Synthesis of chiloglottones - Semiochemicals from sexually deceptive orchids and their pollinators

Article abstract of DOI:10.1039/b912233h

A five-step synthesis of monoalkyl- and 2,5-dialkyl-1,3-cyclohexanediones (1) is described via a sequence involving sequential Birch reductions and alkylations from the readily accessible and inexpensive starting material, 3,5-dimethoxybenzoic acid. Two approaches were considered in which alkylation at C-2 occurs either prior or subsequent to the proposed reduction. The successful route, in which Birch reduction of a 3-alkyl resorcinol derivative (3) precedes alkylation was applied in the synthesis of chiloglottone 1 (1dc), in 58% overall yield. Chiloglottone 1 is a member of a new class of natural products, representing a known sex pheromone of the thynnine wasp Neozeleboria cryptoides and pollinator attractant in the Australian sexually deceptive orchid genus Chiloglottis. The synthetic homologues were assessed for their biological activity via electroantennographic detection.

Full text of DOI:10.1039/b912233h

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of chiloglottones – semiochemicals from sexually deceptive orchids
and their pollinators†
Jacqueline Poldy,a,b Rod Peakallb and Russell Allan Barrow*a
Received 2nd July 2009, Accepted 31st July 2009
First published as an Advance Article on the web 17th August 2009
DOI: 10.1039/b912233h
A five-step synthesis of monoalkyl- and 2,5-dialkyl-1,3-cyclohexanediones (1) is described via a
sequence involving sequential Birch reductions and alkylations from the readily accessible and
inexpensive starting material, 3,5-dimethoxybenzoic acid. Two approaches were considered in which
alkylation at C-2 occurs either prior or subsequent to the proposed reduction. The successful route, in
which Birch reduction of a 3-alkyl resorcinol derivative (3) precedes alkylation was applied in the
synthesis of chiloglottone 1 (1dc), in 58% overall yield. Chiloglottone 1 is a member of a new class of
natural products, representing a known sex pheromone of the thynnine wasp Neozeleboria cryptoides
and pollinator attractant in the Australian sexually deceptive orchid genus Chiloglottis. The synthetic
homologues were assessed for their biological activity via electroantennographic detection.
Table 1 Labelling for dialkyl-1,3-cyclohexanediones and related alky-
lated compounds
Introduction
Australian Chiloglottis orchids are known to release compounds
R1
H
a
Me
b
Et
c
Pra
d
Bu
e
Pent
f
representing an entirely new class of natural product1 that mimic
the female sex pheromones of their thynnine wasp pollinators.
These chemical signals elicit sexual behavior in the male wasps,
which can lead to copulatory attempts resulting in pollination of
the orchid flower. In one example, chiloglottone 1 (1dc) has been
confirmed from both the female insect Neozeleboria cryptoides
and the flower of C. trapeziformis as the single unique chemical
responsible for attraction of the male wasps.2 In related orchid
taxa, theprevalence of chiloglottones is demonstrated bythe recent
disclosure of biologically active analogues, chiloglottone 2 (1fc)
and chiloglottone 3 (1be),1 which differ in their substitution while
conserving the 2,5-dialkyl-1,3-cyclohexanedione skeleton (Fig. 1;
Table 1). It is predicted that these allomones are identical to the
female released sex pheromone of the respective pollinators, but
this has been difficult to confirm for all but one case,2 as female
R2
H
Me
Et
Pr
Bu
Pent
Hex
a
b
c
d
e
f
aa
ab
ac
ad
ae
af
ba
bb
bc
bd
bea
bf
ca
cb
cc
cd
ce
cf
da
db
dc
dd
de
df
ea
eb
ec
ed
ee
ef
fa
fb
fca
fd
fe
ff
g
ag
bg
cg
dg
eg
fg
a Dialkyl-1,3-cyclohexanedione natural products, and analogues with R1 =
Pr, were prepared as representative targets using the synthetic route.
thynnines spend much of their lives underground emerging only
to mate.3,4
As likely products of fatty acid biosynthetic pathways, it is
anticipated that chiloglottones and related compounds represent a
widespread class of new natural products that will be encountered
across diverse groups of living organisms. To date, the known
chiloglottones 1–3 exhibit a substitution pattern characterized
by even numbers of carbons at the 2-position and odd numbers
of carbons at the 5-position, consistent with biosynthesis from
putative fatty acid precursors.1 Dialkylresorcinols, with an ap-
parent similarity to the chiloglottones, have been reported from
Pseudomonas sp. displaying the same pattern of C-2 even, C-5 odd
substitution,5 resulting from head-to-head condensation of two
fatty-acid derived subunits.6 The biosynthesis of chiloglottones in
Chiloglottis and related orchids is currently under investigation in
our laboratories.
Recently we reported a synthetic route delivering a selection
of mono and disubstituted chiloglottone 1 derivatives employing
a cadmium mediated desymmetrisation as the pivotal step in
a comparatively lengthy nine step synthesis.7 As part of our
ongoing interest in chiloglottone analogues, we report here an
approach that substantially improves upon the efficiency of
synthesis allowing rapid access (5 steps) to variably substituted
Fig. 1 Molecular structures of chiloglottones 1–3.
aResearch School of Chemistry, The Australian National University,
Canberra, ACT, 0200, Australia. E-mail: rab@anu.edu.au; Fax: +61 2 6125
0760; Tel: +61 2 6125 3419
bResearch School of Biology, The Australian National University, Canberra,
ACT, 0200, Australia
† Electronic supplementary information (ESI) available: Experimental
procedures and analytical data for all compounds 2d–f, 3a–f, 4a–f and
1da–dg, along with copies of 1H and 13C NMR spectra for natural products
and new compounds 1da–dg, 1fc, 1be, 4c–e. See DOI: 10.1039/b912233h
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Scheme 1 Synthesis of alkyl resorcinol derivatives 3 from 1,3-dimethoxybenzoic acid.
2,5-dialkyl-1,3-cyclohexanediones, exemplified by the preparation
of chiloglottones 1–3 and homologues 1da–dg (Table 1). This
approach will allow the timely preparation of these unstable
molecules, facilitating identification and field studies that will
elucidate the role of this evolving class of semiochemicals in orchid
pollination.
Results and discussion
Treating 3,5-dimethoxybenzoic acid with linear alkyl halides under
Birch reductive-alkylation conditions readily afforded the diene
acids 2b–f in very good yields (77–99%) (Scheme 1).8,9 As indicated
by 1H and 13C NMR data, these compounds were of high purity
and could generally be used without further purification. Where
purification was necessary or desired the crude solids could
be readily recrystallised from CH2Cl2, delivering clear colorless
crystals.
Oxidative decarboxylation of compounds 2b–f using lead
tetraacetate in benzene, with minor modifications on a reported
procedure,8 returned the 5-alkylresorcinol derivatives 3b–f in
excellent yields (91–99%). These mobile oils could be purified
by Kugelrohr distillation or chromatographed on silica (CH2Cl2
as eluent) as required, but generally gave products suitable for
subsequent reaction.
At this point we considered the aromatic substrates 3 might
represent a potential divergence in the synthesis. Thus we envis-
aged Path A (Scheme 2) would proceed with Birch reduction of 3
to give the 3-alkyl-1,5-dimethoxycyclohexadienes 4, which would
be alkylated to the 3,6-dialkyl-1,5-dimethoxycyclohexadienes 5
and then hydrolyzed under mild acid conditions to the target
cyclohexanediones 1. Path B sees an inversion of reduction and
alkylation steps, with an initial directed ortho metalation to give
the 2,5-dialkyl-1,3-dimethoxybenzenes 6, with the anticipated
Birch reduction on this aromatic material converging at the
1,5-dimethoxycyclohexadienes 5.
In our investigations of Path B, directed ortho metalations
of 3 gave the 2,5-dialkyl-1,3-dimethoxybenzenes 6 under mild
conditions (R1 = Pr, R2 = Me, Et, Pr). However, these 1,2,3,5-
tetrasubstituted aromatics failed to undergo Birch reduction under
a variety of conditions, returning unaltered starting material. The
1H NMR spectra showed no trace of the anticipated product
5 when either t-butyl alcohol or ethanol were employed as the
source of protons in conjunction with lithium or sodium as the
electron source. This finding is corroborated by an absence in the
literature of any accounts of substrates of this nature undergoing
Birch reductions. Indeed, we are aware of only one account of
a resorcinol derivative with substitution at both C-2 and C-5
participating in such a reaction. Here the C-2 group is electron
withdrawing,10 in contrast to the mildly donating nature of our
alkyl substituents.
Scheme 2 Two envisaged pathways to 2,5-dialkyl-1,3-cyclohexanediones
1 from 5-alkyldimethoxy benzenes 3.
Progress via Path A was ultimately successful with Birch
reduction of 3b–f, using a large excess of lithium and t-butyl
alcohol, readily affording the alkyl dienes 4b–f (61–100%). The
chemically inequivalent ring methylene protons of these alkyl
dienes (H-6a and H-6b) appear in the 1H NMR spectra as second
order multiplets centred on dH ª 2.8 ppm. An unusual observation
is the extensive degree of coupling of the methine (H-3) resonance
1
at dH ª 3.0 ppm, displaying an AL3X2MN spin system. The H
NMR spectrum of 4b presents a representative multiplet at dH
=
3.04 ppm (Fig. 2). A series of selective irradiations of the coupled
resonances revealed that the thirteen clean constituent peaks
comprise couplings of 3J = 6.9 Hz (quartet) to the adjacent methyl
group (H-1¢), 3J = 3.3 Hz (triplet) to the olefinic protons (H-2,4),
5
and entirely unanticipated couplings of J = 6.9 Hz (doublet of
doublets, pseudotriplet) to the ring methylenes (H-6a and H-6b).
Similar behaviour was observed for the alkyl dienes 4c–f.
Compounds 4 represent a point of diversification in that a
selection of alkyl groups can be incorporated. The six homologues,
4a–f, can be rapidly diverged to give an expansive array of mono
and dialkyl dienes 5aa–fg (Table 1). NMR data show a distribution
of stereoisomers with regard to the relationship between the
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synthetic materials through GC-EAD, using the known attractant
1dc as a positive control. In addition to chiloglottone 1, two
compounds (1db and 1dd) yielded an antennal response (Fig. 3) in
seven of eight GC-EAD trials, while the C-2 unalkylated material
(1da) and the higher homologues (1de–dg) showed no activity
across a total of 15 experiments.
Fig. 2 The highly coupled resonance from H-3 of 4b (500 MHz, CDCl3)
with J values distinguished through selective irradiation of coupled
resonances.
3- and 6-alkyl substituents. We chose not to attempt to isolate
these, recognising that hydrolysis of the vinyl ethers would remove
the stereochemical differences due to epimerization about C-2.
Finally, acid hydrolysis yielded compounds of 1 as white solids,
with spectroscopic data consistent with those of 2,5-dialkylated-
1,3-cyclohexanediones.7 Acquisition of 1H and 13C NMR spectra
in CD3OD simplifies spectral interpretation by confining the
compounds to their vinylogous acids, rather than the tautomeric
distribution present in less polar solvents such as CDCl3.
To demonstrate the effectiveness of the route, we aimed
to use the synthesis in the preparation of biologically active
compounds, including natural products chiloglottones 1, 2 and
3 (1dc, 1fc and 1be, respectively). Synthesis of chiloglottone 1
commenced from commercially available 3,5-dimethoxybenzoic
acid. Birch reductive alkylation with bromopropane delivered
3,5-dimethoxy-1-propyl-2,5-cyclohexadienecarboxylic acid (2d) in
almost quantitative yield (99%). Spectroscopic and physical data
agreed with previously reported values.9 Decarboxylation returned
the aromatic compound 3d as a clear mobile oil, in excellent
yield (99%). 1,5-Dimethoxy-3-propyl-1,4-cyclohexadiene 4d was
furnished in 88% yield when 3d was treated with lithium in
refluxing liquid ammonia (-33 ◦C), using t-butyl alcohol as the
source of protons. Directed metalation using t-butyllithium at
-78 ◦C and iodoethane as alkylating agent gave 6-ethyl-1,5-
dimethoxy-3-propyl-1,4-cyclohexadiene (5dc) as a stereoisomeric
mixture, which was treated with aqueous 2N HCl in acetone. The
resulting white solid (1dc) (82% over two steps) was characterized
on the basis of its 1H and 13C APT NMR, HR EIMS and melting
point. Chiloglottones 2 and 3 were similarly prepared†.
We prepared a selection of chiloglottone 1 homologues, with
substitution at C-2 ranging from the unalkylated homologue
through to the hexyl-substituted compound (1da–dg). These
variably substituted 2-alkyl-5-propyl-1,3-cyclohexanediones were
assessed for their biological activity via electroantennographic
detection (EAD). Compounds that are electrophysiologically
perceived by an insect antenna are indicated by a response in the
electoantennograph. Male wasps belonging to the Neozeleboria
monticola complex (pollinators of Chiloglottis valida) were cap-
tured by baiting with chiloglottone 1 in Kosciuszko National Park
during December of 2008. The excised antennae were exposed to
Fig. 3 Gas chromatographic analysis of synthetic cyclohexanediones
1da–dg with simultaneous flame ionization detection (FID) and electroan-
tennographic detection (EAD) using antennae from male N. monticola
wasps captured when attracted to chiloglottone 1.
An absence of response does not preclude the possibility of a
compound being active in other thynnine species. Equally, EAD
activity does not in itself confirm that a compound will exhibit a
behavioural function in the field.11 While the substitution pattern
of chiloglottones 1–3 characterized by even numbers of carbons at
the C-2 position and odd numbers of carbons at the C-5 position
suggests a probable biosynthesis from fatty acid precursors,1 our
2,5-dialkyl-1,3-cyclohexanedione array includes alternative possi-
bilities (Table 1), some of which display an EAD response but are
less likely to represent behaviourally active natural products. We
are currently completing this array (Table 1) to fill out the range of
2- and 5-alkyl homologues and explore their activity in related
taxa.
Coupled with the recent, systematic investigation of mass spec-
tral data from 2,5-dialkyl-1,3-cyclohexandiones,1 this synthetic
route will advance the discovery of new natural products in this
class.
Conclusions
We have developed a new and general method for the synthesis
of 2,5-dialkylated-1,3-cyclohexanediones 1 from commercially
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available and inexpensive starting materials, via a sequence of
Birch reduction and alkylation steps. Substitution at C-5 of
compound 1 is imparted through an initial in situ Birch reductive
alkylation of 3,5-dimethoxybenzoic acid (Scheme 1), whereas the
C-2 substituent is delivered through a two-step Birch reduc-
tion/metalation process (Scheme 2, Path A). This methodology
allows an efficient approach to construct an extensive array of
analogues of chiloglottone 1 (1dc), which may be simultaneously
female released sex pheromones of thynnine wasp and allomones
in sexually deceptive Chiloglottis orchids to attract their male
pollinators.
was added Pb(OAc)4 (1.3 equiv.). After 30–40 min, by which
time the mixture had become colorless, H2O (approx. equivolume
to benzene) was added and the mixture filtered under vacuum
through a plug of silica. The aqueous phase was extracted with
Et2O and the combined organic extracts washed (sat. aqueous
NaHCO3 solution), dried (MgSO4) and the solvents removed in
vacuo to give 3 as a pale mobile oil.
1,3-dimethoxy-5-propylbenzene 3d was prepared in 99% yield as
a pale yellow oil: 1H NMR (300 MHz, CDCl3): d 6.35 (2H, d, 4J =
4
2.1, H-4,6), 6.30 (1H, t, J = 2.1, H-2), 3.78 (6H, s, 3,5-OCH3),
3
3
3
2.53 (2H, t, J = 7.5, H-1¢), 1.63 (2H, tq, J = 7.5, J = 7.5, H-
2¢), 1.23 (3H, t, 3J = 7.5, H-3¢); 13C NMR APT NMR (75 MHz,
CDCl3): d 160.6 (C, C-1,3), 145.1 (C, C-5), 106.5 (CH, C-4,6), 97.5
(CH, C-2), 55.2 (CH3, 3,5-OCH3), 38.4 (CH2, C-1¢), 24.3 (CH2,
Experimental
∑
C-2¢), 13.9 (CH3, C-3¢); m/z (EI) 180.1149 [M+ C11H16O2 requires
General experimental methods
180.1150 (D = 0.7 ppm)].
1H and 13C NMR spectra were recorded at 300 MHz and
75 MHz respectively, unless indicated otherwise. When acquired
in deuterated chloroform, 1H NMR spectra are referenced to the
resonance from residual CHCl3 at 7.26 ppm, and 13C NMR spectra
to the central peak in the signal from CDCl3, at 77.0 ppm. In d4-
Representative procedure for synthesis of
1,5-dimethoxy-3-alkyl-1,4-cyclohexadienes 4
To a solution of 3 (1 equiv.) in dry THF (approx. 4.5 mL/mmol)
and tBuOH (approx. 4.5 mL/mmol), NH3 (approx. 10–
15 mL/mmol) was condensed. Lithium (17 equiv.) was added in
portions at -33 ◦C and the solution allowed to warm slowly to
room temperature. NH3 was evaporated under a stream of N2 and
the residue partitioned between Et2O and sat. aqueous NH4Cl
solution. The aqueous was reextracted (Et2O), the combined
organics dried (MgSO4) and concentrated under vacuum to return
the diene 4.
1
methanol H NMR spectra are referenced to the central peak
of the resonance from residual CHD2OD at 3.30 ppm, and 13C
NMR spectra to the central peak in the signal from CD3OD, at
49.0 ppm. Peak assignments were established using APT, HMQC,
HMBC and COSY experiments where assignment was otherwise
ambiguous.
Representative proceedure for preparation of
1,5-dimethoxy-3-propylcyclohexa-1,4-diene 4d was prepared in
88% yield as a pale yellow oil. H NMR (300 MHz, CDCl3): d
3,5-dimethoxy-1-alkyl-2,5-cyclohexadienecarboxylic acids 2
1
Reductive alkylations were performed with adaptations to pub-
lished procedures.8,9 To a solution of 3,5-dimethoxybenzoic acid
(1 equiv.) in dry THF (2 mL/mmol) liquid NH3 (approx.
5 mL/mmol) was condensed. Lithium (2.2 equiv.) was added
in portions at -33 ◦C until a deep blue color persisted. The
appropriate alkyl halide (1.2 equiv.) was added dropwise, causing
an immediate reversion of the color change through orange to
colorless. NH3 was evaporated under a stream of N2 overnight. The
residue was partitioned between Et2O and H2O, the aqueous layer
4.60–4.59 (2H, m, H-2,4), 3.56 (6H, s, 1,5-OCH3), 3.03–2.93 (1H,
m, H-3), 2.77–2.75 (2H, m [apparent d], J = 6.9, H-6), 1.37–1.34
(4H, m, H-1¢ and H-2¢), 0.91 (3H, t, 3J = 7.0, H-3¢); 13C APT NMR
(75 MHz, CDCl3): d 151.6 (C, C-1,5), 96.0 (CH, C-2,4), 54.1 (CH3,
1,5-OCH3), 40.6 (CH2, C-1¢), 35.4 (CH2, C-3), 31.3 (CH2, C-6),
19.4 (CH2, C-2¢), 14.3 (CH3, C-3¢); m/z (Electrospray ionization)
183.1383 [(M+H)+. C11H19O2 requires 183.1385 (D = 1.1 ppm)].
Representative procedure for synthesis of
1,5-dimethoxy-3,6-dialkyl-1,4-cyclohexadienes 5
◦
chilled to 0 C and acidified to pH 3–4 with careful addition of
2N HCl. The aqueous layer was reextracted (EtOAc), the organic
phase washed (H2O), dried (MgSO4) and concentrated in vacuo.
The solid residue was recrystallized from CH2Cl2 to return the
diene acid 2.
3,5-dimethoxy-1-propylcyclohexa-2,5-dienecarboxylic acid 2d
was obtained in 99% yield as a colorless solid. 1H NMR (300 MHz,
CDCl3): d 4.68 (2H, s, H-2,6), 3.60 (6H, s, 3,5-OCH3), 2.75 (2H,
s, H-4), 1.73–1.67 (2H, m, H-1¢¢), 1.31–1.20 (2H, m, H-2¢), 0.90
(3H, t, 3J = 7.2, H-3¢); 13C APT NMR (75 MHz, CDCl3): d 182.8
(C, CO2H), 153.0 (C, C-3,5), 94.8 (CH, C-2,6), 54.4 (CH3, 3,5-
OCH3), 49.9 (C, C-1), 43.4 (CH2, C-1¢), 31.1 (CH2, C-4), 17.6 (CH2,
C-2¢), 14.2 (CH3, C-3¢); m/z (Electrospray ionization) 227.1289
[(M+H)+. C12H19O4 requires 227.1283 (D = 2.8 ppm)].
Alkylations were achieved in a similar manner to previously
reported methods.12 A solution of 4 (1 equiv.) in dry THF
(10 mL/mmol) was cooled to -78 ◦C. tBuLi (1.1 equiv., 1.25 M in
pentane) was added dropwise via syringe. The solution was stirred
for 30 min at -78 ◦C before dropwise addition of the required
alkyl halide (1.6 equiv.). After 10–15 min at -78 ◦C the suspension
was slowly warmed to r.t. and quenched with H2O. The aqueous
residue was extracted with Et2O, the combined organic phases
dried (MgSO4) and the solvents removed in vacuo, returning 5,
which was immediately hydrolyzed to 1, without separation of
diastereomers.
Representative procedure for synthesis of
2,5-dialkyl-1,3-cyclohexanediones 1
Representative procedure for synthesis of
1,3-dimethoxy-5-alkylbenzenes 3
With modifications on a reported method,13 crude 5 (1 equiv.)
was dissolved in acetone (5 mL/mmol) and aq. 2N HCl (3 equiv.)
added. The resulting solution was stirred overnight at r.t. The
Following a published account with minor modifications;8 to a
rapidly stirred solution of 2 (1 equiv.) in benzene (20 mL/mmol)
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acetone was evaporated under reduced pressure and the residue
diluted (H2O), basified (aq. 1N NaOH) and washed with Et2O. The
aqueous layer was reacidified (pH 1–3 aq. 2N HCl) and extracted
with EtOAc. The organic phase was dried (MgSO4) and concen-
trated in vacuo to return 1 as a white solid.
outlet was split 1:1 with one outlet directed over a male wasp’s
antenna prepared according to Mant et al.11 The other outlet
was directed to the FID. EAD signals and FID responses were
recorded simultaneously.
2-ethyl-5-propyl-1,3-cyclohexanedione 1dc (chiloglottone 1) was
synthesized in 82% over two steps. m.p. 124–126 ◦C; IR (neat):
2957, 2928, 2872, br. 2640, 1557, 1383, 1263, 1244, 1105 cm-1; 1H
Acknowledgements
This work was supported by an Australian Research Council
grant to RP (DP0451374) and The Australian National University
(ANU). JP acknowledges the ANU for an Australian Postgraduate
Award.
2
3
NMR (500 MHz, CD3OD): d 2.46 (2H, dd, J = 16.5, J = 4.3,
H-4eq, 6eq), 2.25 (2H, q, 3J = 7.5, H-1¢¢), 2.14 (2H, dd, 2J = 16.5,
3J = 11.3, H-4ax, 6ax), 2.07–2.01 (1H, m, H-5), 1.39–1.34 (4H,
m, H-1¢ and H-2¢), 0.94 (3H, t, 3J = 6.5, H-3¢), 0.90 (3H, t, 3J =
7.5, H-2¢¢); 13C APT NMR (125 MHz, CD3OD): d 176.5 (br, C, C-
1,3), 118.5 (C, C-2), 39.3 (CH2, C-4,6), 38.8 (CH2, C-1¢), 34.4 (CH,
C-5), 20.7 (CH2, C-2¢), 16.0 (CH2, C-1¢¢), 14.4 (CH3, C-3¢), 13.6
Notes and references
1 S. Franke, F. Ibarra, C. M. Schulz, R. Twele, J. Poldy, R. A. Barrow, R.
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and W. Francke, Science, 2003, 302, 437–438.
3 R. Peakall, Funct. Ecol., 1990, 4, 159–167.
∑
(CH3, C-2¢¢); m/z (EI) 182.1307 [M+ C11H18O2 requires 182.1307
(D = 0.1 ppm)], 182 (39%), 167 (5), 154 (6), 139 (17), 125 (42), 111
(30), 97 (100), 84 (35), 69 (39), 55 (78).
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13 E. Piers and J. R. Grierson, J. Org. Chem., 1977, 42, 3755–3757.
Electroantennographic studies
Gas chromatography with electroantennographic detection (GC-
EAD) of the synthetic materials 1da–1dg were performed accord-
ing to previously published procedures.2 Solutions of compounds
1da–1dg were prepared at approximately 0.1 mg/mL. 4–5 mL of
each sample were injected splitless (subsequently opened after
1 min, split ratio 1:1) into a gas chromatograph (HP 5890),
equipped with a BP21 column (30 m ¥ 0.25 mm) and a retention
gap represented by a 5 m deactivated wide-bore fused silica column
mounted between injector and analytical column with helium
serving as carrier gas. The temperature profile commenced at 40 ◦C
(1 min), then was ramped at 10 C min-1 to 230 ◦C, and held
◦
for 10 min. Injector, EAD outlet and flame ionisation detector
(FID) temperatures were at 250 ◦C. At the end of the column, the
4300 | Org. Biomol. Chem., 2009, 7, 4296–4300
This journal is
The Royal Society of Chemistry 2009
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