Stereoselective chemical defense in the Drosophila parasitoid Leptopilina
heterotoma is mediated by (-)-iridomyrmecin and (+)-isoiridomyrmecin
Johannes Stökl, John Hofferberth, Maria Pritschet, Michael Brummer, and Joachim Ruther
SUPPLEMENTAL INFORMATION
Table S1: Kovats retention indices of iridomyrmecin and isoiridomyrmecin on polar and
non-polar GC columns.
Compound
Retention index BPX-5
Retention index DB-Wax
Iridomyrmecin sample
1465
2133
Isoiridomyrmecin sample
1477
2146
Iridomyrmecin reference
1465
2132
Isoiridomyrmecin reference
1477
2146
Table S2: Statistical values of Chi² tests (one-constraint vs. expected equal distribution) for the
first choice of Myrmica rubra ants between odor-impregnated and control food items or
between decapitated and intact females and males of Leptopilina heterotoma.
First choice of ants
female extract vs. control
male extract vs. control
female with head vs. female without head
male with head vs. male without head
(-)-iridomyrmecin vs. control
(+)-iridomyrmecin vs. control
(-)-isoiridomyrmecin vs. control
(+)-isoiridomyrmecin vs. control
Chi²
7.2
12.8
10.8
3.3
16.2
1.8
9.8
20
P
0.007
0.003
0.001
0.067
< 0.001
0.179
0.001
< 0.001
N
20
20
30
30
20
20
20
20
Supplement Page 1
Table S3: Statistical values of Wilxocon tests of the time until the first contact of Myrmica
rubra ants with odor-impregnated and control food items.
Time until first contact by ants
female extract vs. control
male extract vs. control
female with head vs. female without head
male with head vs. male without head
(-)-iridomyrmecin vs. control
(+)-iridomyrmecin vs. control
(-)-isoiridomyrmecin vs. control
(+)-isoiridomyrmecin vs. control
Wilxocon W
195
177
405
317
208
175
183.5
210
P
< 0.001
< 0.001
< 0.001
0.082
< 0.001
0.009
< 0.001
< 0.001
N
20
20
30
30
20
20
20
20
Table S4: Statistical values of Chi² tests comparing the decision ratios of ants between
experiments with synthetic iridomyrmecins. Chi² values in lower triangle, P values (not
corrected for ties) in upper triangle.
uncorr. P
(-)iridomyrmecin
Chi²
(-)iridomyrmecin
(+)5.62
iridomyrmecin
(-)1.11
isoiridomyrmecin
(+)1.03
isoiridomyrmecin
(+)iridomyrmecin
0.060
(-)(+)isoiridomyrmecin isoiridomyrmecin
0.573
0.598
0.344
2.13
8.48
0.014 (0.084
corrected)
0.197
3.24
Supplement Page 2
Table S5: Statistical values for pairwise Mann-Whitney U-tests for the time difference until
first contact by ants between odor-impregnated and control food items. Uncorrected P values
in upper triangle, P values corrected for ties (sequential Bonferroni correction) in lower
triangle.
uncorr. P
corr. P
(-)iridomyrmecin
(+)iridomyrmecin
(-)isoiridomyrmecin
(+)isoiridomyrmecin
(-)iridomyrmecin
(+)iridomyrmecin
0.001
(-)(+)isoiridomyrmecin isoiridomyrmecin
< 0.001
0.002
0.409
0.088
0.005
< 0.001
not significant
0.449
0.010
not significant
not significant
Table S6: Statistical values for pairwise Mann-Whitney U-tests for the total amount of
iridomyrmecins (sum of (-)-iridomyrmecin and (+)-isoiridomyrmecin) found in the
headspace analyses of Leptopilina heterotoma females and males with and without ants and
ants alone. Uncorrected P values in upper triangle, P values corrected for ties (Bonferroni
correction) in lower triangle.
uncorr. P
females
corr. P
females
females +
ants
males
males + ants
ants
< 0.001
0.45
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
females +
ants
< 0.001
males
1.000
< 0.001
males + ants
< 0.001
0.001
0.006
ants
0.002
< 0.001
0.006
< 0.001
< 0.001
Supplement Page 3
Figure S1: Total ion current (TIC) chromatograms on a non-polar GC column (PBX-5) of (a)
synthetic (+)-iridomyrmecin, (b) synthetic (±)-isoiridomyrmecin, (c) the polar fraction of a
dichloromethane (DCM) extract of virgin Leptopilina heterotoma females, and (d) the polar
fraction of a DCM extract of L. heterotoma males.
Supplement Page 4
Figure S2: Total ion current (TIC) chromatograms on a polar GC column (RH-Wax) of (a)
synthetic (+)-iridomyrmecin, (b) synthetic (±)-isoiridomyrmecin, (c) the polar fraction of a
dichloromethane (DCM) extract of virgin Leptopilina heterotoma females, and (d) the polar
fraction of a DCM extract of L. heterotoma males.
Supplement Page 5
Figure S3: Chromatograms on a chiral GC column (Beta-Dex 225) of (a) synthetic
iridomyrmecin and isoiridomyrmecin, (b) the polar fraction of a dichloromethane (DCM)
extract of virgin Leptopilina heterotoma females, (c) the epimerized polar fraction of a DCM
extract of virgin L, heterotoma females, and (d) the polar fraction of a dichloromethane (DCM)
extract of virgin L. heterotoma males. * indicate contaminations originating from the
epimerization procedure.
Supplement Page 6
Figure S4: Mass spectra (electron impact ionization) of (a) (-)-iridomyrmecin and (b) (+)isoiridomyrmecin from dichloromethane extracts of Leptopilina heterotoma females and males,
respectively.
Supplement Page 7
Figure S5: Total ion current (TIC) chromatograms of separate extract of (a) the head, ( b) the
thorax, and (c) the abdomen of Leptopilina heterotoma females (CHCs = cuticular
hydrocarbons).
Supplement Page 8
Synthesis Information:
All NMR data were acquired on a Bruker Avance-300 instrument and chemical shifts
were determined relative to the solvent residue signal. Optical rotations were
measured on an AUTOPOL IV Automatic Polarimeter. Solvents were dried over
molecular sieves using an MBRAUN Auto SPS. IBX was prepared using the
procedure of Frigerio et al. (1999). All other reagents were used as received. The silica
gel for chromatographic purification was ‘Synthetic Silica Gel for Chromatography,
Pharma. Research’ from Silicycle. EI mass spectra of nepetalactol and intermediate 1
were obtained on an Agilent Technologies 5975 VL MSD.
Detailed experimental procedures and complete analytical data for intermediates in
the synthesis of nepetalactol from citronellal have been recently reported by us and
are not duplicated here (Beckett et al., 2010). In order to corroborate that nepetalactol
was indeed the starting material for subsequent steps, a 1H NMR was acquired
(representative spectrum given below) and the LRMS was confirmed (M+, m/z calc
168.1, obsvd 168.1).
Intermediates 1 and 2 represent new compounds to science. For intermediate 1, full
NMR spectra (1H, 13C, COSY, HSQC, NOESY) and a EI mass spectrum are provided
here (see below) to support the structure indicated in the text. As described in the
text, intermediates 2 represent four diastereomers which can only be partially
resolved into two pairs of two diasteroemers (2a and 2b). Because these intermediates
could not be isolated, full annotation of the NMR spectra was not possible. In
addition, solutions 2a and 2b were found to decompose at room temperature over a
few hours and recourse was made to converting them into isoiridomyrmecin and
iridomyrmecin, respectively, immediately following chromatography. As such, the
structures we posit for 2a and 2b should be considered tentative assignments.
However, the structure of their precursor (intermediate 1) and the products of their
oxidation (isoiridomyrmecin and iridomyrmecin) are well substantiated and strongly
suggest, by inference, the structures of 2a and 2b.
Iridomyrmecin and isoiridomyrmecin are well-known compounds and the identities
of the samples generated here were corroborated by comparison to published spectra
(Schöllhorn and Mulzer, 2006; Beckett et al., 2010). Representative 1H NMR spectra
are provided below as are measurements of the specific rotation for the
enantiomerically pure samples produced using the protocol described herein.
Specific rotation measurments for entiomerically pure iridomyrmecin and
isoiridomyrmecin:
Supplement Page 9
(+)-iridomyrmecin: [ ]D25 +106 (CHCl3, c = 0.13)
(-)-isoiridomyrmecin: [ ]D25 -36.5 (CHCl3, c = 0.17)
(-)-iridomyrmecin: [ ]D25 -112 (CHCl3, c = 0.13)
(+)-isoiridomyrmecin: [ ]D25 +34.5 (CHCl3, c = 0.24)
References
BECKETT, J. S., BECKETT, J. D. and HOFFERBERTH, J. E. 2010. A Divergent Approach to the
Diastereoselective Synthesis of Several Ant-Associated Iridoids. Org. Lett. 12:1408–1411.
FRIGERIO, M., SANTAGOSTINO, M. and SPUTORE, S. 1999. A User-Friendly Entry to 2Iodoxybenzoic Acid (IBX). The Journal of Organic Chemistry. J. Org. Chem. 64:4537–4538.
SCHÖLLHORN, B. and MULZER, J. 2006. Stereocontrolled Formation of Three Contiguous
Stereogenic Centers by Free Radical Cyclization – Synthesis of (+)-Iridomyrmecin and (–)Iso-iridomyrmecin – Formal Synthesis of δ-Skythantine. Eur. J. Org. Chem. 2006:901–908.
Supplement Page 10
Supplement Page 11
Supplement Page 12
EI-LRMS (M+ m/z: calc 152.1, obsvd 152.1)
Supplement Page 13
Supplement Page 14
Supplement Page 15