1
SUPPLEMENTARY MATERIAL
New oxygenated himachalenes in male-specific odor of the Chinese windmill
butterfly, Byasa alcinous alcinous
Hisashi Ômuraa*, Taro Noguchia and Tatsuo Nehirab
a
Graduate School of Biosphere Science, Hiroshima University, Higashihiroshima
739-8528, Japan; bGraduate School of Integrated Arts and Sciences, Hiroshima
University, Higashihiroshima 739-8521, Japan
*Corresponding author. E-mail: homura@hiroshima-u.ac.jp
Abstract
Male adults of the Chinese windmill Byasa alcinous alcinous (Papilionidae) are well
known to have a strong musk-like odor, in which two oxygenated himachalene
compounds, together with six sesquiterpene hydrocarbons, were newly discovered.
-Himachalen-4-yl acetate (1) was the predominant compound isolated from the solvent
extract of the males. The structure of 1 was determined using MS and NMR, and its
relative configuration was established as 1S*,4R*,6R* by NOE analysis with the help of
quantum mechanical computation. Interestingly, the amount of 1 in males increased
until 7 days after eclosion, suggesting that this compound is involved in sexual
maturation for mating. Another new compound was identified as -himachalen-4-ol (2)
by comparison with the retention time and mass spectrum of the hydrolysate of 1. Since
males of other papilionid species have general volatiles omnipresent in plants and
insects, the presence of species-specific volatiles in males is characteristic of B. alcinous
alcinous.
Keywords: Adult butterfly, Papilionidae, Byasa alcinous alcinous Male-specific
volatiles, -Himachalene derivatives, Simulation-aided conformational analysis
2
Contents:
Table S1. Chemical composition of sesquiterpene volatiles in the male extract of Byasa
alcinous alcinous
Table S2. Relevant distances between selected protons of -himachalen-4-yl acetate
Table S3. Retention indices of sesquiterpene volatiles in the male extract of Byasa
alcinous alcinous
Figure S1. EI-Mass spectrum (70 eV) of -himachalen-4-yl acetate
Figure S2. 1H NMR spectrum (400 MHz) of -himachalen-4-yl acetate
Figure S3. 13C NMR spectrum (100 MHz) of -himachalen-4-yl acetate
Figure S4. 1H–1H COSY spectrum (600 MHz) of -himachalen-4-yl acetate
Figure S5. HMBC spectrum (600 MHz) of -himachalen-4-yl acetate
Figure S6. 1H−1H COSY and the key HMBC correlations of -himachalen-4-yl acetate
Figure S7. Selected NOE difference spectra (600 MHz) of -himachalen-4-yl acetate
within the region between 2.0 and 6.0 ppm
Figure S8. Selected NOE difference spectra (600 MHz) of -himachalen-4-yl acetate
within the region between 0.9 and 5.5 ppm
Figure S9. Experimental ECD spectrum of -himachalen-4-yl acetate ( in L mol-1
cm-1, c = 3.9 x 10-5 mol L-1) in acetonitrile
Figure S10. Total ion chromatograms of the crude extract of male adult Byasa alcinous
alcinous and the corresponding hydrolysate
Figure S11. EI-Mass spectrum (70 eV) of -himachalen-4-ol
Figure S12. 1H NMR spectrum (400 MHz) of -himachalen-4-ol
Figure S13. 13C NMR spectrum (100 MHz) of -himachalen-4-ol
Figure S14. EI-Mass spectra (70 eV) of -copaene, unknown C15H24, -himachalene,
-himachalene, -himachalene, and -cadinene
3
Table S1. Chemical composition of sesquiterpene volatiles in the male extract of Byasa
alcinous alcinous
No.a
Compound
Male old
Amount/individual
(g, mean±SD)
3
-Copaene
3 day
0.28±0.16
4
unknown C15H24
3 day
0.37±0.23
5
-Himachalene
3 day
0.38±0.26
6
-Himachalene
3 day
0.41±0.32
7
-Himachalene
3 day
0.40±0.25
8
-Cadinene
3 day
0.46±0.30
2
-Himachalen-4-ol
3 day
0.90±0.83
1
-Himachalen-4-yl acetate
0 day
2.63±1.05
3 day
22.93±12.34
7 day
88.12±44.26
10 day
83.23±5.42
a
The number of compound corresponds to the peak number in Figure 2.
4
Table S2. Relevant distances between selected protons of -himachalen-4-yl acetate
All the stable conformations were obtained from DFT optimizations at B3LYP/6-31G(d) level under presence of chloroform with PCM
method on Gaussian 09 after a standard conformational search with CONFLEX7/MMFF94S. The populations were estimated by the
Boltzmann distribution at 298 K.
Relative configuration
(1S *,4R *,6R *)
Conformer-ID
c01
c02
c03
c04
(1S *,4S *,6R *)
c01
c02
c03
(1S *,4S *,6S *)
c01
c02
c03
c04
(1S *,4R *,6S *)
c01
Experimental NOE
Population (%)
12.7
10.3
59.0
18.1
21.3
10.0
68.7
24.7
14.7
27.5
33.1
100.0
H1 <-> H6
2.27
2.33
2.89
2.30
2.33
2.27
2.29
3.01
3.02
3.01
3.01
3.01
Observed
Distance (angstroms)
H4 <-> H6
H6 <-> H14
3.87
2.54
3.64
3.67
3.67
3.79
3.85
2.55
2.64
3.91
4.23
2.56
2.62
3.93
3.77
3.83
3.78
3.83
3.76
3.88
3.77
3.88
2.63
2.41
None
Very weak
H6 <-> H15
2.36
2.32
2.37
2.33
2.32
2.37
2.37
2.41
2.42
2.42
2.41
3.78
Observed
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Table S3. Retention indices of sesquiterpene volatiles in the male extract of Byasa alcinous alcinous
No.a
Compound
RIb measured
HP-5MS DB-1
RI from litarature
Authentic sample
DB-1
1366
Reference
Pala-Paul, J., Velasco-Negueruela, A., Perez-Alonso, M.J., Sanz, J.
J . Chromatogr. A 2001, 923, 295-298.
present in essential oil
Ylang ylang oil
(Cananga odorata )
Iranshahi, M., Amin, G., Sourmaghi, M.S., Shafiee, A., Hadjiakhoondi, A.
Flavour Fragr . J . 2006, 21, 260-261.
Pala-Paul, J., Perez-Alonso, M.J., Velasco-Negueruela, A., Vadare, J., Villa,
A.M., Sanz, J., Brophy, J.J. J . Chromatogr . A 2005, 1074, 235-239.
Pala-Paul, J., Brophy, J.J., Perez-Alonso, M.J., Usano, J., Soria, S.C.
J . Chromatogr . A 2007, 1175, 289-293.
Abella, L., Cortella, A.R., Velasco-Negueruela, A., Perez-Alonso, M.J.
Pharm . Biol . 2000, 38, 3, 197-203.
Atlas cedar oil
(Cedrus atlantica )
Atlas cedar oil
(Cedrus atlantica )
Atlas cedar oil
(Cedrus atlantica )
Atlas cedar oil
(Cedrus atlantica )
3
-Copaene
1387
1366
4
unknown C15H24
1437
1409
5
-Himachalene
1472
1439
1438
6
-Himachalene
1499
1467
1461
7
-Himachalene
1522
1490
1495
8
-Cadinene
1524
1495
1501
2
-Himachalen-4-ol
1716
1662
1
-Himachalen-4-yl acetate
1830
1780
a
The number of compoud corresponds to the peak number in Figure 2.
b
Retention index on a capillary column.
c
The components present in essential oils (Pranarôm International, Ghislenghien, Belgium) were used as authetic samples.
c
6
Figure S1. EI-Mass spectrum (70 eV) of -himachalen-4-yl acetate
[m/z (%)]: 262 (M+, 0.06), 220 (7), 202 (36), 187 (28), 173 (3), 159 (24), 146 (39), 145 (40), 133 (19), 132 (37), 131 (100), 119 (38), 117
(18), 115 (14), 105 (34), 93 (15), 91 (42), 79 (16), 77 (23), 60 (30), 55 (23), 53 (16), 45 (60), 43 (84), 41 (56).
7
(CH3CH2)2O
☓
(CH3CH2)2O
☓
CHCl3
☓
Figure S2. 1H NMR spectrum (400 MHz) of -himachalen-4-yl acetate
8
CDCl3
Figure S3. 13C NMR spectrum (100 MHz) of -himachalen-4-yl acetate
9
Figure S4. 1H–1H COSY spectrum (600 MHz) of -himachalen-4-yl acetate
10
Figure S5. HMBC spectrum (600 MHz) of -himachalen-4-yl acetate
11
Figure S6. 1H−1H COSY and the key HMBC correlations of -himachalen-4-yl acetate
12
Figure S7. Selected NOE difference spectra (600 MHz) of -himachalen-4-yl acetate within the region between 2.0 and 6.0 ppm
Trace 1: 1H-NMR spectrum (CDCl3) within the region between 2.0 and 6.0 ppm. Trace 2: NOE spectrum with saturation of the H-4
resonance (5.29 ppm). Trace 3: NOE spectrum with saturation of the H-6 resonance (2.53 ppm). Trace 4: NOE spectrum with saturation
of the H-1 resonance (2.23 ppm).
13
Figure S8. Selected NOE difference spectra (600 MHz) of -himachalen-4-yl acetate within the region between 0.9 and 5.5 ppm
Trace 1: 1H-NMR spectrum (CDCl3) within the region between 0.9 and 5.5 ppm. Trace 2: NOE spectrum with saturation of the H-4
resonance (5.29 ppm). Trace 3: NOE spectrum with saturation of the H-6 resonance (2.53 ppm). Trace 4: NOE spectrum with saturation
of the H-1 resonance (2.23 ppm).
14
Figure S9. Experimental ECD spectrum of -himachalen-4-yl acetate ( in L mol-1 cm-1, c = 3.9 x 10-5 mol L-1) in acetonitrile
The solution concentration was speculated from the UV absorption at 192 nm supposing the molar absorption coefficient  28,000 of a
typical diene molecule.
15
Figure S10. Total ion chromatograms of the crude extract of male adult Byasa alcinous alcinous and the corresponding hydrolysate
Peak 1: -himachalen-4-yl acetate, peak 2: -himachalen-4-ol.
16
Figure S11. EI-Mass spectrum (70 eV) of -himachalen-4-ol
[m/z (%)]: 220 (M+, 2), 202 (34), 187 (31), 171 (10), 159 (31), 157 (20), 146 (40), 145 (54), 143 (16), 133 (21), 132 (47), 131 (100), 129
(21), 119 (46), 117 (24), 115 (24), 107 (16), 105 (51), 93 (21), 91 (50), 79 (18), 77 (27), 67 (14), 65 (17), 55 (26), 53 (19), 43 (20), 41
(61).
17
☓
☓
☓
Figure S12. 1H NMR spectrum (400 MHz) of -himachalen-4-ol
18
☓
Figure S13. 13C NMR spectrum (100 MHz) of -himachalen-4-ol
☓
19
Figure S14. EI-Mass spectra (70 eV) of -copaene, unknown C15H24, -himachalene, -himachalene, -himachalene, and -cadinene
The number of compound corresponds to the peak number in Figure 2.