SUPPLEMENTARY INFORMATION
Hydrocarbon synthesis
General methods
All solvents and reagents were obtained from commercial vendors (Fluka, Aldrich or Acros)
and used without further purification. All reactions involving air-sensitive reagents were
performed under N2 using syringe septum cap techniques and flame-dried glassware. Flash
column chromatography was performed using silica gel (40-63μmol, Merck) or Isolute SiO2
columns from Biotage. Celite (coarse) filtrations were performed using plastic filters.
Compounds were visualised on TLC (silica gel 60 F254 plates) using UV light, DNP, H2SO4 or
KMnO4 stain. 1H and 13C NMR spectra were recorded on either a 300 Bruker Avance
instrument or a 300 Varian Mercury instrument (abbreviated “Varian” below), with CDCl 3 as
an internal reference standard (δ 7.26, 77.16ppm). GC-MS analyses were performed with a
Shimadzu GC-17A gas- chromatograph (capillary column: MDN-5S, 30m x 25μm x 0.25μm,
carrying gas Helium) coupled with a Shimadzu GCMS-QP 5050 (electron ionization).
Synthesis of 1-Bromohexacosane (3)
1-Bromohexacosane (previously synthesised by (Kutsumizu et al. 2000)) was prepared using
a procedure adapted from (Dickschat et al. 2004). Bromine (0.045ml, 0.87mmol) was added
to PPh3 (226mg, 0.86mmol) in DCM (6ml) at room temperature (RT) and stirred for 5
minutes. Hexacosan-1-ol (242mg, 0.63mmol) was added slowly, and the mixture was heated
slightly to dissolve the alcohol and left stirring for 2 hours at RT. Et2O (6ml) was then added,
followed by aqueous Na2SO3 (1M, 3ml). The organic phase was dried with MgSO4, filtered
and concentrated in vacuo. The compound was absorbed on Celite using Et2O and purified
by flash column chromatography (Isolute, 20g, pentane/Et2O 7:0 – 7:1), yielding 282mg of 1bromohexacosane. 1H NMR (CDCl3) δ 0.88 (t, J=7Hz, CH3, 3H), 1.26-1.54 (m, (CH2)n, 46H),
1.81-1.90 (m, CH2, 2H), 3.41 (t, J=7Hz, CH2Br, 2H). 13C NMR (CDCl3) δ 14.3, 22.9, 28.4, 28.9,
29.5, 29.6, 29.7, 29.9, 32.1, 33.0.
Synthesis of 5-Hexacosyl-2,3-dimethylthiophene (4)
n-BuLi in hexanes (0.85ml, 1.3mmol, 1.6M) was added to 2,3-dimethylthiophene (154mg,
1.37mmol) dissolved in THF (4ml) at RT. The mixture was stirred while 3 (282mg, 0.63mmol)
in THF (5ml) was added dropwise, then left to stir over night at RT. Saturated aqueous NH 4Cl
was added and the mixture extracted with Et2O (3x), washed with brine, dried with Na2SO4
and concentrated in vacuo. Column chromatography (Isolute SiO2, 5g, 100% pentane) gave
two major fractions: Fraction 1 (217mg) with 3 present (~ 5mol%) and fraction 2 (50mg)
with pure 4 estimated by 1H NMR and GC-MS and visualised by TLC (H2SO4). TLC (100%
pentane) Rf 0.86. (1-bromo-n-hexacosane: Rf 0.94). 1H NMR of fraction 2 (CDCl3) δ 0.88 (t,
J=7Hz, CH3, 3H), 1.25 (m, (CH2)n, 48H). 2.06 (s, ArCH3, 3H), 2.28 (s, ArCH3, 3H), 2.67 (t, J=7Hz,
ArCH2, 2H), 6.44 (s, ArH, 1H). 13C NMR (Varian, CDCl3) δ 13.2, 13.8, 14.4, 23.0, 29.4, 29.6,
29.7, 29.8, 30.0, 30.2, 32.0, 32.2, 126.9, 129.8, 132.3, 141.0. GC-MS 125, 476.
Synthesis of (R/S)-3-Methylhentriacontane (1)
3-Methylhentriacontane (3-MeC31) has previously been described from alkane mixtures
from various natural sources (e.g. Kaneda 1967; FinidoriLogli et al. 1996; Akino 2006; Dreier
& d'Ettorre 2009) and synthesised as part of a mixture of compounds (Cope et al. 1965). A
solution of 4 (48mg, 0.1mmol) in dioxane (1ml) was bubbled through with N2 for five
minutes, whereupon aqueous Raney Ni (0.5ml of a 50% slurry) was added. The slurry was
refluxed overnight, cooled to RT and filtered through a Celite column using dioxane and
pentane. The clear filtrate was concentrated in vacuo (43mg) and checked by NMR to see
disappearance of aromatic protons. The resulting desulfurised product (43mg) in a 1:1
mixture of EtOH/heptane (5ml) was stirred with 5% Pd/C (19mg) under an atmosphere of H 2
at an oil bath temperature of 40ºC over night. The mixture was filtered through Celite using
pentane. The filtrate was concentrated in vacuo and filtered through an Isolute SiO2 column
(5g) with pentane giving 1 as a white wax (39mg, 95%). 1H NMR (CDCl3) δ 0.82-0.90 (m,
3xCH3, 9H), 1.05-1.52 (m, (CH2)n, 57H). 13C NMR (CDCl3) δ 11.6, 14.3, 19.4, 22.9, 27.3, 29.5,
29.7, 29.8, 29.9, 30.2, 32.1, 34.6, 36.8. GC-MS 57, 71, 85, 99, 113, 141, 155, 169, 183, 197,
211, 225, 253, 267, 281, 421 [M-C2H5]+.
(R/S)-1-(3-Methylthiophen-2-yl)tricosan-1-ol (5)
1-Bromodocosane (5.82g, 15mmol) in Et2O (8ml) was added to magnesium (547mg,
22.5mmol) suspended in Et2O (10ml). Approximately 0.1ml of 1,2-dibromoethane was
added and the mixture was heated for 1h, before 3-methylthiophene-2-carbaldehyde
(1.20g, 9.5mmol) was added. The mixture was left stirring overnight at RT. Saturated
aqueous NH4Cl (5ml) was added and the mixture was left stirring for 15 min followed by
addition of H2O (10ml). The solvent was decanted and the filter cake was washed several
times with Et2O. The mixture was concentrated followed by purification using flash column
chromatography giving 5 (765mg, 18%). TLC (heptane/EtOAc 4:1) Rf 0.4. 1H NMR (CDCl3) δ
0.88 (t, J=7Hz, CH3, 3H), 1.25-1.88 (br, (CH2)n, OH, 42H), 2.23 (s, CH3, 3H), 4.97 (t, J=7Hz,
ArCHOH, 1H), 6.79 (d, J=5Hz, ArH, 1H), 7.15 (d, J=5Hz, ArH, 1H). 13C NMR (Varian, CDCl3) δ
14.1, 14.4, 23.0, 26.2, 29.6, 29.7, 29.77, 29.82, 29.88, 29.91, 30.0, 32.2, 39.4, 68.6, 123.0,
130.0, 133.4, 142.0. GC-MS 111, 124, 137, 418 [M-H2O]+.
3-Methyl-2-tricosylthiophene (6)
Compound 6 was prepared using a procedure adapted from (Lau et al. 1986). ZnI2 (511mg,
1.6mmol) was added to a solution of 5 (436mg, 1.0mmol) in 1,2-DCE (30ml) at RT,
whereupon the mixture turned slightly pink.NaBH3CN (471mg, 7.5mmol) was added and the
mixture turned colourless. After two days stirring at RT, the mixture was filtered on Celite
and the filter cake was washed with additional DCM. Flash column chromatography
(heptane/Et2O 100:0 – 20:1) yielded 6 as a solid wax (320mg, 76%). TLC (100% heptane) Rf
0.57. 1H NMR (CDCl3) δ 0.88 (t, J=7Hz, CH3, 3H), 1.26-1.64 (m, (CH2)n, 42H), 2.15 (s, CH3, 3H),
2.70 (t, J=8Hz, ArCH2, 2H), 6.77 (d, J=5Hz, ArH, 1H), 7.00 (d, J=5Hz, ArH, 1H). 13C NMR (CDCl3)
δ 13.7, 14.3, 22.9, 28.0, 29.45, 29.52, 29.6, 29.7, 29.82, 29.86, 31.7, 32.0, 32.1, 120.9, 130.0,
132.4, 139.1. GC-MS 111, 420 [M]+.
(R/S)-3-Methylheptacosane (2)
This compound (3-MeC27) has been previously described from a mixture of alkanes isolated
from natural sources or synthesised as the R- or S-enantiomer (FinidoriLogli et al. 1996;
Marukawa et al. 2001). Compound 2 was prepared according to the procedure described for
compound 1 starting with 6 (198mg, 0.5mmol), dioxane (4ml) and Raney Nickel (4.2g of 50%
slurry) giving a desulphurised mixture (175mg), which was further treated with Pd/C (57mg)
and H2 (1 atm) at 40ºC in a 1:1 mixture of heptane/EtOH (20ml) overnight. Flash column
chromatography (100% pentane) of the filtered compound yielded 2 as a white wax (156mg,
84%). 1H NMR (CDCl3) δ 0.88 (m, 3xCH3, 9H), 1.07-1.55 (m, (CH2)n, 49H). 13C NMR (CDCl3) δ
11.7, 14.4, 19.5, 23.0, 27.4, 29.6, 29.7, 29.9, 30.3, 32.2, 34.6, 36.9. GC-MS 57, 71, 85, 99,
113, 127, 141, 155, 169, 183, 197, 211, 225, 253, 267, 281, 337, 365 [M-C2H5]+.
SUPPLEMENTARY INFORMATION REFERENCES
Akino, T. 2006 Cuticular hydrocarbons of Formica truncorum (Hymenoptera: Formicidae):
Description of new very long chained hydrocarbon components. Applied Entomology
and Zoology 41, 667-677.
Cope, A. C., Burrows, E. P., Derieg, M. E., Moon, S. & Wirth, W. D. 1965 Rimocidin I:
Carbon skeleton partial structure and absolute configuration at C-27. Journal of the
American Chemical Society 87, 5452-&.
Dickschat, J. S., Wenzel, S. C., Bode, H. B., Muller, R. & Schulz, S. 2004 Biosynthesis of
volatiles by the myxobacterium Myxococcus xanthus. Chembiochem 5, 778-787.
Dreier, S. & d'Ettorre, P. 2009 Social context predicts recognition systems in ant queens.
Journal of Evolutionary Biology 22, 644-649.
FinidoriLogli, V., Bagneres, A. G., Erdmann, D., Francke, W. & Clement, J. L. 1996 Sex
recognition in Diglyphus isaea Walker (Hymenoptera: Eulophidae): Role of an
uncommon family of behaviorally active compounds. Journal of Chemical Ecology
22, 2063-2079.
Kaneda, T. 1967 Biosynthesis of long-chain hydrocarbons I: Incorporation of L-valine, Lthreonine, L-isoleucine and L-leucine into specific branched-chain hydrocarbons in
tobacco. Biochemistry 6, 2023-&.
Kutsumizu, S., Ichikawa, T., Yamada, M., Nojima, S. & Yano, S. 2000 Phase transitions of 4
'-n-hexacosyloxy-3 '-nitrobiphenyl-4-carboxylic acid (ANBC-26): Two types of
thermotropic cubic phases. Journal of Physical Chemistry B 104, 10196-10205.
Lau, C. K., Dufresne, C., Belanger, P. C., Pietre, S. & Scheigetz, J. 1986 Reductive
deoxygenation of aryl aldehydes and ketones and benzylic, allylic, and tertiary
alcohols by ZNI2-NaCNBH3. Journal of Organic Chemistry 51, 3038-3043.
Marukawa, K., Takikawa, H. & Mori, K. 2001 Synthesis of the enantiomers of some methylbranched cuticular hydrocarbons of the ant, Diacamma sp. Bioscience Biotechnology
and Biochemistry 65, 305-314.
Table S1: Composition of the chemical profile of workers, queens and queen-laid eggs of
Lasius niger. The stars denote compounds that make up a significantly different proportion
of the profile of queens and workers (ANOVA; n = 20 workers and 20 queens); * = p < 0.05,
** = p < 0.01, *** = p < 0.001.
Peak
C25
C27:1
C27
3-MeC27
C29:1
C29
7-, 9-, 11- and 13-MeC29
5-MeC29
7,11-, 9,15- and 11,15-diMeC29
3-MeC29
5,15-diMeC29
5,9,13- and 7,11,15-triMeC29
12- and 14-diMeC30
10,14-diMeC30
C31:1
C31
7-, 9-, 11-, 13- and 15-MeC31
5-MeC31
7,17-, 9,15-, 9,17-,11,15- and 13,17-diMeC31
3-MeC31
5,13- and 5,17-diMeC31
7,13,17-triMeC31
3,9-, 3,11-, 3,13-, 3,15- and 3,19-diMeC31
10-, 14- and 15-MeC32
4-MeC32 and 10,14-diMeC32
C33:1
7-, 9-, 11-, 13-, 15- and 17-MeC33
11,15-, 13,17- and 15,19-diMeC33
9,15- and 11,17-diMeC33
3-MeC33 and 5,17- and 5,19-diMeC33
7,13,15- and 7,13,19-triMeC33
3,9-, 3,11- and 3,13-diMeC33
9,17-, 11,19-, 13,21- and 15,23-diMeC35
Workers
% of profile
Queens
0.4
0.2
0.9***
0.2
0.9
2.1*
1.7**
7.6**
1.1
1.8***
2.1***
1.3***
2.0
2.2
1.0
13.3***
1.1
19.9***
1.9
3.5***
2.6***
3.3***
2.1***
2.1
1.1
2.9
2.5
5.5
2.3
2.0***
1.7***
7.7***
3.2***
1.7
1.0
5.9
2.3***
0.7
1.0
0.7
2.1
8.0***
3.3***
6.5
1.6
14.1
11.9***
0.6
1.9
1.5
1.3
2.5
5.6***
2.9
2.9
6.3
2.6
1.3
1.0
3.9
Eggs
0.7
0.6
5.5
1.3
2.6
7.1
1.2
8.2
2.3
8.4
2.6
1.8
0.4
0.9
11.7
3.4
8.2
4.3
5.2
17.3
0.7
1.0
1.3
0.4
0.4
2.3
SUPPLEMENTARY INFORMATION FIGURE LEGEND
Figure S1: Ovary activation (on a scale of 1-4) in queenless groups of workers following
supplementation with 3-MeC31, C31 or pentane. This is the same dataset as in Figure 1B, but
split into the 6 individual colonies.