Communication
doi.org/10.1002/chem.202001356
Chemistry—A European Journal
Table 1. Incorporation of deuterium labelling in sex pheromone collected from female pea aphids, Acyrthosiphon pisum.
Deuterium-labelled compound applied to female A. pisum
Evidence of deuterium incorporation
in nepetalactol 1
Evidence of deuterium incorporation
in nepetalactone 2
[8,8,8,10,10,10-2H6]-labelled compounds
2H6À Geraniol (14a)
×
×
×
×
×
×
×
×
×
×
×
×
*
2H6À Nerol (14b)
*
2H6-(S)-Citronellol (14c)
*
2H6À Geranial (15a)
*
2H6À Neral (15b)
*
2H6-(S)-Citronellal (15c)
*
[8,8,10,10,10-2H5]-labelled compounds
p
p
p
p
2H5-8-Hydroxygeraniol (20a)
*
2H5-8-Hydroxynerol (20b)
*
2H5-(S)-8-Hydroxycitronellol (20c)
×
×
*
p
p
2H5-8-Hydroxygeranial (24a)
*
p
p
2H5-8-Hydroxyneral (24b)
*
2H5-(S)-8-Hydroxycitronellal (24c)
×
×
*
[8,10,10,10-2H4]-labelled compounds
p
p
p
p
2H4-8-Oxogeraniol (17a)
*
2H4-8-Oxonerol (17b)
*
2H4-8-(S)-Oxocitronellol (17c)
×
×
*
p
p
2H4-8-Oxogeranial (18a)
*
p
p
2H4-8-Oxoneral (18b)
*
2H4-(S)-8-Oxocitronellal (18c)
×
×
*
Biosynthesis of nepetalactones from 8-oxogeranial in plants
is thought to be a multi-step process involving reduction and
cyclization to generate an activated non-isolable enol inter-
mediate, followed by oxidation to a corresponding lactone.
Recently, Lichman et al suggested that these steps are
uncoupled and catalysed by different enzymes (ISY, NEPS) with
the enol intermediate diffusing between enzyme active sites.[14]
Our data suggest that female A. pisum aphids are not only able
to incorporate 8-oxogeranial as a substrate for nepetalactone
production, similar to that observed for plants, but are also able
to utilize closely related compounds which only differ in their
oxidative state. Incorporation of neryl-derived substrates in
nepetalactone biosynthesis as described in our work has not
been reported in vivo elsewhere, even in plants although in vitro
activity has been observed,[8] suggesting that further work on
enzyme acceptability of substrates in this biosynthetic pathway
needs to be undertaken, as well as confirmation of the
stereochemistry of the resulting nepetalactol/nepetalactone.
The unacceptability of [2H6]-alcohols and [2H6]-aldehydes sug-
gests that A. pisum is unable to use non-ω-oxygenated
monoterpenoid precursors for sex pheromone biosynthesis.
Given the high degree of deuterium incorporation observed for
probes that are accepted, unacceptability could be due to
either a lack of oxygenase in A. pisum limiting hydroxylation of
the [2H6]-alcohols and [2H6]-aldehydes, inability of compounds
to pass through the haemolymph, or, less likely, aphid depend-
ence on the host plants for availability of the intermediates.
Lichman et al.[14] reported that (S)-8-oxocitronellal is not
incorporated as a substrate for NEPS enzymes, and that high
concentrations of buffer are required for the formation of the
enol intermediate which either undergoes cyclization sponta-
neously or acts as a substrate for NEPS3, a multi-functional
cyclase-dehydrogenase. In our experiments, none of the
citronellyl-based labelled compounds (including the 8-oxo
variants) resulted in production of labelled nepetalactones,
despite the fact that they were performed in vivo, with potential
for the presence of all biosynthetic enzymes utilized by the
insect. This implies that either (i) unlike for plants, nepetalac-
tone biosynthesis in A. pisum occurs via tandem reduction/
cyclization of 8-oxogeranial, as postulated in Figure S2 or (ii) if
nepetalactone biosynthesis occurs via a non-concerted process,
as for plants, then exogenously added 8-oxocitronellal cannot
insert into the pathway, possibly due to the absence of
spontaneous enol formation. Interestingly, Lichman et al.
showed that cyclization of (S)-8-oxocitronellal i.e. via the enol in
high buffer conditions leads to formation of nepetalactols with
relative stereochemistry similar to 3 and 4, which comprises the
sex pheromone for P. humuli. It could be that P. humuli
possesses the enzymology for incorporating citronellyl-derived
compounds in pheromone biosynthesis whereas A. pisum does
not.
Assuming that scenario (ii) above is correct, as A. pisum
does not produce nepetalactol 3, one could reasonably expect
that citronellyl derivatives will not be accepted. Our early work
on sex pheromone identification in aphids detected the
presence of (S)-citronellol in pheromone collections, with the
implication being that it was involved in the biosynthesis of 1
and 2.[16] However, its presence in collections alongside 1 and 2
could also be explained by a lack of enzyme acceptability/
suitable enzyme to catalyse cyclization. Further work is required
to confirm if P. humuli, which utilises 3 and 4 as a sex
pheromone, can incorporate any of the citronellyl derivatives
used in this study in sex pheromone production, and to confirm
the enzymology involved in aphid sex pheromone biosynthesis.
In summary, we provide evidence for the biosynthesis of
(1R,4aS,7S,7aR)-nepetalactol 1 and (4aS,7S,7aR)-nepetalactone 2
Chem. Eur. J. 2021, 27, 7231–7234
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