revealed an electrophysiological response to a single
compound.
esterification (DCC, DMAP, and acetic acid) was con-
ducted on an aliquot of the female wasp head extract,
analysis of the reaction mixture showed the disappearance
of the EAD active compound from the sample and the
appearance of a hitherto unseen compound, with a peak
producing an EI-MS spectrum consistent with the forma-
tion of an acetate. These results provided evidence for the
presence of a hydroxyl group within the EAD active
compound.
After synthetic preparation of several compounds
matching these criteria (i.e., trisubstituted pyrazines with
primary or secondary hydroxyl groups including all six
isomers of hydroxymethyl-(3-methylbutyl)-methylpyrazine))
and comparison of the retention times and fragmentation
patterns, the natural product was tentatively identified as
the new molecule 2-hydroxymethyl-3-(3-methylbutyl)-5-
methylpyrazine.
Concurrent analyses showed that the same EAD active
constituent identified from flowers of D. livida was a major
component eluting from the GC in extracts prepared from
the dissected heads of female Z. nigripes. Furthermore,
using SPME fibers, this same compound was detected in
the headspace surrounding females exhibiting courtship
behavior. Electrophysiological activity was confirmed for
both samples of female head extracts and SPME
headspace.
Owing to the small quantity of sample available in the
natural extracts (∼1ꢀ10 ng/flower and wasp), further
identification of the physiologically active compound re-
lied solely on GC-MS analysis. Using extracted ion chro-
matograms, a single peak and mass spectrum representing
the unknown compound was identified. The tailing in the
abundance of larger m/z fragments in the EI-MS made
identification of the molecular ion difficult (Figure 1).
However, utilizing the softer ionization technique avail-
able through chemical ionization (CI-MS) allowed con-
fident assignment of the molecular ion.
The putative compound was prepared from 2,5-di-
methylpyrazine (1) as a starting material in five steps
(Scheme 1). 2,5-Dimethylpyrazine (1) was oxidized with
7
hydrogen peroxide in acetic acid to return the N-oxide 2
8
which was then chlorinated utilizing phosphorus oxy-
chloride in the presence of a catalytic amount of sulfuric
acid to yield 3. Appendage of the 3-methylbutyl side
chain was achieved efficiently by employing the Kumadaꢀ
9
Corriu cross-coupling which provided access to 2,5-
dimethyl-3-(3-methylbutyl)pyrazine (4). The pyrazine 4
was then subjected to a second N-oxidation to form the
N-oxide 5 which was hydroxylated in a Boekelheide
7
procedure to yield the desired alcohol 6.
Scheme 1. Synthetic Route to 2-Hydroxymethyl-3-(3-
methylbutyl)-5-methylpyrazine
Figure 1. Mass spectrum (70 eV EI) of the electrophysiologically
active compound in Drakaea livida.
þ
CI-HRMSconfirmedthe quasi-molecularion (Mþ H)
of m/z 195.1504 supporting a molecular formula of
C H N O.
1
1
18
2
Careful analysis of lower m/z fragments in conjunction
with database searching supported a pyrazine skeleton.
Consistent with this hypothesis, the even m/z ratio (m/z
In addition to the observed match of the mass spectra,
coinjection of the synthetic product with the natural
sample showed peak enhancement on two separate GC
columns. 1D NOESY NMR experiments were used to
confirm the substitution pattern of the product by irradiating
the aryl proton and observing enhancement of the C5 methyl
proton resonance. This was supported by the reciprocal
experiment. GC-EAD confirmed the electrophysiological
1
38) observed for the daughter ion representing the base
peak of the EI-MS spectrum (Figure 1) was indicative of a
McLafferty type rearrangementcommonly observedinthe
mass spectra of pyrazines possessing three alkyl side chains
6
where one is able to rearrange. An additional clue to the
structure was provided by HRMS data for this fragment,
which indicated a molecular formula of C H N O. A
7
10
2
number of structures were proposed including pyrazinols,
alcohols, and ethers. In an effort to broadly discriminate
between these structural isomers microscale derivatization
experiments were performed. Notably, when a simple
(7) Cheng, X.-C.; Liu, X.-Y.; Xu, W.-F. J. Chem. Res. 2006, 2006,
77.
5
(
(
8) Karmas, G.; Spoerri, P. E. J. Am. Chem. Soc. 1952, 74, 1580.
9) Ohta, A.; Masano, S.; Iwakura, S.; Tamura, A.; Watahiki, H.;
(
6) Brophy, J. J.; Cavill, G. W. K. Heterocycles 1980, 14, 477.
Tsutsui, M.; Akita, Y.; Watanabe, T. J. Heterocycl. Chem. 1982, 19, 465.
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