to remove any residual organic material. Dichlor-
omethane (Fluka, residue analysis grade) was used for extraction of
1366
dx.doi.org/10.1021/np1008366 |J. Nat. Prod. 2011, 74, 1364–1369

Journal of Natural Products
ARTICLE
Table 1. Constituents Identified in the Generation Gland Secretion of the Male Sungazer, C. giganteus
quantity (ng/mg)c
relative amountsd
number in Figure 3,a
constituent
ident.b
fore-leg
hind-leg
fore-leg (n = 10)
hind-leg (n = 10)
Alkenes
5
1-tetradecene
A, B
A, B
5
4
2
4
0.09 ( 0.06
0.09 ( 0.03
0.02 ( 0.01
0.04 ( 0.02
7
1-pentadecene
Acids
3
nonanoic acid
A, B
A, B
A, B
A, B
A, B
A, B
A, B
A, B
A, B
A, B
A, B
A, C
A, B
A, B
A, B
2
1
3
2
0.01 ( 0.00
0.01 ( 0.00
0.19 ( 0.08
0.06 ( 0.03
2.70 ( 0.54
1.36 ( 0.71
12.16 ( 6.92
39.32 ( 3.54
0.59 ( 0.26
2.31 ( 1.11
6.72 ( 2.04
0.01 ( 0.01
0.45 ( 0.18
0.05 ( 0.02
0.10 ( 0.07
0.01 ( 0.01
<0.005
4
decanoic acid
8
dodecanoic acid
33
31
0.23 ( 0.12
0.06 ( 0.07
2.27 ( 0.56
0.84 ( 0.33
13.75 ( 4.27
38.30 ( 3.57
0.42 ( 0.18
2.80 ( 0.69
5.92 ( 1.36
<0.005
11
tridecanoic acid
40
16
15
tetradecanoic acid
pentadecanoic acid
(Z)-9-hexadecenoic acid
hexadecanoic acid
heptadecanoic acid
(Z)-9-octadecenoic acid
octadecanoic acid
nonadecanoic acid
icosanoic acid
483
220
838
3479
88
440
164
1488
3971
77
18
24
26
27
32
160
501
34
378
715
29
34
37
40
88
132
21
0.53 ( 0.19
0.06 ( 0.01
0.12 ( 0.05
42
henicosanoic acid
docosanoic acid
15
44
28
39
Alcohols
6
1-dodecanol
A, B
A, B
A, B
A, B
13
16
45
21
63
6
0.57 ( 0.51
0.13 ( 0.03
0.03 ( 0.01
0.03 ( 0.02
1.06 ( 0.55
0.05 ( 0.02
0.03 ( 0.01
0.01 ( 0.01
12
1-tetradecanol
1-pentadecanol
1-hexadecanol
16
23
18
20
Ketones
9
2-tetradecanone
2-pentadecanone
2-heptadecanone
A, C
A, B
A, B
4
12
15
3
28
15
0.01 ( 0.01
0.09 ( 0.04
0.08 ( 0.04
0.01 ( 0.00
0.02 ( 0.01
0.05 ( 0.01
13
22
Aldehydes
1
nonanal
A, B
1
2
1
1
0.02 ( 0.01
0.01 ( 0.01
0.02 ( 0.01
0.03 ( 0.02
0.03 ( 0.07
0.57 ( 0.86
0.01 ( 0.01
0.02 ( 0.04
0.01 ( 0.00
0.03 ( 0.03
0.01 ( 0.00
0.12 ( 0.16
2
decanal
A, B
10
tetradecanal
pentadecanal
hexadecanal
2-heptadecenal
A, B
31
36
28
145
12
12
8
14
A, C
A, B
17
19
A, C, E
192
Esters
23
methyl hexadecanoate
A, B
A, B
A, B
19
29
2
17
17
0.02 ( 0.01
0.03 ( 0.02
0.07 ( 0.12
0.02 ( 0.02
0.10 ( 0.06
0.08 ( 0.07
47
dodecyl (Z)-9-hexadecenoate
dodecyl hexadecanoate
48
102
Nitriles
21
hexadecanitrile
octadecanitrile
A, B
A, B
133
22
62
24
1.84 ( 0.92
0.18 ( 0.06
0.41 ( 0.24
0.06 ( 0.04
30
Amides
28
pentadecanamide
(Z)-9-hexadecenamide
hexadecanamide
heptadecanamide
octadecanamide
nonadecanamide
icosanamide
A, B
A, B
A, B
A, C
A, B
A, C
A, B
16
46
15
80
288
24
78
8
0.12 ( 0.17
0.79 ( 0.16
0.25 ( 0.14
0.12 ( 0.10
0.45 ( 0.16
0.02 ( 0.01
0.24 ( 0.09
0.05 ( 0.02
0.73 ( 0.09
0.19 ( 0.12
0.05 ( 0.04
0.32 ( 0.11
0.02 ( 0.00
0.33 ( 0.12
33
35
575
34
38
41
83
43
11
45
32
26
Steroids
49
5β-cholestan-3R-ol
A, D
609
1099
3.81 ( 1.18
5.16 ( 1.69
1367
dx.doi.org/10.1021/np1008366 |J. Nat. Prod. 2011, 74, 1364–1369

Journal of Natural Products
ARTICLE
Table 1. Continued
quantity (ng/mg)c
fore-leg hind-leg
relative amountsd
number in Figure 3,a
constituent
ident.b
fore-leg (n = 10)
hind-leg (n = 10)
50
5R-cholestan-3R/β-ol
5R-cholestan-3R/β-ol
3-ethoxycholestane
cholestan-2/3-one
5R-cholest-7-en-3β-ol
5β-ergostanol
A, D
A, D
A
607
445
25
770
712
18
1.86 ( 0.73
1.13 ( 0.35
0.10 ( 0.08
0.07 ( 0.03
0.06 ( 0.05
0.12 ( 0.05
0.16 ( 0.11
0.11 ( 0.10
0.03 ( 0.01
0.14 ( 0.07
2.02 ( 0.73
1.90 ( 0.71
0.08 ( 0.05
0.17 ( 0.13
0.07 ( 0.03
0.21 ( 0.07
0.32 ( 0.20
0.17 ( 0.14
0.05 ( 0.02
0.31 ( 0.16
51
52
53
A
107
57
17
54
A, D
A, D
A, D
A
54
55
45
27
56
ergost-5-en-3β-ol
cholest-4-en-3-one
5β-stigmastanol
94
90
57
66
169
38
58
A, D
A, B, D
109
60
59
lanosterol
184
Miscellaneous
29
hexadecane-4-olide
squalene
A, B
A, B
43
51
20
0.14 ( 0.07
0.28 ( 0.17
0.10 ( 0.09
0.69 ( 0.45
46
123
Unknown constituents
25
31
36
39
m/z 39, 42, 57, 69, 71, 83, 85, 239
m/z 41, 43, 55, 57, 69, 71, 239
m/z 41, 43, 55, 57, 69, 71, 239
m/z 41, 43, 55, 60, 101, 114
A
A
A
A
1088
80
1389
130
40
12.63 ( 1.14
0.12 ( 0.04
0.06 ( 0.04
0.35 ( 0.20
12.30 ( 1.15
0.05 ( 0.04
0.03 ( 0.04
0.19 ( 0.16
48
99
58
a Compound classes given in order of elution from GC column. b Identification method(s). A, GC-MS analysis; B, retention time comparison with
authentic synthetic compounds; C, retention time increment comparison; D, EI-mass spectra of TMS derivatives; E, E/Z-isomerism unknown.
c Amount in ng of individual constituents per mg of secretion, calculated proportional to the amount of internal standard (octadecane) using their
chromatographic peak area ratios. d Relative amounts calculated as a percentage of the 168 compounds detected by GC-FID. The mean ( standard
deviation is reported.
Histology of the Generation Glands. Skin biopsies were ob-
tained from specimens collected for studies regarding the reproductive cycle
of these lizards.20 Biopsies were subjected to routine histological procedures
using paraffin wax (Merck, Histosec, 56ꢀ58 °C) as embedding medium.
Sections (6ꢀ10 μm) were cut with a rotary microtome and stained with
hematoxylin (Merck) and eosin yellowish (Saarchem).21
Collection and Sample Preparation. Generation gland secre-
tions were collected individually from the fore- and hind-legs of 10 adult
male lizards in a study population on a farm in the Lindley district (Free
State Province, South Africa; 28°010 S, 28°050 E) during April 1998. The
secretions were collected by removing the external layers (reminiscent of
shed skin) of the glandular scales on the fore- and hind-legs of the lizards,
using a pair of sharp-tipped stainless steel forceps. The collected material
was transported to Stellenbosch at ꢀ10 °C and stored at ꢀ70 °C until
processed for analysis. The organic constituents were extracted from
individually collected secretions with 100 μL of DCM. The extracts were
subsequently centrifuged and concentrated to ca. 10 μL by slow
evaporation of the solvent at room temperature in a N2 atmosphere.22
Samples of 0.2 to 1.0 μL of these concentrated extracts were used per
Trimethylsilyl (TMS) Derivatives. An extract of the generation
gland material was concentratedina Reacti-Vial leftuncapped in a purified
N2 atmosphere to yield approximately 1 mg of solvent-free material. TMS
derivatizations24 were performed as previously described.3
Reference Compounds. Most of the reference compounds used
to confirm the identity of the constituents of the generation gland
secretions by retention time comparison were obtained commercially.
Hexadecanitrile (21), octadecanitrile (30), pentadecanamide (28), (Z)-
9-hexadecenamide (33), hexadecanamide (35), octadecanamide (41),
icosanamide (45), dodecyl (Z)-9-hexadecenoate (47), and dodecyl
hexadecanoate (48) were synthesized from authentic starting materials
according to procedures exemplified by the syntheses of 21, 28, and 47.
(For other syntheses and the spectroscopic data of other compounds,
see the Supporting Information.)
Hexadecanitrile (21) was synthesized from 1-bromopentadecane
by substitution of the bromine with cyanide.25,26 A mixture of 1-bro-
mopentadecane (0.48 g, 1.65 mmol), NaCN (0.101 g, 2.06 mmol), and
triethyleneglycol (ca. 1 mL) was stirred and slowly heated to 140 °C.
After stirring the reaction mixture at this temperature for another 3 h, the
mixture was added to H2O (10 mL), and the organic product was
extracted with CHCl3. The extract was washed with H2O to neutral pH
and dried on anhydrous MgSO4, and the organic product was isolated in
the usual manner to yield pure (GC-MS) hexadecanitrile (0.25 g, 64%):
13C NMR (100 MHz, CDCl3) δ 14.06 (C-16), 17.06 (C-2), 22.65 (C-
15), 25.35 (C-3), 28.63 (C-4), 28.73 (C-5), 29.27 (C-13), 29.33 (CH2),
29.47 (CH2), 29.56 (CH2), 29.61 (CH2), 29.62 (CH2), 29.64 (CH2),
29.65 (CH2), 31.89 (C-14), 119.75 (C-1); EIMS m/z 222 (2), 208 (12),
194 (17), 180 (13), 166 (11), 152 (13), 138 (21), 124 (41), 110 (70), 97
(82), 82 (42), 69 (44), 57 (86), 43 (100), 29 (45).
analysis for qualitative GC-MS analyses.
Quantitative GC determination of the relative abundance of the
constituents of the secretions of the group of males was performed using
solventless sample introduction23 of the raw secretion. For the determina-
tion of the absolute abundance of each constituent in the generation gland
secretion, extracts of the secretions from the fore- and hind-legs of a male
lizard were prepared by extracting 11.3 and 10.3 mg of the respective
secretions with four portions of 200 μL of DCM each. Each extract was
slowly evaporated to dryness in an inert atmosphere at 22 °C, and the
residual material was dissolved in 20 μL of an internal standard solution
containing 35.9 μg of octadecane/mL in DCM. Quantities of 2 μL of each
of these solutions were used for quantitative GC determinations. Absolute
constituent amounts were calculated proportional to that of the internal
standard, using their chromatographic peak area ratios.
Pentadecanamide (28) was prepared from pentadecanoic acid
via the corresponding acid chloride.25,26 Pentadecanoic acid (0.20 g,
0.825 mmol) was refluxed with thionyl chloride (1 mL, 13.8 mmol) for
1368
dx.doi.org/10.1021/np1008366 |J. Nat. Prod. 2011, 74, 1364–1369

Journal of Natural Products
ARTICLE
30 min, after which the remaining thionyl chloride was distilled off. The
acid chloride was cooled in an ice bath and treated with concentrated
ammonia solution (3 mL, 25%). The resulting solid material was filtered
and washed with H2O containing a small percentage of EtOH. The
product was dried at room temperature on filtration paper, yielding the
pure (GC-MS) pentadecanamide (0.18 g, 90%): 1H NMR (400 MHz,
CDCl3) δ 0.87 (3H, t, J = 6.8, CH3), 1.24 (22H, m), 1.62 (2H, m, H-3),
2.21 (2H, t, J = 7.8, H-2), 5.45 (1H, s, NH), 5.82 (1H, s, NH); 13C NMR
(CDCl3, 100 MHz) δ 14.14 (C-15), 22.71 (C-14), 25.55 (C-3), 29.25
(C-4), 29.35 (C-5), 29.38 (C-12), 29.49 (CH2), 29.63 (CH2), 29.67
(CH2), 29.67 (CH2), 29.69 (CH2), 29.71 (CH2), 31.95 (C-13), 36.00
(C-2), 176.17 (C-1); EIMS m/z 241 (1, Mþ), 212 (2), 198 (3), 184 (2),
170 (2), 156 (3), 142 (3), 128 (5), 114 (5), 97 (7), 86 (8), 72 (36), 59
(100), 43 (35), 29 (8).
(10) Van Wyk, J. H.; Mouton, P. l. N. Amphib. Reptil. 1992, 13, 1–12.
(11) Cordes, I. G.; Mouton, P. l. N.; Van Wyk, J. H. South Afr. J. Zool.
1995, 30, 187–196.
(12) Du Toit, D. A.; Mouton, P. l. N.; Flemming, A. F.; Van Niekerk,
A. J. Herpetol. 2005, 39, 384–388.
(13) Dujsebayeva, T. N.; Ananjeva, N. B.; Miroschnichenko, L. V.
Amphib. Reptil. 2007, 28, 537–546.
(14) McLafferty, F. W.; Turecek, F. Interpretation of Mass Spectra;
University Science Books: Mill Valley, CA, 1993.
(15) Zaretskii, Z. V. I.; Curtis, J. M.; Brenton, A. G.; Beynon, J. H.;
Djerassi, C. Org. Mass Spectrom. 1988, No. 23, 453–459.
(16) Zaretskii, Z. V. I.; Curtis, J. M.; Brenton, A. G.; Beynon, J. H.;
Djerassi, C. Org. Mass Spectrom. 1988, 23, 460–468.
(17) Simpson, J. T.; Sharp, T. R.; Wood, W. F.; Weldon, P. J. Z.
Naturforsch. (C) 1993, 48, 953–955.
Dodecyl (Z)-9-hexadecenoate (47) was synthesized by reflux-
ing a mixture of (Z)-9-hexadecenoic acid (25.4 mg, 0.01 mmol),
dodecanol (18.4 mg, 0.01 mmol), and pTSA monohydrate (1 mg) in
benzene (20 mL) for 30 h.25 The product was washed twice with H2O
and dried, and the solvent was removed on a rotary evaporator to yield
practically pure (GC-MS) dodecyl (Z)-9-hexadecenoate in quantitative
(18) Weldon, P. J.; Ortiz, R.; Sharp, T. R. . In Biology of the Pitvipers;
Campbell, J. A.; Brodie, E. D., Eds.; Selva: Tyler, TX, 1992; pp 309ꢀ319.
(19) Salamon, M.; Davies, N. W. J. Chem. Ecol. 1998, 24, 1659–1676.
(20) Van Wyk, J. H. J. Herpetol. 1995, 29, 522–535.
(21) Bancroft, J. D.; Stevens, A. B. Theory and Practice of Histological
Techniques; Churchill Livingstone: Edinburg, 1977.
13
yield: C NMR (100 MHz, CDCl3) δ 14.13 (C-120), 14.15 (C-16),
(22) Reiter, B.; Burger, B. V.; Dry, J. J. Chem. Ecol. 2003,
29, 2235–2252.
22.69 (C-15), 22.72 (C-110), 25.06 (C-3), 25.97 (C-30), 27.20 (C-11),
27.26 (C-8), 28.69 (C-20), 29.02 (C-4), 29.14 (CH2), 29.17 (CH2),
29.21 (CH2), 29.29 (CH2), 29.38 (CH2), 29.56 (CH2), 29.61
(CH2), 29.67 (CH2), 29.68 (CH2), 29.72 (CH2), 29.77 (CH2), 31.82
(C-100), 31.95 (C-14), 34.44 (C-2), 64.45 (C-10), 129.78 (C-9), 130.01
(C-10), 174.03 (C-1); EIMS m/z 422 (1, Mþ), 255 (8), 236 (40), 194
(14), 152 (31), 125 (19), 111 (43), 98 (57), 97 (77), 83 (85), 71 (60),
69 (95), 57 (90), 43 (79), 41 (57), 29 (24).
(23) Burger, B. V.; Munro, Z.; Smit, D.; Schmidt, U.; Wu, C.-L.;
Tien, F.-C. J. Chromatogr. 1990, 518, 207–214.
(24) Thenot, J.-P.; Horning, E. C. Anal. Lett. 1972, 5, 21–33.
(25) Vogel, A. I. Vogel’s Textbook of Practical Organic Chemistry,
5th ed.; Longman Group UK Limited, 1989.
(26) Hazzard, B. J. Organicum: Practical Handbook of Organic
Chemistry; Pergamon Press Ltd.: Oxford, 1973.
’ ASSOCIATED CONTENT
S
Supporting Information. Mass spectrometric data of
b
identified constituents and spectroscopic data of synthesized
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: stefanlouw@gmail.com. Tel: þ2721-406-6453.
’ ACKNOWLEDGMENT
This research was supported by Stellenbosch University and
the National Research Foundation, Pretoria, South Africa. L.
Ruddock is thanked for the collection of secretions.
’ REFERENCES
(1) Mason, R. T. In Biology of Reptilia: Hormones, Brain, and
Behavior; Gans, C., Ed.; The University of Chicago Press: Chicago
and London, 1992; Vol. 18, Physiology E, p 115.
(2) Weldon, P. J.; Flachsbarth, B.; Schulz, S. Nat. Prod. Rep. 2008,
25, 738–756.
(3) Louw, S.; Burger, B. V.; Le Roux, M.; Van Wyk, J. H. J. Chem.
Ecol. 2007, 33, 1806–1818.
(4) Lopez, P.; Martin, J. Biochem. Syst. Ecol. 2009, 37, 304–307.
(5) Kopena, R.; Lopez, P.; Martin, J. Z. Naturforsch. (C) 2009,
64, 434–440.
(6) Gabirot, M.; Lopez, P.; Martin, J.; de Fraipont, M.; Heulin, B.;
Sinervo, B.; Clobert, J. Biochem. Syst. Ecol. 2008, 36, 539–544.
(7) Martin, J.; Lopez, P. Chem. Senses 2010, 35, 253–262.
(8) Maderson, P. F. A. Copeia 1967, 743–752.
(9) Maderson, P. F. A. Breviora 1968, 288, 1–35.
1369
dx.doi.org/10.1021/np1008366 |J. Nat. Prod. 2011, 74, 1364–1369