[18O]-oxygen incorporation reveals novel pathways in spiroacetal biosynthesis by Bactrocera cacuminata and B. cucumis

Article abstract of DOI:10.1021/ja026215l

The origins of the oxygen atoms in 1,7-dioxaspiro[5.5]undecane (1) and hydroxyspiroacetal (2) from Bactrocera cacuminata, and in 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (3) and hydroxyspiroacetal (4) from B. cucumis, have been investigated by incorporati

Full text of DOI:10.1021/ja026215l

Published on Web 06/05/2002
[18O]-Oxygen Incorporation Reveals Novel Pathways in Spiroacetal
Biosynthesis by Bactrocera cacuminata and B. cucumis
Mary T. Fletcher,† Barry J. Wood,† Ian M. Brereton,‡ Jeanette E. Stok,† James J. De Voss,*,† and
William Kitching*,†
Department of Chemistry and Centre for Magnetic Resonance,
The UniVersity of Queensland, Brisbane 4072, Australia
Received March 17, 2002
Although spiroacetals represent a growing and important class
of insect-derived semiochemicals,1,2 the very limited information
on their biosynthesis3-5 hinders development of control strategies
based on suppression of spiroacetal formation. Monooxygenase-
mediated pathways have previously been implicated in the bio-
synthesis of Bactrocera fruit fly spiroacetals,4,5 and in this context
we now reveal disparate patterns of [18O]-oxygen incorporation into
the spiroacetals of two Australian Bactrocera species. These
unanticipated incorporation patterns demonstrated surprisingly
divergent origins for spiroacetals 1-4 and require novel pathways
in their construction.
dioxygen and 18O-labeled water were conducted with B. cucumis.
The major glandular component (>90%) from B. cacuminata is
1,9 along with low levels of the dihydropyran (5), amide (6),
tetrahydropyranol (7), and hydroxyspiroacetal (2) and minor
positional isomers.6,9 In B. cucumis, the major compound in the
released volatiles, and also in the glandular secretion, is spiroacetal
(E,E)-(3), along with its (E,Z) and (Z,Z) diasteromers.10 The
spiroacetals 4, 8, and 9 are also present along with amide (6) and
1,3-nonanediol (10).2,10
A general paradigm devised4,5 for Bactrocera spiroacetal bio-
synthesis involves monooxygenase-mediated hydroxylation of an
intermediate alkyltetrahydropyranol (or a biological equivalent) in
the penultimate step. Figure 1 illustrates the postulated route to
1,7-dioxaspiro[5.5]undecane (1) in B. cacuminata and 2,8-dimethyl-
1,7-dioxaspiro[5.5]undecane (3) in B. cucumis, with further hy-
droxylation yielding the accompanying hydroxyspiroacetals,2,6 2 and
4, respectively. Monooxygenase involvement in these pathways was
investigated by using [18O]-oxygen incorporation from both dioxy-
gen and water.
The presence of amide 6 in each of the species provided an
internal monitor for these experiments. Formation of this amide
by established routes from the corresponding acid would require
the amide carbonyl to be derived ultimately from water. In
agreement with this, amide 6 from flies exposed to 99.8% [18O2]-
dioxygen incorporated 18O at a low level (∼5%) compared with
incorporation into the accompanying spiroacetals 1 (∼45%) or 3
(∼75%). In flies exposed to 20% [18O]-water, a significantly higher
level of [18O] incorporation into 6 was seen (11% incorporation or
>50% uptake of available [18O]). Besides supporting our hypothesis
of the unexceptional origin of 6, these results clearly demonstrate
minimal metabolic mixing of the dioxygen- and water-derived pools
of oxygen atoms in these flies.
Figure 1. Proposed biosynthesis of Bactrocera spiroacetals.
All spiroacetals from both species of flies had significant
incorporation of 18O from [18O2]-dioxygen. In addition, the hydroxyl
moiety of the hydroxyspiroacetals (2 and 4) from both species was
extensively labeled by [18O2]-dioxygen and derived no label from
[18O]-H2O. These observations are consistent with the postulate4,5
that alkyltetrahydropyranols are the penultimate precursors to
spiroacetals in these Dipteran species, with side chain oxidation
by a monooxygenase being followed by cyclization to the spiro-
acetal. Monooxygenation of the parent compound then produces
the observed hydroxy derivatives. e.g. 2 and 4 (Figure 1).
However, the patterns of oxygen labeling in the spiroacetals from
the two fly species were strikingly different. GC-MS comparisons
of spiroacetal (1) generated normally with that produced in an
[18O2]-atmosphere demonstrated that both oxygen atoms of 1
were labeled and hence dioxygen derived. This follows because
the molecular ion of 1 (M+ ) 156) is now displaced to M+ ) 160
and identifiable fragment ions also require the incorporation of two
[18O] atoms. The extremely low level of material with M+ ) 158
confirms the near absence of monolabeled 1. In contrast, the
Specimens of B. cacuminata7 were placed in an atmosphere of
20% 18O2 (99.8 atom % 18O) and 80% N28 with a sugar and water
diet. Headspace volatiles were regularly sampled by the Solid-Phase
Micro-Extraction technique8 (SPME) and 18O-incorporation was
monitored by GC-MS analyses. This sampling demonstrated a
smooth dilution of endogenous metabolites with material generated
in the [18O2] atmosphere. After ca. 5 days, the flies were sacrificed
and the rectal glandular components examined by combined GC-
MS methods that had been employed previously2,9,10 for the
unambiguous identification of all the components. In addition, mass
spectral analysis allowed quantitative assessment of 18O incor-
poration into these molecules and, in many cases, identification of
the site(s) of incorporation. Complementary labeling experiments
were carried out with normal air in the presence of [18O]-water
(20% 18O enrichment). Similar experiments with both [18O2]-
* Corresponding author. E-mail: devoss@chemistry.uq.edu.au.
† Department of Chemistry.
‡ Centre for Magnetic Resonance.
9
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J. AM. CHEM. SOC. 2002, 124, 7666-7667
10.1021/ja026215l CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
The incorporation of two dioxygen-derived oxygen atoms into
both nonane-1,3-diol (10) in B. cucumis and spiroacetal 1 in B.
cacuminata was totally unexpected. Without this knowledge,
precedent would suggest a fatty acid/polyketide origin12 for both 1
and 10 in which one (in 1) or both (in 10) oxygen atoms were
derived from water. The presence of these two C9 metabolites, with
unexpected oxygen incorporation from [18O2]-dioxygen, implies that
insect biosynthesis may utilize unique biosynthetic processes.
Among several possibilities, an oxidative cascade achieving C-C
bond cleavage, perhaps via 1,2-diol creation, to furnish a function-
alized C9 system, appears economical.14,15 Other possibilities such
as oxidative fission of an R-ketoacid are also possible (Figure 2B).
The present results, while confirming the salience of alkytetra-
hydropyranols in spiroacetal formation, demonstrate divergent
biosynthesis of these precursors in two Dipteran species. Ongoing
studies of spiroacetal biosynthesis in other insect orders will define
the generality of monooxygenase activity in the production of such
metabolites.
Figure 2. Incorporation of water (H2b) and dioxygen (O2) into spiroacetals
of (A) B. cucumis and (B) B. cacuminata. The symbol k indicates oxygen
of undetermined origin.
spiroacetals 3, 8, and 9 isolated from B. cucumis incorporated only
one oxygen atom from [18O2]-dioxygen. When 18O-labeled water
was employed, there was no incorporation into 1, but one labeled
oxygen atom appeared in each of 3, 8, and 9. Consistent with the
unexpected results for 1, the dihydropyran (5) from B. cacuminata
was also found to incorporate one 18O-label when produced in an
[18O2]-dioxygen-enriched atmosphere, but no label from [18O]-water.
Strikingly, in 1,3-nonanediol (10) from B. cucumis, both oxygen
atoms derive from [18O2]-dioxygen.
Acknowledgment. The authors are grateful to the Australian
Research Council for support of this work.
Supporting Information Available: Table of 18O incorporations
in both species, mass spectra analysis for [18O2]-oxygen uptake into 1,
3, 8, and 9, and 13C NMR spectra for [18O2]-(1) (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
Evidence for the remarkable double incorporation from [18O2]-
dioxygen into 1 is also provided by 187 MHz 13C NMR measure-
ments of the glandular components of 15 flies, directly extracted
into CDCl3. With the high spectral resolution obtained at this
frequency, 18O-isotope effects (∆ ppm, all to higher field) on shifts
of four of the five different carbon atoms in the molecule (1) were
References
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(3) Reference 2a, pp 798-799, contains a summary of the situation as of
shifts of C2 (0.028 ppm) and (C3 and C5) (0.005 and 0.006 ppm)
are in the normal range for oxygen atoms singly bonded to carbon.11
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These incorporation results reveal not only the generality of
monooxygenase mediation of spiroacetal formation, but also an
unexpected complexity in their biosynthesis. We propose that two
distinct but convergent pathways operate in these Bactrocera
species.
(6) Fletcher, M. T.; Jacobs, M. F.; Kitching, W.; Krohn, S.; Drew, R. A. I.;
Haniotakis, G. E.; Francke, W. Chem. Commun. 1992, 1457.
(7) These specimens were obtained from a culture maintained by the
Department of Primary Industries, Queensland. We are grateful to Dr.
Annice Lloyd for her cooperation in the provision of fruit flies.
(8) Dioxygen (18O2) of >99 atom % 18O was obtained from Isotech Inc.,
Miamisburg, OH. 18O-enriched H2O (20 atom % 18O) was manufactured
by Yeda R & D Co. Ltd, Israel. A Carboxen PDMS fibre (Supelco) was
employed for SPME analysis.
With respect to the components from B. cucumis,2,10 a modified
fatty acid/polyketide construction of the tetrahydropyranol or
equivalent system (Figure 2A) is proposed, resulting in the ether
oxygen being water derived. Such a specialized pathway has been
implicated previously in insect pheromone biosyntheses.12 Mono-
oxygenase-mediated (ω - 1) oxidation would introduce an oxygen
atom from dioxygen, so that, upon dehydrative cyclization, the
singly 18O-labeled 2,8-dimethyl-1,7-dioxaspiro[5.5.]undecane sys-
tem (3) would result. GC-MS analyses confirmed that the [18O2]-
derived label was located only in the ethyl-bearing ring in each of
the (E,E) and (E,Z) isomers of 8, and almost certainly in the seven-
membered ring of 9. We propose that the less favored (ω - 2) or
ω hydroxylation of the side chain of the tetrahydropyranol would
lead to the 2-ethyl-1,6-dioxaspiro[4.5]decanes (8) and 2-methyl-
1,7-dioxaspiro[5.6]dodecane (9), respectively. The co-occurring,
hydroxy derivatives, e.g. 4, would then result from monooxygenase-
mediated oxidation of the initially formed 3. These proposals, and
the predicted oxygenation patterns for the use of 18O-labeled
dioxygen and water, are summarized in Figure 2A and are in
harmony with the experimental outcomes described here.13
(9) Krohn, S.; Fletcher, M. T.; Kitching, W.; Drew, R. A. I.; Moore, C. J.;
Franke, W. J. Chem. Ecol. 1991, 17, 485.
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Biochem. Biophys, 1992, 293, 333.
(13) Experiments with 2H-labeled precursors demonstrate that the dioxygen-
derived oxygen atom is the second of the two oxygen atoms to be installed
in the spiroacetals 8 and 9.
(14) Analogous oxidative cleavage pathways have been proposed for the
monooxygenase-catalysed formation of pregnenolone (Ortiz de Montel-
lano, P. R. Cytochrome P450 Structure, Mechanism and Biochemistry,
2nd ed.; Plenum Press: New York, 1995) and pimelic acid (Stok, J. E.;
De Voss J. J. Arch. Biochem. Biophys. 2000, 384, 351-60).
(15) For a proposal of enantioselective hydroxylation of a fatty acid in
pheromone biosynthesis see: Leal, W. S.; Zarbin, P. H. G.; Wojtasek,
H.; Ferreira, J. T. Eur. J. Biochem. 1999, 259, 175.
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