A Specific Blend of Drakolide and Hydroxymethylpyrazines: An Unusual Pollinator Sexual Attractant Used by the Endangered Orchid Drakaea micrantha

Article abstract of DOI:10.1002/anie.201911636

Bioactive natural products underpin the intriguing pollination strategy used by sexually deceptive orchids. These compounds, which mimic the sex pheromones of the female insect, are emitted in particular blends to lure male insect pollinators of specific species. By combining methods from field biology, analytical chemistry, electrophysiology, crystallography, and organic synthesis, we report that an undescribed β-hydroxylactone, in combination with two specific hydroxymethylpyrazines, act as pollinator attractants in the rare hammer orchid Drakaea micrantha. This discovery represents an unusual case of chemically unrelated compounds being used together as a sexual attractant. Furthermore, this is the first example of the identification of pollinator attractants in an endangered orchid, enabling the use of chemistry in orchid conservation. Our synthetic blend is now available to be used in pollinator surveys to locate suitable sites for plant conservation translocations.

Full text of DOI:10.1002/anie.201911636

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Angewandte
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Accepted Article
Title: A specific blend of drakolide and hydroxymethylpyrazines – an
unusual pollinator sexual attractant used by the endangered
orchid Drakaea micrantha
Authors: Björn Bohman, Monica Tan, Ryan Phillips, Adrian Scaffidi,
Alexandre Sobolev, Stephen Moggach, Gavin Flematti, and
Rod Peakall
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201911636
Angew. Chem. 10.1002/ange.201911636
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10.1002/anie.201911636
Angewandte Chemie International Edition
COMMUNICATION
A specific blend of drakolide and hydroxymethylpyrazines
– an unusual pollinator sexual attractant used by the
endangered orchid Drakaea micrantha
Björn Bohman*[a,b], Monica M. Y. Tan[a], Ryan D. Phillips[b,c,d], Adrian Scaffidi[a], Alexandre N.
Sobolev[a], Stephen A. Moggach[a], Gavin R. Flematti[a] and Rod Peakall[a,b]
Abstract:
hydroxymethylpyrazines in Drakaea,[1e] (methylthio)phenols in
Caladenia[1h] and tetrahydrofuranyl acids in Cryptostylis.[4] Most
systems involve multiple compounds, usually derived from similar
Bioactive natural products underpin the intriguing pollination strategy
used by sexually deceptive orchids. These compounds, which mimic
the sex pheromones of the female insect, are emitted in particular
blends to lure male insect pollinators of specific species. By combining
methods from field biology, analytical chemistry, electrophysiology,
crystallography, and organic synthesis, we report that an undescribed
biosynthetic origins. Only one example of
a
mixed
pheromone/pollinator attractant system originating from distinct
biosynthetic pathways has been reported: the orchid Caladenia
plicata attracts its Zeleboria sp. C thynnine wasp pollinator with a
specific mixture of the terpene citronellol and a likely polyketide-
derived acetophenone.[1h]
β-hydroxylactone,
in
combination
with
two
specific
hydroxymethylpyrazines, act as pollinator attractants in the rare
hammer orchid Drakaea micrantha. This discovery represents an
unusual case of chemically unrelated compounds being used together
as a sexual attractant. Furthermore, this is the first example of the
identification of pollinator attractants in an endangered orchid,
enabling the use of chemistry in orchid conservation. Our synthetic
blend is now available to be used in pollinator surveys to locate
suitable sites for plant conservation translocations.
Drakaea (hammer orchids) is an Australian genus with flowers
characterized by highly reduced tepals and a hinged insectiform
labellum, which are pollinated by sexual deception of thynnine
wasps. Previous studies have shown that hydroxymethylpyrazines
1c]
in D. livida are detected by the wasp pollinators.[1b,
In D.
glyptodon, another set of alkyl- and hydroxymethylpyrazines were
confirmed as pollinator attractants in field bioassays.[1e] Five of the
ten known species of Drakaea are endangered, with one
additional species poorly known and possibly extinct.[5] The rarity
of hammer orchids, in combination with their reliance on specific
species of thynnine wasp pollinators, potentially raises great
conservation challenges.
Pollination by sexual deception is a highly specialized pollination
strategy used by hundreds of plant species, where the flower
typically attracts male insects with chemical compounds that
mimic the female sex pheromone of the pollinator species.[1] Most
examples of this pollination strategy are from orchids, where
sexual deception is known from over 20 genera.[1a] So far, there
has only been a few studies where the compounds involved in
orchid pollination have been elucidated and their biological
function confirmed. The active compounds include n-alkanes and
alkenes, along with hydroxy- and keto acids in Ophrys,[2]
Here, we investigate the pollination chemistry of the rare Drakaea
micrantha, the smallest species (flower size ca. 4 mm x 20 mm),
and the only member of the genus pollinated by a Zeleboria
thynnine wasp. We demonstrate that pollination of D. micrantha
requires
trimethylpyrazine
a
specific
(1),
blend
of
2-hydroxymethyl-3,5,6-
2-hydroxymethyl-3,5-dimethyl-6-
Chiloglottis,[3]
alkyl-
and
ethylpyrazine (2) and the unique β-hydroxylactone 4-hydroxy-3-
methyl-6S-(pentan-2S-yl)-5,6-dihydro-2H-pyran-2-one (3c, here
termed drakolide). Key tools facilitating the identification of the
compounds included gas chromatography-electroantennography
(GC-EAD), where insect antennae were employed as the detector,
and semi-preparative gas chromatography. GC-EAD repeatedly
cyclohexane-1,3-diones
in
[a]
Dr B. Bohman, M. M. Y. Tan, Dr A. Scaffidi, Dr A. N. Sobolev, Dr S.
A. Moggach, Dr G. R. Flematti, Prof. R. Peakall
School of Molecular Sciences
The University of Western Australia
Crawley, WA 6009, Australia
detected two compounds
(Figure 1), identified as
hydroxymethylpyrazines 1 and 2 by synthesis,[6] GC-MS[7] and co-
injection on two GC columns. The most abundant EAD-active
compound 1, has also previously been reported as EAD-active to
the Catocheillus wasp that pollinates some populations of D.
livida.[1b] The homologue 2 (in lower abundance than 1 in D.
micrantha) is identified here for the first time as a natural product,
having already been synthesized as part of a mass spectrometry
study.[7] Compounds 1 and 2, although EAD-active, did not elicit
strong attraction of the Zeleboria pollinator in preliminary field tests
Email: bjorn.bohman@uwa.edu.au
Dr B. Bohman, Dr R. D. Phillips, Prof. R. Peakall
Research School of Biology
Australian National University
Canberra, ACT 2600, Australia
[b]
[c]
[d]
Dr R. D. Phillips
Department of Biodiversity, Conservation and Attractions
Kings Park Science, 1 Kattidj Close
West Perth, WA 6005, Australia
Dr R. D. Phillips
Department of Ecology, Environment and Evolution
La Trobe University Melbourne
Victoria 3086, Australia
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201911636
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COMMUNICATION
(SI, Figure S1). Therefore, to find the missing component(s) of the
semiochemical blend not detected by GC-EAD, bioassay-guided
semi-preparative GC of floral extracts was conducted.
the main compound 3 in the bioactive fraction relied on electron
impact mass spectrometry (EIMS, mass spectrum, Figure 2b), as
NMR was not possible due to the limited floral material available.
High resolution GC-MS (GC-HRMS) revealed a compound with
molecular formula C11H18O3 (m/z: 198.1260, calcd. 198.1256).
The base peak, with formula C6H7O3 (m/z = 127.0393, calcd.
127.0395) indicated 3 double bond equivalents (DBE) and a loss
of a saturated hydrocarbon fragment of C5H11, supporting the
presence of at least one ring in the molecule along with a high
degree of oxygenation.
Since our mass spectrometry data (Figure 2b) did not align with
any compounds related to the semiochemicals previously
identified from Drakaea,[1a] we searched for clues from bioactive
compounds in other pollination systems involving sexual
deception of thynnine wasps. Most similar to the unsaturated
oxygen containing semiochemical in D. micrantha, are the
cyclohexane-1,3-dione (chiloglottone) sex pheromones of
Neozeleboria thynnine wasps,[3] which pollinate Chiloglottis
Figure 1. GC-FID chromatogram (top trace) of the floral extract
(dichloromethane) of Drakaea micrantha. GC-EAD (three replicates, lower
traces) of the antennal responses of the Zeleboria pollinator to the floral extract
of D. micrantha. Hydroxymethylpyrazines 1 and 2 are indicated with arrows.
orchids,
a genus closely related to Drakaea. However,
comparisons of mass fragmentations of various chiloglottones[3]
revealed no obvious similarities to 3. A search of the literature
revealed some synthesised β-hydroxylactones with mass spectra
similar to our unknown 3. In particular, 4-hydroxy-6-pentyl-5,6-
dihydro-2H-pyran-2-one[9] showed several common mass losses
observed at m/z ions that were 14 mass units (i.e. -CH2) lower than
in 3. Ion fragments of m/z 113, 129 and 184, in similar ratios,
matched those of m/z 127, 143 and 198 of our unknown. The base
peaks of m/z 113 and 127, corresponding to a loss of C5H11 from
the molecular ion, were also in good agreement, collectively
guiding us to explore this class of compounds further.
As D. micrantha is an endangered species, we used a protocol to
minimize the number of flowers collected. We first confirmed that
an extract of a single D. micrantha flower in dichloromethane
added to a dressmaker pin head, a method successfully used for
D. glyptodon,[1e] attracted and elicited pseudocopulation by the
pollinator in field experiments. Next, we prepared an extract from
20 flowers (combined from several populations, collected over two
seasons to comply with permit requirements), which was
subjected to semi-preparative GC using a bioassay-guided
fractionation approach. This extract was separated in increasingly
smaller fractions, which were tested together with synthetic
compound 1 (the main EAD-active compound, Scheme 1).
Following the activity in field trials, three active fractions,
containing the same one main peak, remained.
First, we investigated a methyl group in the 3-position of 4-
hydroxy-6-pentyl-5,6-dihydro-2H-pyran-2-one, to yield 4-hydroxy-
3-methyl-6-pentyl-5,6-dihydro-2H-pyran-2-one. After synthesis
and comparison of GC retention data and mass spectra, it was
evident that this compound showed strong mass spectral
similarities to the natural product, although the data were not
identical. Next, all 3-methyl-6-(branched pentyl)-isomers were
subsequently synthesised via condensation reactions of β-oxoacid
esters with aldehydes following a modified procedure from Lokot
et al. (Figure 2a).[10] By evaluating GC-MS data, the natural
product was confirmed as a stereoisomer of 4-hydroxy-3-methyl-
6-(pentan-2-yl)-5,6-dihydro-2H-pyran-2-one, by co-injection on
two GC columns and mass spectral comparisons.
D.micrantha
extract
(Dichloromethane)
0-8
min
8-10
min
10-11
min*
11-12
min
12-13
min
13-14
min
14-16
min
10.00-10.33 10.33-10.66 10.66-11.00 13.00-13.33 13.33-13.66 13.66-14.00
min min min min min min
13.32-13.38 13.38-13.44 13.44-13.50 13.50-13.56 13.56-13.62 13.62-13.68
min min min min min min
Scheme 1. Semi-preparative GC separation of a 20–flower dichloromethane
extract of the flowers of D. micrantha. Fractions in grey boxes elicited pollinator
responses in field experiments. (* = weakly active fraction. No activity when the
fraction was further separated.)
A mixture of the four stereoisomers 3a-3d was prepared from
racemic 2-methylpentanal and ethyl-2-methyl-3-oxobutanoate
(Figure 2a). Field bioassays confirmed that this product, in a
specific blend with the hydroxymethylpyrazines 1 and 2, elicited
pseudo-copulation by the male thyninne wasps. With the use of
chiral-phase GC fitted with a cyclodextrin γ column, we separated
the four stereoisomers 3a - 3d. To determine the absolute
configuration of the natural isomer, an asymmetric synthesis was
conducted, employing enantio-enriched aldehydes in the
condensation reaction, thereby fixing the stereocenter in the
pentan-2-yl side chain.
Aside from the rarity of the plant, the identification of bioactive
compounds was also complicated by the difficulty in locating
females of the pollinator, an undescribed species referred to as
Zeleboria sp. A, currently unknown from museum collections.[5]
Despite extensive searches for pairs in copula, we were unable to
locate any females. Thus, we had no means of comparing floral
attractants and female sex pheromones (which are often identical,
or very similar to the active compounds in sexually deceptive
flowers) to help pinpoint candidate compounds. Identification of
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10.1002/anie.201911636
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Co-injection of the floral extract with the epimer-enriched products,
confirmed that the sidechain configuration was S (Figure 2c).
All stereoisomers from the racemic synthetic mixture were also
separated by chiral-phase HPLC, using a semi-preparative
cellulose dimethylphenyl carbamate (DMP) column (Figure 2d).
The four separated stereoisomers were analysed by chiral-phase
GC and compared with the natural product. The fraction matching
the natural product was the first eluting compound by chiral-phase
HPLC. This and the epimer (eluting last), also with S-side chain
configuration, were collected and recrystallised for X-ray
crystallography studies (Figure 3 and SI, Table S1).
Figure 3: Structure of a single molecule from the crystal structure for drakolide
3c. Colour scheme: O, red; C, grey; H, white. Ellipsoids for C and O are drawn
at 50% probability. H-atoms are drawn as solid spheres.
By combining the results from the analysis of the asymmetrically
synthesised products and the relative configuration obtained by X-
ray crystallography, the natural product was confirmed to be 4-
hydroxy-3-methyl-6S-(pentan-2S-yl)-5,6-dihydro-2H-pyran-2-one
(drakolide, 3c). Under the crystallization conditions used, the enol-
tautomer of 3c was obtained (Figure 3). NMR studies were in
agreement when using deutero-methanol as solvent. However,
when deutero-chloroform was used, the keto-tautomer was
exclusively obtained (SI, Figure S3-S6).
Under suitable conditions, male thynnine wasps respond rapidly
to orchid flowers and synthetic attractants in field bioassays.
Typically, the responses decline within minutes of initial
presentation, but a renewed response is obtained after moving to
other positions in the landscape.[11] To confirm semiochemical
activity, we employed a sequential two-phase bioassay design in
which phase 1 involves a single test blend, whereas phase 2
provides a dual-choice test against a control orchid flower or
known active semiochemical blend.[1a,1e,1h]
Figure 2 (a) Synthesis of 4-hydroxy-3-methyl-6-(pentan-2-yl)-5,6-dihydro-2H-
pyran-2-one stereoisomers 3a - 3d. [10] (b) Electron impact mass spectrum of the
bio-active natural product 3 with fragments for major ions presented. (c) Chiral-
phase GC-MS analysis; synthetically prepared 3a - 3d, co-injection of 3a -3d
We commenced with tests involving blends of one of the two
hydroxymethylpyrazines (1 or 2) with drakolide (as a mixture of
stereoisomers 3a-3d) at a one-to-one ratio (Figure 4a). Blends of
1 or 2 with drakolide lead to frequent lands and attempted
copulation (L+C) by the male Zeleboria wasps in phase 1.
with D. micrantha solvent extract, and D. micrantha solvent extract alone (
unrelated compound). (d) Chiral-phase HPLC chromatogram of 3a - 3d.
=
*
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10.1002/anie.201911636
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(1+2+3) elicited the strongest response of 77% L+C (Figure 4aiii),
compared with 58% each for the other two blends (Figure 4ai - aii,
see also SI Figure 2 for the results at test blends 5:5:10 and 2:0:18
with L+C of 50% and 62%, respectively).
Next, it was tested whether all three components were necessary
for full copulatory behaviour (Figure 4b). These bioassays
confirmed that 1 and 2 in combination were barely attractive, and
never enticed any wasps to land (Figure 4bi). In phase 2, the
control blend (1:1:18) was significantly more attractive than the
pyrazines only (Figure 4bi). Conversely, drakolide alone led to
more than 40% of the total responses in phase 1, but most of these
responses were approaches only, with L+C comprising just 5% of
responses (Figure 4bii). In phase 2, the control blend (1:1:18) was
significantly more attractive than the drakolide alone (Figure 4 bii).
In total, more than 400 wasp visits were recorded to synthetic
blends (Figure 4, SI Figure 1, SI Figure 2, SI Video), with the
overall results showing that neither 1 nor 2 were strongly active on
their own or in combination, and that a strong sexual response
required the combination of 1 and/or 2 with 3.
We report here the first case where pollinator attractants of a
threatened species of orchid have been identified. Locating sites
with high pollinator availability can be critical for the success of
plant conservation translocations, where new populations are
established to reduce the risk of extinction.[12] While sexually
deceived thynnine wasps can be readily surveyed using orchid
flowers as baits,[12,13] our synthetic blend can now be used to
survey for pollinators of the rare D. micrantha without requiring any
picked flowers. Synthetic attractants will also allow study of the
pollinators outside of the orchid flowering season, and with
controlled quantities of attractant. Given that several genera of
sexually deceptive orchids are characterised by a high incidence
of threatened species,[14] this approach may prove broadly
applicable to a large number of species.
The combination of hydroxymethylpyrazines and drakolide is only
the second known example of a mixed sex pheromone/pollinator
attractant system where the compounds likely arise from different
biosynthetic precursors. While hydroxymethylpyrazines 1 and 2
are structurally similar, drakolide is to our knowledge not closely
related to any known natural product. The most similar are the 3-
Figure 4. Outcomes of sequential two-phase bioassays showing the response
of male Zeleboria thynnine wasps to three different blends of synthetic
compounds 1:2:3 in phase 1, with Drakaea micrantha flowers as the control in
the dual-choice phase 2. (ai) Compounds 1 and 3 (10:0:10); (aii) Compounds 2
and 3 (0:10.10) (aiii): Compounds 1, 2, and 3 (1:1:18). Also shown are wasp
responses to 1 and 2 only (bi), or 3 only in phase 1 (bii), with the active blend
(1:1:18) as the control in the dual-choice phase 2. Compound 3 was always
tested as a mixture of stereoisomers 3a-3d. In the left panels, wasp responses
are partitioned into approaches (A, black bars) versus lands only, or lands
followed by attempted copulations (L+C grey bars with % given above the
columns), and shown as the proportion of the total of phase 1 and 2 responses,
averaged across replicate experiments. The right panels show the total counts
across replicate experiments for phase 2, and the G-test outcomes. All ratios are
given in the order of compounds (1:2:3).
acyl-substituted
4-hydroxy-5,6-dihydro-2H-pyran-2-ones
(podoblastins) from the May Apple Podophyllum peltatum[15] and
Serratia plymuthica bacterial cultures,[16] and the 3,6-dialkyl-4-
hydroxy-2-pyrones from various Pseudomonas spp.,[17] both of
which are bioactive antibiotics.
Our discovery highlights the variety of bioactive compounds
involved in pollination among Australian sexually deceptive
orchids. Within two Zeleboria pollinated orchids alone, citronellol
and 2-hydroxy-6-methylacetophenone in C. plicata,[1h] and now
two hydroxymethylpyrazines and 4-hydroxy-3-methyl-6-(pentan-
2-yl)-3,6-dihydro-2H-pyran-2-one in D. micrantha, have been
identified. These discoveries raise some fascinating but
challenging theoretical questions: First, is this cross-kingdom use
of unusual semiochemicals underpinned by convergence at the
biosynthetic and molecular levels? Second, what are the
mechanisms that underpin the evolution of pheromone systems
In phase 2, the (1+3) and (2+3) blends were equally or more
attractive than the D. micrantha flower. Given that the ratios of 1,
2 and 3 varied among floral extracts, we also tested blends with
proportionally less pyrazines, while holding the total amount
constant (Figure 4aiii, SI Figures 1-2). In phase 1, the 1:1:18 blend
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10.1002/anie.201911636
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COMMUNICATION
comprising combinations of biosynthetically unrelated compounds
that are shared among kingdoms? While some common
secondary metabolites are known to be produced by both plants
and insects, examples of the cross-kingdom convergent evolution
of semiochemical use are few.[18] The exceptions include bark
beetles and symbiotic fungi, where both produce bark beetle
aggregation signals,[19] and plant cyanogenic glucoside anti-
feedants sequestered by caterpillars for their own anti-predator
defence[20] or even synthesised de novo by the burnet moth for the
same purpose.[21] Nonetheless, the shared use of the highly
unusual compounds uncovered in the Zeleboria wasp/orchid
systems appears to be unprecedented.[1h]
Acknowledgements
BB, GRF, RDP, and RP acknowledge the Australian Research
Council for funding (DE160101313, DE15010720, LP130100162,
DP150102762, DP16010288). D. Bainbridge is gratefully
acknowledged for designing and fabricating the preparative GC
collector used in this study. The authors acknowledge the facilities,
and the scientific and technical assistance of the Australian
Microscopy & Microanalysis Research Facility at the Centre for
Microscopy, Characterization & Analysis, The University of
Western Australia, a facility funded by the University, State and
Commonwealth Governments.
In summary, for the first time we have identified pollinator
attractants of an endangered orchid. With the use of semi-
preparative GC, GC-EAD, chiral-phase GC, chiral-phase HPLC,
and single crystal X-ray crystallography, we identified the novel
drakolide and two hydroxymethylpyrazines, of which one is a new
natural product, from sub-microgram quantities in floral extracts.
In field bioassays, a blend of these compounds attracted sexually
excited pollinators at a similar level as the orchid flowers, and is
now available for pollinator surveys at candidate conservation
translocation sites for D. micrantha.
Keywords: Sexual deception • drakolide • semiochemicals •
natural products • pollination
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10.1002/anie.201911636
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Björn Bohman*, Monica M.Y. Tan, Ryan
D. Phillips, Adrian Scaffidi, Alexandre
N.Sobolev, Stephen A. Moggach , Gavin
R. Flematti and Rod Peakall
Page No. – Page No.
A specific blend of drakolide and
hydroxymethylpyrazines – an
unusual pollinator sexual attractant
used by the endangered orchid
Drakaea micrantha
Duping Drakolide: The novel natural product Drakolide 3c, together with the two
hydroxymethylpyrazines 1 and 2, act as pollinator attractants in the endangered
sexually deceptive hammer orchid Drakaea micrantha.
This article is protected by copyright. All rights reserved.