Spiroacetal biosynthesis: (±)-1,7-dioxaspiro[5.5]undecane in Bactrocera cacuminata and Bactrocera oleae (olive fruit fly)

Article abstract of DOI:10.1021/ol050143w

A biosynthetic scheme rationalizing the formation of (±)-1,7- dioxaspiro[5.5]undecane (5) in the fruit fly species Bactrocera cacuminata and Bactrocera oleae (olive fruit fly) is presented. Incorporation studies with deuterium-labeled keto aldehyde (10), 1,5-nonanediol (11), and 1,5,9-nonanetriol (12), and our previous finding that both oxygen atoms of 5 originate from dioxygen, are strongly evidentiary. The racemic condition of the natural spiroacetal 5 is accounted for, and inter alia, it is demonstrated that dihydropyran (18) is not an important intermediate en route to 5.

Full text of DOI:10.1021/ol050143w

ORGANIC
LETTERS
2005
Vol. 7, No. 6
1173-1176
Spiroacetal Biosynthesis:
)-1,7-Dioxaspiro[5.5]undecane in
(±
Bactrocera cacuminata and Bactrocera
oleae (Olive Fruit Fly)
Brett D. Schwartz,† Christopher S. P. McErlean,† Mary T. Fletcher,†
Basilis E. Mazomenos,‡ Maria A. Konstantopoulou,‡ William Kitching,† and
James J. De Voss*,†
Department of Chemistry, The UniVersity of Queensland, Brisbane, Australia 4072,
and Institute of Biology, NCSR “D”, 15310 Ag. ParaskeVi Attikis, Greece
j.deVoss@uq.edu.au
Received January 21, 2005
ABSTRACT
A biosynthetic scheme rationalizing the formation of (±)-1,7-dioxaspiro[5.5]undecane (5) in the fruit fly species Bactrocera cacuminata and
Bactrocera oleae (olive fruit fly) is presented. Incorporation studies with deuterium-labeled keto aldehyde (10), 1,5-nonanediol (11), and 1,5,9-
nonanetriol (12), and our previous finding that both oxygen atoms of 5 originate from dioxygen, are strongly evidentiary. The racemic condition
of the natural spiroacetal 5 is accounted for, and inter alia, it is demonstrated that dihydropyran (18) is not an important intermediate en route
to 5.
Spiroacetals are a fascinating class of insect-derived
semiochemicals,1-4 and recently we proposed a general
paradigm for spiroacetal biosynthesis in Bactrocera sp. that
involved monooxygenase-mediated hydroxylation of an
intermediate alkyltetrahydropyranol in the penultimate step.5-7
Specific incorporation of [2H]-labeled alkyltetrahydropyra-
nols was observed, and the patterns of [18O]-oxygen incor-
poration from both dioxygen and water into spiroacetals from
B. oleae, B. cacuminata, and B. cucumis were in harmony
with this proposal.8,9 With respect to B. cacuminata and B.
oleae, both oxygen atoms of the predominant volatile
component, (()-1,7-dioxaspiro[5.5]undecane,9,10 5 originated
† The University of Queensland.
‡ Institute of Biology.
(1) Francke, W.; Kitching, W. Curr. Org. Chem. 2001, 5, 233.
(2) Fletcher, M. T.; Kitching, W. Chem. ReV. 1995, 95, 789.
(3) Tu, Y. Q.; Hu¨bener, A.; Zhang, H.; Moore, C. J.; Fletcher, M. T.;
Hayes, P.; Dettner, K.; Francke, W.; McErlean, C. S.; Kitching, W. Synthesis
2000, 13, 1956.
(4) Hayes, P.; Fletcher, M. T.; Moore, C. J.; Kitching, W. J. Org. Chem.
2001, 66, 2530.
(5) Stok, J. E.; Lang, C.-S.; Schwartz, B. D.; Fletcher, M. T.; Kitching,
W.; De Voss, J. J. Org. Lett. 2001, 3, 397.
(6) Hungerford, N. L.; Mazomenos, B. E.; Konstantopoulou, M. A.;
Krolcos, F. D.; Haniotakis, G. E.; Hu¨bener, A.; Fletcher, M. T.; Moore, C.
J.; Kitching, W. Chem. Commun. 1998, 863.
(7) Hayes, P.; Fletcher, M. T.; Chow S.; McGrath, M. J.; Tu, Y. Q.;
Zhang, H.; Hungerford, N. L.; McErlean, C. S. P.; Stok, J. E.; Moore, C.
J.; De Voss, J. J.; Kitching, W. Chirality 2003, 15, 116.
(8) Fletcher, M. T.; Wood, B. J.; Brereton, I. M.; Stok, J. E.; De Voss,
J. J.; Kitching, W. J. Am. Chem. Soc. 2002, 124, 7666.
(9) Fletcher, M. T.; Mazomenos, B. E.; Georgakopoulos, J. H.; Kon-
stantopoulou, M. A.; Wood, B. J.; De Voss, J. J.; Kitching, W. Chem.
Commun. 2002, 1302.
(10) For a description of the apparatus utilized in these studies, see:
Fletcher, M. T.; Wood, B. J.; Schwartz, B. D.; Rahm, F.; Lambert, L. K.;
Brereton, I.; Moore, C. J.; DeVoss, J. J.; Kitching, W. ArkiVoc 2004, 10,
109.
10.1021/ol050143w CCC: $30.25
© 2005 American Chemical Society
Published on Web 02/16/2005

from [18O]-dioxygen. Likely precursors to this oxygenated
nine-carbon unit were considered to be those incorporating
the oxidatively susceptible O-C-C-O moiety, as present
in 1,2-diols, and R-hydroxy or R-ketoacids. A generalized
pathway accommodating these7-9 observations was devel-
oped and is summarized below (Scheme 1), although we
Scheme 2
Scheme 1
[2H4]-11, this was not the case for the dihydropyran 18, which
retained one or two deuterium atoms, but not three. (One
2H must be lost in the dehydration step to form the
dihydropyran [2H3]-18 from the putative dihydropyranol
[2H4]-16.) This is shown in Scheme 3b. The gas chromato-
graphic trace and the mass spectra make this clear, as shown
in Figure 1. These results demonstrate that no significant
opportunity can be available for H-D exchange on the major
pathway to the spiroacetal from nonane-1,5-diol. In contrast,
emphasized that the order of oxidative events and the level
of oxidation of the oxygenated carbons was not clear.11
Longer chain fatty acids (C14-C18) frequently occur in the
glandular secretions of fruit fly species, and certain observa-
tions led to the view that fatty acids were probably the source
of the nine-carbon unit of 5, as mono-oxygenated nine-carbon
units, including nonanoic acid, 1-nonanol, nonanal, 5-non-
anone, and 5-nonanol, were not incorporated into 5 when
2H-labeled versions were administered to B. cacuminata.12
Consequently, we hypothesized that hydroxylation of a
longer chain fatty acid would furnish an oxidatively suscep-
tible unit (e.g., 1,2-diol) that, after cleavage, would yield a
1,5-dioxygenated nine-carbon unit. This sequence is shown
below (Scheme 2) and is seen to be accommodated, other
than for oxidation levels, within Scheme 1.
Scheme 2 identifies the hydroxyaldehyde (10) as the
initially available nine-carbon unit for transformation to 5.
Consistent with this, [2H4]-10 (see Scheme 3) was very
efficiently incorporated into 5 in B. cacuminata. As reduction
must be achieved if aldehyde 10 is a bona fide intermediate
on the route to 5, [2H4]-nonane-1,5-diol, [2H4]-11, was then
administered and also shown to incorporate very efficiently
into the spiroacetal 5 in both B. cacuminata and B. oleae.
This experiment was informative in other ways, as labeled
dihydropyran (18) was also produced. (Under natural condi-
tions, the dihydropyran 18 and tetrahydropyranol (16) co-
occur in the glandular extract of B. cacuminata with the
spiroacetal 5 and some other minor components.)2
2
the dihydropyran has lost more than one H-atom, and this
requires that it cannot be part of the major pathway to the
spiroacetal.
It was unsurprising that the dihydropyran 18 was not an
intermediate on the pathway to 5, as we have previously
observed that, although it can be processed to the spiroacetal,
it is less readily incorporated than the corresponding tet-
rahydropyranol 16.5 However, as deuterium loss in the
dihydropyran can only occur as part of the dehydration-
hydration (D-H exchange) that interconverts the dihydro-
pyran and the tetrahydropyranol or by prior exchange of
deuterium in the tetrahydropyranol, it follows also that the
tetrahydropyranol is not part of the major pathway to the
spiroacetals (see Scheme 3b).
As we now believe that 1,5-nonanediol 11 is a bona fide
intermediate, two oxidative events are then required prior
to formation of the spiroacetal 5, namely, oxidation at C5
and hydroxylation at C9. The tetrahydropyranol 16, or its
open-chain form, the hydroxyketone (15), both differ in
oxidation level at C-5 compared with 11, and the labeling
results above suggest that these are not intermediates.
However, hydroxylation of the diol 11 at C-9 would
provide 1,5,9-nonanetriol (12). Thus, we synthesized
[2H4]-12 and found that it is very efficiently incorporated
into 5 in both B. cacuminata and B. oleae. This triol, on
secondary alcohol oxidation, would provide ketodiol (13),
which would spontaneously cyclize to spiroacetal 5.
Interestingly, wherever this spiroacetal 5 has occurred
naturally (in B. olea, B. cacuminata, and B. umbrosa), it is
strictly racemic,2 whereas it is known that the enantiomers
(shown in Scheme 3a) are optically stable (at pH ∼7)2 and
Although the spiroacetal 5 retained all four deuter-
ium atoms from the tetradeuterated [2H4]-nonane-1,5-diol,
(11) McErlean, C. S. P.; Fletcher, M. T.; Wood, B. J.; De Voss, J. J.;
Kitching, W. Org. Lett. 2002, 4, 2775.
(12) Hungerford, N. L. Ph.D. Thesis, The University of Queensland,
Brisbane, Australia, 1998.
1174
Org. Lett., Vol. 7, No. 6, 2005

Scheme 3
easily separable by enantioselective gas chromatography.13
In contrast to the racemic nature of the spiroacetal 5, we
now predict that a chiral intermediate 11 is an intermediate
in the biosynthesis. To investigate any enantioselectivity
inherent in the pathway, [2H4]-labeled enantiomers of 1,5-
nonane diol 11 were synthesized as shown in Scheme 4.14,15
Enantioselective gas chromatography of the diols and their
trifluoroacetates using cyclodextrin-based phases established
the ee values to be at least 95%.16 Administration of these
enantiomers to B. cacuminata was conducted in the normal
This finding is consistent with the intermediacy of the achiral
1,5,9-nonanetriol 12, and ketodiol 13. Nonenzymatic dehy-
drative spirocyclization of 13 must afford (()-5. Labeled
1,5-nonanediol and 1,5,9-nonanetriol are also very efficiently
processed by female B. oleae to give a product and
incorporation profile very similar to that of B. cacuminata.
It is likely that the pathway in Scheme 3 is employed also
by the olive fly in generating (()-5.
Figure 1. Gas chromatographic trace showing the formation of [2H]-labeled dihydropyran 18 and labeled spiroacetals 5 after administering
[2H4]-nonane-1,5-diol 10. [2H4]-5 elutes prior to unlabeled 5. EIMS shows that the dihydropyran 18 (M ) 140) consists of predominantly
the [2H1] and [2H2] isotopomers (M ) 141, 142). In contrast, 5 (M ) 156) is overwhelmingly [2H4]-5, (M ) 160).
Org. Lett., Vol. 7, No. 6, 2005
1175

facilitate incorporation of rapidly metabolized polyketide
precursors.18 Labeled hydroxy fatty acids such as 12-
hydroxyhexadecanoic acid (2H at C11, C13) (and its methyl
ester) and 7,8-dihydroxyhexadecanoic acid (2H at C10,C11)
(alone and with the inhibitor, 2-fluorostearic acid)19 were
administered. These two hydroxy-substituted C16 acids on
further hydroxylation and cleavage could provide appropri-
ately oxygenated C9 units. However, once again no incor-
poration was detected. There may be several reasons for this.
Extensive dilution with endogenous fatty acids would occur,
and with the fatty acid at the “front end” of a lengthy
sequence, there could be competition for it from other
processing opportunities. More specific, advanced fatty acid
precursors are currently being investigated in the hope that
they will be directed to spiroacetal formation rather than
general metabolism.
Scheme 4
way, but GCMS showed essentially no discrimination
between the two enantiomers, with both being equally and
efficiently incorporated into 5. It was possible that this
apparent lack of selectivity was the result of strong enzymic
selection of the minor enantiomer present in the unnatural
isomer 11. Thus, we undertook feeding experiments in which
the [2H4]-(S)-11 was mixed with an equimolar amount of
unlabeled [2H4]-(R)-11 and vice versa. In these experi-
ments, enzymic selection would lead to unlabeled 5 when
the [2H4]-11 that was not a precursor was administered to
the flies. However, once again, no discrimination was
observed between the enantiomers of 11. This may not be
indicative of the natural pathway but may simply reflect the
remoteness of the site of hydroxylation (the methyl group)
from the existing C5 stereogenic center to give achiral 12.
Incorporation studies were also conducted in an attempt
to provide support for the proposal that a fatty acid was the
immediate precursor of the hydroxyaldehyde 10. Dietary
Thus, the general paradigm advanced previously (Scheme
1) for spiroacetal biosynthesis appears to be valid, although
the oxidative events for the formation of 5 differ from those
originally proposed.5 Mono-oxygenation of the methyl group
of the C9 precursor preceded C5 oxo formation, and nonane-
1,5-diol rather than the corresponding tetrahydropyranol is
an intermediate in the biosynthesis of 5. Investigation of
spiroacetal biosynthesis in other species is continuing, as are
inhibition studies of the oxidative events central to these
processes.
Acknowledgment. The authors are grateful to the Aus-
tralian Research Council for support and to Dr. Annice Lloyd
(DPIF, QLD) for B. cacuminata specimens.
administration of H-labeled octadecanoic acid (2H at C9,
2
C10, C12, C13) and hexadecanoic acid (2H at C12, C13) and
their methyl esters led to no discernible 2H-incorporation into
5 in B. cacuminata. The coadministration of the â-oxidation
inhibitor 3-tetradecylthiopropionic acid17 did not change this,
although such compounds have previously been reported to
Supporting Information Available: Precursor adminis-
tration methods; synthesis and characterization of [4,4,6,6-
2H4]-10, [4,4,6,6-2H4]-11, (R)-11, (S)-11, (S)-[2,2,3,3-2H4]-
11, (R)-[2,2,3,3-2H4]-11, and [4,4,6,6-2H4]-12. This material
is available free of charge via the Internet at http://pubs.acs.org.
(13) Ko¨nig, W. A. Gas Chromatographic Enantiomer Separation with
Modified Cyclodextrins; Huthig: Heidelberg, 1992; p 76.
(14) Procedures for reduction of propargylic and homopropargylic
alcohols with deuterium gas, to avoid scrambling and also possible
racemization and formation of side-products, will be discussed elsewhere
(Schwartz, B.; Hayes, P.; Kitching, W.; De Voss, J. J. J. Org. Chem. In
press, jo0477547).
(15) Paddon-Jones, G. C.; McErlean, C. S. P.; Hayes, P.; Moore, C. J.;
Ko¨nig, W.; Kitching, W. J. Org. Chem. 2001, 66, 7487.
OL050143W
(17) Hovik, R.; Osmundsen, H.; Berge, R.; Aarsland, A.; Bergseth, S.;
Bremer, J. Biochem. J. 1990, 270, 167.
(18) Li, Z.; Martin, F. M.; Vederas, J. C. J. Am. Chem. Soc. 1992, 114,
1531.
(16) We are grateful to Dr. Melanie Junge and Prof. Dr. W. Ko¨nig for
the ee determinations of the H-labeled 1,5-nonanediols.
(19) Plettner, E.; Slessor, K. N.; Winston, M. L.; Oliver, J. E. Science
1996, 271, 1851.
2
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Org. Lett., Vol. 7, No. 6, 2005