Synthetic 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acid substrates for C-C hydrolase BphD: Investigation of a general base catalytic mechanism

Article abstract of DOI:10.1039/b410322j

A chemical synthesis of the 2-hydroxy-6-ketohexa-2,4-dienoic acid intermediates on bacterial meta-cleavage pathways has been established, using a Heck coupling strategy. Coupling of ethyl 3-bromo-2-acetoxyacrylate with 1-aryl vinyl ketals or 1-aryl allylic alcohols proceeded in 70-90% yield. Heck coupling with an alkyl vinyl ketal was also successful, allowing the synthesis of an alkyl-substituted ring fission intermediate. The synthetic ring fission intermediates were used to investigate the enzymatic reaction catalysed by C-C hydrolase BphD from Pseudomonas LB400. A reduced substrate analogue 2,6-dihydroxy-6-phenylhexa-2,4-dienoic acid was processed enzymatically to benzaldehyde by C-C hydrolase BphD, consistent with a catalytic mechanism involving general base-catalysed attack of water to give a gem-diol intermediate, and not consistent with a nucleophilic mechanism. A series of para-substituted 2-hydroxy-6-keto-6-phenylhexa-2,4-dienoic acid substrates were assayed against BphD, and the derived Hammett plot (ρ = -0.71) is consistent with a departing carbanion in the transition state for C-C cleavage.

Full text of DOI:10.1039/b410322j

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A R T I C L E
Synthetic 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acid substrates
for C–C hydrolase BphD: investigation of a general base catalytic
mechanism
Damian M. Speare,a Sarah M. Fleming,b Martin N. Beckett,a Jian-Jun Lia and
Timothy D. H. Bugg*a
a
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
E-mail: T.D.Bugg@warwick.ac.uk; Fax: +44 (0)2476 524112; Tel: +44 (0)2476 573018
Syngenta, Jealott’s Hill Research Centre, Bracknell, Berkshire, UK RG42 6ET
b
Received 7th July 2004, Accepted 16th August 2004
First published as an Advance Article on the web 16th September 2004
A chemical synthesis of the 2-hydroxy-6-ketohexa-2,4-dienoic acid intermediates on bacterial meta-cleavage
pathways has been established, using a Heck coupling strategy. Coupling of ethyl 3-bromo-2-acetoxyacrylate with
1-aryl vinyl ketals or 1-aryl allylic alcohols proceeded in 70–90% yield. Heck coupling with an alkyl vinyl ketal was
also successful, allowing the synthesis of an alkyl-substituted ring fission intermediate. The synthetic ring fission
intermediates were used to investigate the enzymatic reaction catalysed by C–C hydrolase BphD from Pseudomonas
LB400. A reduced substrate analogue 2,6-dihydroxy-6-phenylhexa-2,4-dienoic acid was processed enzymatically
to benzaldehyde by C–C hydrolase BphD, consistent with a catalytic mechanism involving general base-catalysed
attack of water to give a gem-diol intermediate, and not consistent with a nucleophilic mechanism. A series of para-
substituted 2-hydroxy-6-keto-6-phenylhexa-2,4-dienoic acid substrates were assayed against BphD, and the derived
Hammett plot (q = −0.71) is consistent with a departing carbanion in the transition state for C–C cleavage.
triazacyclononane, which yields the methyl ester of the meta-
Introduction
cleavage product.3
The bacterial degradation of aromatic compounds by soil bac-
teria such as Pseudomonas and Acinetobacter commonly pro-
ceeds via aerobic pathways, involving the oxidative cleavage of
catechol intermediates.1 Intradiol oxidative cleavage of catechol,
catalysed by non-haem iron(III) dependent catechol 1,2-
dioxygenase, yields cis,cis-muconic acid ring cleavage products;
whereas extradiol oxidative cleavage, catalysed by non-haem
iron(II) dependent catechol 2,3-dioxygenase, yields 2-hydroxy-
muconic acid semialdehyde.1 Extradiol cleavage of 3-substituted
catechols likewise gives 6-substituted 2-hydroxy-6-ketohexa-2,4-
dienoic acids, which have been produced enzymatically, but for
which no synthetic route has been reported (see Fig. 1).
This family of meta-ring fission products are substrates for
a hydrolytic C–C cleavage reaction, catalysed by C–C hydrolases
that belong to the ab-hydrolase family (see Fig. 1).4 Crystallo-
graphic studies on Rhodococcus sp. RHA1 C–C hydrolase
BphD have revealed that this family of enzymes contain a
serine–histidine–aspartate triad at their active site.5 The exis-
tence of a keto-intermediate, formed by the initial keto/enol
tautomerisation of the dienol substrate (see Fig. 2), has been
verified by pre-steady state kinetics and by isotope exchange
studies on hydrolase MhpC from Escherichia coli.6,7 Cleavage
of the C–C bond could proceed either by nucleophilic attack of
the active site serine, followed by C–C fragmentation to give an
acyl enzyme intermediate, or by base-catalysed attack of water
to give a gem-diol intermediate, as shown in Fig. 2. Despite the
presence of the serine triad at the active site of these enzymes,
repeated attempts to trap a covalent acyl enzyme intermediate
using a 14C-labelled substrate yielded very low stoichiometries
(<1%) of enzyme-bound 14C label.8 In contrast, 18O incorpora-
tion experiments have demonstrated 4–6% incorporation of 2
atoms of 18O from H218O in the enzyme-catalysed reaction, and
have shown that MhpC catalyses the exchange of 18O into the
C-4 ketone of a non-cleavable substrate analogue.8 These data
are consistent with the formation of a gem-diol intermediate
in the MhpC-catalysed reaction, arising from a general base
mechanism.
The dienol substrates for these enzymes have previously been
available only by enzymatic2 or biomimetic3 catechol oxidative
cleavage. In this paper we report the investigation of two retro-
synthetic disconnections of this target molecule (see Fig. 3),
and the first chemical synthesis of this family of compounds.
Disconnection of the 5,6-bond (A) requires the c-alkylation of
a silyl dienol ether with an acylium or oxonium ion equivalent,
while disconnection of the 3,4-bond (B) requires the Heck coup-
ling of a bromoenol acetate with an a,b-unsaturated ketone. We
then describe the application of these synthetic compounds to
investigate the catalytic mechanism of Pseudomonas sp. LB400
C–C hydrolase BphD, found on the biphenyl degradative path-
way. A communication describing a part of this work has previ-
ously been published.9
Fig. 1 Bacterial meta-cleavage pathway for degradation of 3-substi-
tuted catechols (R = H, CH3, CH2CH2CO2H, Ph), via extradiol catechol
oxidative cleavage, to give a 6-substituted 2-hydroxy-6-ketohexa-2,4-
dienoic acid, followed by C–C hydrolytic cleavage.
We have previously reported the isolation and chemical
characterisation of 2-hydroxy-6-ketonona-2,4-dienoic acid, the
meta-ring fission product on the phenylpropionate catabolic
pathway of Escherichia coli, produced via enzymatic ring cleav-
age.2 This compound was found to exist in the trans,transoid
conformation, and was observed only as the dienol tautomer.2
We have also recently reported a model reaction for extradiol
oxidative cleavage, involving an iron(II) complex of 1,4,7-
2 9 4 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 4 2 – 2 9 5 0
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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c-alkylation product 4a, in 33% yield, with none of the a-
alkylation product observed. Reaction of 2 with acylium ion
equivalents was found to give none of the desired c-acylation
product, but in two cases the a-acylation product 5, containing
a hydrated ketone (dC 137.5 ppm), was isolated: by reaction of 2
with propionic anhydride in the presence of SnCl4 (34% yield),
or by reaction with propionyl chloride in the presence of FeCl3
(30% yield).
The c-alkylation of 2 in the presence of BF3·OEt2 was
investigated further using several additional acetals of
propionaldehyde (3b–e), which could be subjected to subsequent
deprotection. Only in the case of the diallyl acetal 3b was the
c-alkylation product 4b obtained, in 26% yield, in the presence
of BF3·OEt2. Unfortunately, deprotection of the allyl protecting
group of 4b proved unsuccessful using a range of Pd0, RhI, or IrI
catalysts, giving either no reaction, or decomposition of 4b.
Heck coupling of bromoenol acetate to a,b-unsaturated ketone
equivalents (disconnection B)
The palladium-catalysed Heck coupling of alkenyl halides with
a range of alkene acceptors, including a,b-unsaturated ketones,
has been used to synthesise a wide range of dienes.12 Disconnec-
tion of the diene portion of the target molecule, namely the 3,4
bond, requires the coupling of phenyl vinyl ketone (6) with a
bromoenol acetate (7).
Fig. 2 Nucleophilic and general base mechanisms for C–C hydrolases
BphD (R = Ph) and MhpC (R = CH2CH2CO2H).
Bromoalkene
7
was synthesised from ethyl 3-
bromopyruvate by reaction with acetic anhydride.13 Palladium-
catalysed couplings of 7 with tolyl vinyl ketone 6b were
attempted under a variety of conditions, however the optimum
yield of the coupled product 8b using palladium(II) acetate was
only 10% (see Scheme 2). Therefore, alternative equivalents for
the alkene acceptor were tested. Tolyl vinyl ketone 6b could be
converted into the corresponding ketal 9b upon treatment with
ethylene glycol and pyridinium tosylate,14 in 67% yield. Treat-
ment of bromide 7 with ketal 9b, in the presence of palladium(II)
acetate (0.2 equiv.) and silver(I) carbonate (1 equiv.), in refluxing
THF for 16 h, cleanly gave the coupled ketal 10b in 78% yield.
Ketal 10b was converted quantitatively to the desired ketone 8b
by treatment with pyridinium tosylate in acetone/HCl. 1H NMR
signals for the diene portion of 8b were observed at 7.47, 7.24,
and 7.06 ppm. The Heck coupling reaction appeared to take
place selectively at the terminal b-carbon of alkene 9b, since
none of the regio-isomer arising from attack at the a-carbon
was detected.
Fig. 3 Retrosynthetic disconnections A and B for meta-ring fission
product.
Results
Lewis acid-catalysed coupling of a silyl dienol ether
(disconnection A)
Disconnection A (see Fig. 3) requires the c-alkylation of a
dienol ether substrate by an acylium ion equivalent. The selec-
tive c-alkylation of trimethylsilyl dienol ethers, catalysed by
Lewis acids, has previously been reported by Fleming and Lee.10
Thus, silyl dienol ether 2 was prepared, by reaction of diethyl
oxalate with allyl magnesium bromide at −90 °C in diethyl ether/
tetrahydrofuran (1:1),11 to give ester 1, followed by treatment
with trimethylsilyl bromide and triethylamine in anhydrous
dimethylformamide, in 66% overall yield (see Scheme 1).
Reaction of silyl dienol ether 2 with propionaldehyde diethyl
acetal (3a) in the presence of BF3.OEt2 was found to give the
Scheme 2 Synthetic route based on disconnection B, via Heck diene
coupling. X = H (a), CH3 (b), OCH3 (c). Reaction conditions: a.
Pd(OAc)2 (0.1 equiv.), Ag2CO3, toluene, 80 °C; b. 2 M HCl/H2O. Yields
described in text.
Synthesis of other para-substituted ketals 9a–f directly from
vinyl ketones 6a–f was found to be problematic, therefore
an alternative route was developed, as shown in Scheme 3.
Friedel–Crafts acylation of benzene with 3-chloropropionyl
chloride, in the presence of AlCl3, gave aryl ketone 11a. Ketal
formation with ethylene glycol and pyridinium tosylate,
followed by elimination by treatment with triethylamine, gave
ketal 9a in 79% yield. Ketals 9b (X = CH3) and 9c (X = OCH3)
were also synthesised as shown in Scheme 3, and were tested in
Scheme 1 Synthetic route based on disconnection A, via c-alkylation
of silyl enol ether 2. Acetals 3a–e: 3a, R = Et; 3b, R = allyl; 3c, R = p-
nitrobenzyl; 3d, R = CH2CCl3; 3e, R = COCH3.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 4 2 – 2 9 5 0
2 9 4 3

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7.23 ppm, indicating that 16a exists in the trans,transoid confor-
mation, as found previously for an enzymatically-generated ring
fission intermediate.2 Diesters 8b–f were similarly deprotected
by alkaline hydrolysis, and were stored as their mono-sodium
salts 16b–f.
Scheme 3 Synthetic route for preparation of ketals 9a–c. X = H (a),
CH3 (b), OCH3 (c). Reaction conditions: a. ethylene glycol, pyridinium
tosylate; b. Et3N, 1,2-dichloroethane, reflux, 2 h. Yields described in
text.
BphD-catalysed conversion of para-substituted 2-hydroxy-6-
keto-6-phenylhexa-2,4-dienoic acids
C–C hydrolase enzyme BphD from Pseudomonas sp. LB400 was
overexpressed from E. coli BL21(DE3)[pLysS]/pAIA51 con-
taining the bphD gene,15 and was purified to >90% homogeneity
(specific activity 6.4 units mg−1). Treatment of a solution of 16a
in 50 mM potassium phosphate buffer pH 7.0 with BphD gave a
linear decrease in absorbance at 430 nm, and an appearance of
the 2-hydroxypentadienoic acid product at 270 nm, consistent
with the BphD reaction (see Fig. 4).
the Heck coupling. Reaction of ketals 9a and 9c with bromide 7
proceeded in 78% and 81% yield respectively.
Ketal formation as shown in Scheme 3 was unsuccessful for
aryl ketones bearing electron-withdrawing substituents, hence
the corresponding allylic alcohols 12 were also synthesised
and tested under the Heck reaction conditions. Alcohols 12a
(X = H), 12d (X = Cl), 12e (X = CN) and 12f (X = CF3) were
synthesised by reaction of the para-substituted benzaldehyde
with allyl magnesium bromide, in 90–95% yield. Reaction of
12a with bromide 7, in the presence of palladium(II) acetate
(0.1 equiv.) and silver(I) carbonate (2.5 equiv.), was found to
give the desired alcohol 13a in 87% yield, as shown in Scheme 4.
Alcohol 13a was oxidised to ketone 8a using CrO3/H2SO4 in
91% yield. Heck couplings of alcohols 12d–f with bromide 7
also gave the desired diene as the major product, although in
these cases other alkene by-products were visible in the crude
Fig. 4 Hydrolytic cleavage of substrates 16a–f catalysed by C–C
hydrolase BphD.
1
reaction product by H NMR spectroscopy. Oxidation of the
Theseriesof para-substituted2-hydroxy-6-keto-6-phenylhexa-
2,4-dienoic acids (X = H, CH3, OCH3, Cl, CF3, CN) was used
to investigate of the effect of introducing electron-withdrawing
or electron-donating substituents on the rate of the reaction
catalysed by BphD.15,16 Each of the substrates was processed by
purified BphD, as judged by the disappearance of the substrate
chromophore at 430 nm. In each case, the reaction products
were monitored by HPLC, and the appropiate para-substituted
benzoic acid was detected in each case. The extinction coefficient
for each substrate was calculated by calibration of the para-sub-
stituted benzoic acid by HPLC upon 100% conversion, by com-
parison with stock solutions of authentic materials (see Table 1).
The steady-state parameters for conversion of each substrate
were then measured, using Eadie–Hofstee plots. The Km and kcat
values are shown in Table 2. A plot of log(kcat) versus Hammett
coefficient r, shown in Fig. 5, gives a slope q of −0.71.17 The kcat
parameter was used for this plot (rather than kcat/Km), because
the Km values for BphD are extremely low (<1 lM),16 giving rise
to more scatter in plots involving kcat/Km.
coupled alcohol product to the corresponding ketone, fol-
lowed by column chromatography, gave the desired ketones
8d (X = Cl, 74%), 8e (X = CN, 65%), and 8f (X = CF3, 69%) in
good overall yield.
Scheme 4 Heck coupling of allylic alcohols 12a,d–f with bromoalkene
7. X = H (a), Cl (d), CN (e), CF3 (f). Reaction conditions: a. Pd(OAc)2
(0.1 equiv.), Ag2CO3, toluene, 80 °C; b. CrO3, HCl. Yields described in
text.
Hydrolysis of diester 8a to the corresponding acid was car-
ried out under aqueous alkaline conditions. Treatment of
8a with aqueous sodium hydroxide gave within minutes the
dienolate 14a (kmax 435 nm) arising from acetyl ester cleavage.
Hydrolysis of the ethyl ester proceeded more slowly, but was
complete after 2 h at room temperature, giving the disodium
salt 15a (kmax 420 nm), which was neutralised to pH 7 and
freeze-dried as the monosodium salt 16a, as shown in Scheme
5. The 1H NMR spectrum of 16a showed signals for the diene
portion at 7.07 (d, J = 11 Hz, H-3), 7.47 (dd, J = 15, 11 Hz) and
Fig. 5 Hammett plot of log(kcat) vs. substituent coefficient r for pro-
cessing of substrates 16a–f by C–C hydrolase BphD. Gradient of least
squares fit line q = −0.71 ± 0.1.
Scheme 5 Ester hydrolysis of 8a–f. X = H (a), CH3 (b), OCH3 (c), Cl
(d), CN (e), CF3 (f).
2 9 4 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 4 2 – 2 9 5 0

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Table 1 Conversion of para-substituted BphD substrates 16a–f to
para-substituted benzoic acids by BphD, and calculation of molar
extinction coefficients for 16a–f
Table 2 Kinetic parameters measured for processing of substrates
16a–f by C–C hydrolase BphD
Substrate
Substituent
constant r
Km/lM
vmax/DA
kcat/s−1
Substituent (X)
Retention time of
product/min
Molar extinction
(substituent X)
min−1
coefficient/M−1 cm−1
16a (H)
0.00
−0.14
−0.12
+0.34
+0.71
+0.53
0.19 ± 0.10
0.39 ± 0.2
1.06 ± 0.5
0.13 ± 0.07
0.71 ± 0.3
0.23 ± 0.1
0.0745
0.0996
0.0857
0.0621
0.0149
0.0438
4.31 ± 0.4
5.85 ± 0.5
5.04 ± 0.5
3.65 ± 0.4
0.88 ± 0.1
2.57 ± 0.3
H
3.73
3.12
3.03
4.05
2.70
3.08
26,300
25,900
25,500
26,700
27,100
26,700
16b (CH3)
16c (OCH3)
16d (Cl)
CH3
OCH3
Cl
CN
CF3
16e (CN)
16f (CF3)
Synthesis of 6-ethyl ring fission product using Heck coupling
Attempts to deprotect ethyl 2-acetoxy-6-hydroxy-6-
phenylhexa-2,4-dienoate (13a) using aqueous sodium hydroxide
or potassium carbonate gave none of the desired product 19,
the only characterised reaction product being 2,6-diketo-6-
phenylhexanoic acid (20). The formation of 20 under basic con-
ditions can be rationalised by deprotonation at C-6, followed
by re-protonation at C-4, and tautomerisation to the diketo
tautomer. In order to avoid alkaline conditions, enzymatic
hydrolysis of 13a was attempted. Treatment of 13a with 5 units
of pig liver esterase in 50 mM potassium phosphate buffer pH
7.0, followed by acidification and extraction, gave a product
containing 19 as judged by 1H NMR spectroscopic analysis (dH
7.29, 6.97, 6.72, 6.41, 5.19 ppm), but attempts to purify 19 were
unsuccessful.
Therefore, the enzymatic processing of 19 by C–C hydrolase
BphD was tested by addition of 1 unit of BphD to the PLE-ca-
talysed hydrolysis of 13a to 19. Aliquots of the reaction mixture
were removed during the reaction, the products extracted into
chloroform, and the chloroform extract analysed by GC-MS
for the appearance of the cleavage product, benzaldehyde (see
Fig. 6). A new peak of m/z 106 was observed from incuba-
tions containing BphD, which matched authentic samples of
benzaldehyde. The benzaldehyde peak increased in size with
increasing incubation time, and control incubations lacking
either pig liver esterase or BphD showed no formation of
benzaldehyde by GC-MS. By calibration against authentic
benzaldehyde, the kcat for production of benzaldehyde was cal-
culated to be 0.043 s−1.
Since most aromatic degradation pathways involve alkyl-substi-
tuted catechol intermediates,1 it was of wider interest to examine
whether this synthetic route could be used to prepare an alkyl-
substituted ring fission product.
Heck reaction of bromoalkene 7 with the alkyl-substituted
allylic alcohol 3-hydroxyhex-1-ene under the above reaction
conditions gave none of the desired reaction product. Therefore,
an alkyl-substituted vinyl ketal, 3-(ethylenedioxy)pent-1-ene
(shown in Scheme 6), was prepared using a literature method,18
and was reacted with 7 under the standard Heck reaction condi-
tions. The reaction proceeded successfully to give the coupled
product 17, which was isolated in 42% yield, and was deprot-
ected to give ketone 18 in 80% yield. The identity of the product
was confirmed by alkaline hydrolysis to the corresponding acid,
which showed identical UV/vis spectra to material generated
enzymatically from 3-ethyl catechol (kmax 390 nm), and which
was processed efficiently as a substrate by C–C hydrolase MhpC
from Escherichia coli.8 This synthetic sequence indicates that the
Heck coupling of 7 can be performed successfully using alkyl
vinyl ketals.
Discussion
In this paper we describe the first chemical synthesis of the ring
fission intermediates on bacterial meta-cleavage pathways, using
a Heck coupling strategy. It is noteworthy that the Heck coup-
ling of bromide with the a,b-unsaturated ketone 6 proceeded
in <10% yield, whereas much higher yields (70–90%) were ob-
tained using the ketals 9a–f or the allylic alcohols 12a–f. Higher
regio-selectivity in coupling was observed using the ketals 9a–f,
perhaps due to the steric bulk of the ketal substituent. Coupling
of alkyl-substituted reagents was successful using a ketal alkene
substrate, but not using an allylic alcohol substrate. This syn-
thetic route is therefore amenable to the synthesis of aryl- and
alkyl-containing meta-ring fission intermediates. This route
could also be used to synthesise intermediates found on the
degradation pathways of xenobiotics such as polychlorinated
biphenyls, and could be used to access isotopically labelled
derivatives.
Scheme 6 Heck coupling of an alkyl vinyl ketal. Reaction conditions:
a. Pd(OAc)2 (0.1 equiv.), Ag2CO3, toluene, 80 °C; b. 2 M HCl/H2O;
c. NaOH/H2O. Yields described in text.
Assay of reduced substrate 2,6-dihydroxy-6-phenylhexa-2,4-
dienoic acid
In view of our previous studies on C–C hydrolase MhpC,6–8 it
was of interest to examine whether C–C hydrolase BphD would
accept a reduced substrate containing an alcohol at C-6, in place
of a ketone (see Fig. 6). Enzymatic processing of 19 would be
consistent with a general base catalytic mechanism, but not con-
sistent with a nucleophilic mechanism (see Discussion).
Previous investigations of the catalytic mechanism of the C–C
hydrolase family of enzymes have focussed upon nucleophilic
vs. general base mechanisms of catalysis.6–8 The amino acid
sequence and structure of these enzymes clearly demonstrates
that they are members of the ab-hydrolase family, containing a
serine–histidine–aspartate triad at their active sites. The normal
function of Ser-110 in such a catalytic triad would be to act as a
nucleophile, forming an acyl enzyme intermediate. Surprisingly,
previous studies have failed to implicate such an intermedi-
ate; moreover, evidence has been obtained from 18O isotope
Fig. 6 Hydrolytic cleavage of reduced substrate 19 catalysed by C–C
hydrolase BphD.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 4 2 – 2 9 5 0
2 9 4 5

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exchange experiments, consistent with a gem-diol intermediate
arising from base catalysed attack of water.8
acetates as substrates. The opposite sign of q is further confirma-
tion of a difference in mechanism from the serine hydrolases. For
comparison, q values for base-catalysed and acid-catalysed ester
hydrolysis are +2.55 and −0.57, respectively.17
In this paper we have shown that hydrolase BphD catalyses the
C–C cleavage of reduced substrate 19 to give benzaldehyde, in a
time-dependent fashion. The kcat for benzaldehyde production is
approximately 1% of kcat for turnover of the natural substrate by
BphD. The processing of a reduced substrate is consistent with
a general base mechanism of catalysis, illustrated in Fig. 7, since
the intermediate gem-diol would be replaced with an alcohol
functional group, which is able to undergo retro-aldol cleavage
at a reduced rate. This observation is not, however, consistent
with a nucleophilic mechanism, since the reduced substrate 19
contains no incipient carbonyl group for attack by Ser-110, and
would therefore not be able to access the oxyanion intermedi-
ate required for reaction. The enzyme-catalysed processing of
analogue 19 therefore provides a further piece of experimental
evidence in favour of a general base mechanism for C–C cleav-
age. We note that there may be enantioselectivity (at C-6) in the
PLE-catalysed hydrolysis of 13a to 19, and especially in the
BphD-catalysed processing of 19, but that the chemical instabil-
ity of 19 has precluded an analysis of this detail.
Several experimental data now favour a non-nucleophilic
mechanism for this class of C–C hydrolases: 1) the very low
stoichiometry of trapping of a covalent intermediate in the
MhpC-catalysed reaction; 2) the catalytic activity of MhpC
at pH 4–5; 3) 18O exchange from H218O catalysed by MhpC;
4) processing of a reduced substrate analogue by BphD; 5) ob-
servation of a negative q value for processing of p-substituted
substrates by BphD. The implication is therefore that the role
of the active site serine residue is not to act as a nucleophile in
these C–C hydrolases: a fundamental difference in mechanism
from the serine hydrolases. There are other reports in the lit-
erature that indicate alternative roles for the active site serine
in other ab-hydrolase enzymes. A general base mechanism has
been proposed for hydroxynitrile lyase from Hevea brasiliensis,
based on high resolution crystal structures.21 Aldolase activity
has been reported for a S105A mutant of lipase CALB from
Candida antarctica.22 Structure determination of E. coli MhpC
is currently in progress, and preliminary analysis reveals a longer
distance between catalytic residues His-263 and Ser-110, consis-
tent with the proposed mechanism;23 details will be published in
due course.
Experimental
Materials
Propionaldehyde diallyl acetal (3b) was prepared from
propionaldehyde, allyl alcohol and CaCl2, as described by
Mutterer et al.24 Propionaldehyde di(nitrobenzyl) acetal
(3c) and propionaldehyde di(trichloroethyl) acetal (3d) were
prepared from propionaldehyde, the requisite alcohol, and
toluenesulfonic acid, using the method of Schilling et al.25 1,1-
Diacetoxypropane (3e) was prepared from propionaldehyde,
acetic anhydride, and FeCl3, using the method of Kochbar et al.26
Ethyl 2-acetyloxy-3-bromo-2-propenonate (7) was prepared by
the method of Fryzuk et al.13 3-Chloro-1-phenylpropan-1-ones
(11a–c) were prepared by reaction of 3-chloropropionyl chloride
andaluminiumtrichloridewiththeappropriatemonosubstituted
benzene, and were isolated in 75–82% yield. Phenyl vinyl ketone
(6) was prepared by elimination of 3-chloro-1-phenylpropan-
1-one (11a) in the presence of triethylamine, in 92% yield. 1-
Phenylprop-2-en-1-ols (12a,d–f) were prepared by reaction of
vinyl magnesium bromide with the appropriate p-substituted
benzaldehyde, in tetrahydrofuran, and were isolated in 75–95%
yield. E. coli strain BL21(DE3)[pLysS]/pAIA51 was a gift from
Dr Bernd Hofer (GBF, Braunschweig). All other chemicals and
biochemicals were purchased from Sigma-Aldrich.
Preparation of ethyl 2-ketopent-4-enoate (1)
Fig. 7 Proposed non-nucleophilic mechanism for C–C hydrolase
A solution of diethyl oxalate (3.0 mL, 60 mmol) in anhydrous
tetrahydrofuran/diethyl ether (1:1, 240 mL), under a nitrogen
atmosphere, was cooled to −85 °C. Allyl magnesium bromide
(60 mL of 1.0 M solution in diethyl ether) was added dropwise,
and the reaction was stirred for a further 30 min at −85 °C.
A further aliquot of allyl magnesium bromide (15 mL) was
added, and the reaction stirred for a further 10 min. The reac-
tion was then warmed to −60 °C, and quenched by addition of
1 M H2SO4 (100 mL). The product was extracted into diethyl
ether (2 × 100 mL), and the combined extracts washed with
brine (2 × 100 mL), dried (MgSO4), and evaporated under re-
duced pressure to give the product as a yellow oil (6.53 g, 97%).
Rf 0.37 (85:15 petroleum ether/ethyl acetate); IR (liquid film)
3081(w), 2982(m), 1751(s), 1730(s), 1643(w) cm−1; dH (300 MHz,
CDCl3) 6.77 (1H, dt, J = 17.4, 10.3, 10.3 Hz, H-4), 6.20 (1H, d,
J = 10.3 Hz, H-3), 5.39 (1H, dq, J = 17.4, 2.1 Hz, H-5Z), 5.23
(1H, dd, J = 10.3, 2.1, H-5E), 4.30 (2H, q, J = 7.0 Hz, OCH2CH3),
1.33 (3H, t, J = 7.0 Hz, OCH2CH3) ppm; dC (75 MHz, CDCl3)
BphD, illustrating a dissociative transition state for C–C bond cleavage.
The availability of a synthetic route to this class of compounds
has enabled us to synthesise a range of para-substituted deriva-
tives, in order to carry out a Hammett analysis of the enzyme-
catalysed reaction. A plot of log(kcat) versus Hammett coefficient
r (see Fig. 5) gives a reaction coeffcient q of −0.71, indicating
that the reaction is favoured by electron-donating substituents.
The two larger substituents OCH3 and CN both give relatively
low values of kcat, probably due to steric effects upon substrate
binding. A negaitive value of q is consistent with a mechanism
involving C–C cleavage of a gem-diol intermediate, where the
departing carbon atom (C-5) bears a d− charge (stabilised by the
adjacent a,b-unsaturated ketone), and C-6 bears a d+ charge.
Hammett coefficients of +1.8 and +0.9 have been measured for
serine hydrolases a-chymotrypsin19 and subtilisin,20 which pro-
ceed via a nucleophilic mechanism, using p-substituted phenyl
2 9 4 6
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 4 2 – 2 9 5 0

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165.7, 139.6, 129.9, 120.2, 112.4, 62.4, 13.9 ppm; m/z (EI) 142
dichloromethane (5 mL) was added tin(IV) tetrachloride
(2 drops). The mixture was stirred for 2 h at room temperature,
then water (10 mL) was added. The dichloromethane layer was
washed with water (3 × 10 mL), brine (10 mL), dried (Na2SO4),
and evaporated under reduced pressure. The product was
purified by silica column chromatography, eluting with 85:15
petroleum ether/ethyl acetate, to give 5 as a yellow oil (81 mg,
34%). Rf 0.32 (85:15 petroleum ether/ethyl acetate); IR (liquid
film) 2982(m), 2942(w), 1767(s), 1726(s), 1649(m) cm−1; dH
(300 MHz, CDCl3) 6.91 (1H, d, J = 11.5 Hz, H-3), 6.51 (1H, ddd,
J = 17.5, 11.5, 10.5 Hz, CHCHCH2), 5.63 (1H, ddd, J = 17.5,
1.5, 0.5 Hz, CHCHCH2), 5.47 (1H, ddd, J = 10.5, 1.5, 0.5 Hz,
CHCHCH2), 4.25 (2H, q, J = 7.0 Hz, COOCH2CH3), 2.58
(2H, q, J = 7.5 Hz, H-5), 1.31 (3H, t, J = 7.0 Hz, COOCH2CH3),
1.23 (3H, t, J = 7.5 Hz, H-6) ppm; dC (75 MHz, CDCl3) 172.4,
162.3, 137.5, 128.2, 128.2, 125.6, 61.7, 27.3, 14.3, 9.2 ppm; m/z
(CI) 216 (M − H2O + NH4+, 24%), 199 (M − H2O + H+, 100%);
HRMS calc. ([M − H2O]+, C10H14O4) 198.0892, obs. 198.0887.
(M+, 5%).
Preparation of ethyl 2-(trimethylsiloxy)penta-2,4-dienoate (2)
To a solution of sodium bromide (2.4 g, 27 mmol) in distilled
dimethylformamide (12 mL) was added distilled trimethylsilyl
chloride (5.5 mL, 27 mmol), and the mixture stirred for 20 min
at room temperature, forming a precipitate of sodium chloride.
Ethyl 2-ketopent-4-enoate (1, 3.6 g, 25 mmol) and distilled
triethylamine (3.8 mL, 27 mmol) were added, and the reaction
stirred for 18 h at room temperature. The reaction mixture was
added to pentane (90 mL), and the pentane layer washed with
sat. sodium bicarbonate solution (3 × 40 mL), brine (40 mL),
dried (Na2SO4), and evaporated under reduced pressure to give
a yellow oil (3.62 g, 68%); IR (liquid film) 3087(w), 2979(s),
2957(s), 1720(s), 1629(s) cm−1; dH (300 MHz, CDCl3) 6.64 (2H,
m, H-3 and H-4), 5.44 (1H, dd, J = 16.5, 2.0 Hz, H-5E), 5.26 (1H,
dd, J = 10.0, 2.0 Hz, H-5Z), 4.24 (2H, q, J = 7.5 Hz, OCH2CH3),
1.33 (3H, t, J = 7.5 Hz, OCH2CH3), 0.25 (9H, s, SiMe3) ppm; dC
(75 MHz, CDCl3) 165.1, 140.5, 130.5, 121.2, 120.8, 61.3, 14.4,
0.6 ppm; m/z (EI) 214 (M+, 57%); HRMS calc. (C10H18O3Si)
214.1025, obs. 214.1013.
General procedure for 2-phenyl-2-vinyl-[1,3]dioxolanes (9a–c)
To the ketone (11a–c) (10 mmol), dissolved in toluene (25 mL),
was added ethylene glycol (2 mL, excess) and pyridinium
tosylate (0.1 g, cat.). The solution was then refluxed under Dean–
Stark conditions overnight. The solvent was removed under re-
duced pressure, and diethyl ether (20 mL) added. The solution
was then washed with sat. sodium hydrogen carbonate solution
(20 mL), and brine (20 mL). The organic layer was dried
(MgSO4), and the solvent removed under reduced pressure to
give the crude ketal as a pale yellow oil. The ketal was dissolved
in 1,2-dichloroethane (30 mL), and triethylamine (3 mL) was
added dropwise. The reaction was then refluxed for 2 h before
cooling to room temperature and quenching with water (25 mL).
The aqueous layer was separated, extracted with dichlorometh-
ane (2 × 20 mL) and the organic layers were combined, dried
(MgSO4) and evaporated under reduced pressure. Purification
by silica column chromatography (eluent 9:1 Et2O/petroleum
ether) gave 9 as a dark yellow oil.
Preparation of ethyl 2-keto-6-ethoxyocta-3-enoate (4a)
To a stirred solution of silyl dienol ether 2 (128 mg, 0.6 mmol)
and propionaldehyde diethyl acetal (3a, 0.1 mL, 0.6 mmol) in
anhydrous dichloromethane (5 mL) was added boron trifluoride
etherate (2 drops). The mixture was stirred for 4 h at room
temperature, then water (5 mL) was added. The dichloromethane
layer was washed with water (3 × 5 mL), brine (5 mL), dried
(Na2SO4), and evaporated under reduced pressure. The product
was purified by silica column chromatography, eluting with 9:1
petroleum ether/ethyl acetate, to give 4a as a yellow oil (46 mg,
33%). Rf 0.15 (9:1 petroleum ether/ethyl acetate); IR (liquid
film) 2973(s), 2933(s), 2876(s), 1737(s), 1701(m), 1678(m),
1624(m) cm−1; dH (300 MHz, CDCl3) 7.19 (1H, dt, J = 16.0,
7.5 Hz, H-4), 6.69 (1H, dt, J = 16.0, 1.5 Hz, H-3), 4.33 (2H, q,
J = 7.0 Hz, COOCH2CH3), 3.49 (2H, q, J = 7.0 Hz, OCH2CH3),
3.35 (1H, qui, J = 6.0 Hz, H-6), 2.47 (2H, td, J = 7.5, 1.5 Hz,
H-5), 1.51 (2H, qd, J = 7.5, 2.0 Hz, H-7), 1.36 (3H, t, J = 7.0 Hz,
COOCH2CH3), 1.17 (3H, t, J = 7.0 Hz, OCH2CH3), 0.91 (3H, t,
J = 7.5 Hz, H-8) ppm; dC (75 MHz, CDCl3) 183.2, 162.3, 151.6,
126.8, 79.1, 62.3, 37.5, 27.0, 15.5, 14.0, 9.5 ppm; m/z (CI) 246
(M + NH4+, 6%), 229 (MH+, 5%); HRMS calc. (MH+, C12H21O4)
229.1440, obs. 229.1408.
2-Phenyl-2-vinyl-[1,3]dioxolane (9a, X = H). Yield 0.70 g
(65%). dH (300 MHz, CDCl3) 7.50 (2H, d, 2H, J = 8.0 Hz),
7.34 (3H, m), 6.01 (1H, dd, J = 16.5, 10.5 Hz), 5.33 (1H, dd,
J = 16.5, 1.5 Hz), 5.21 (1H, dd, J = 10.5, 1.5 Hz), 3.99 (4H,
m); dC (75 MHz, CDCl3) 141.5, 138.5, 129.1, 128.5, 126.4,
116.9, 108.0, 65.1; m/z (EI) 176 (M+); HRMS calc. (C11H12O2)
176.0834, obs. 176.0837.
2-p-Tolyl-2-vinyl-[1,3]dioxolane (9b, X = CH3). Yield 0.83 g
(73%). dH (300 MHz, CDCl3) 7.29 (2H, d, J = 8.2 Hz), 7.09 (2H,
d, J = 8.2 Hz), 5.95 (1H, dd, J = 16.5, 10.5 Hz), 5.25 (1H, dd,
J = 16.5, 1.5 Hz), 5.14 (1H, dd, J = 10.5, 1.5 Hz), 3.95 (4H, m),
2.27 (3H, s); dC (75 MHz, CDCl3) 141.5, 138.5, 132.7, 129.3,
128.7, 116.8, 108.1, 65.1, 20.1; m/z (CI) 191 (MH+); HRMS
calc. (C12H15O2) 191.1068 obs. 191.1070.
Preparation of ethyl 6-allyloxy-2-ketoocta-3-enoate (4b)
The same method was used to prepare 4b from silyl dienol
ether 2 and propionaldehyde diallyl acetal (3b). 4b was
isolated as a yellow oil after Kugelrohr distillation, in 26% yield.
Rf 0.18 (85:15 petroleum ether/ethyl acetate); IR (liquid film)
3078(w), 2967(s), 2936(s), 2876(s), 1731(s), 1701(m), 1678(m),
1625(m) cm−1; dH (300 MHz, CDCl3) 7.20 (1H, dt, J = 16.0,
7.5 Hz, H-4), 6.70 (1H, td, J = 16.0, 1.5 Hz, H-3), 5.90 (1H, ddt,
J = 16.5, 10.5, 5.5 Hz, allyl C-2), 5.27 (1H, ddd, J = 16.5, 3.0,
1.5 Hz, allyl C-3), 5.16 (1H, ddd, J = 10.5, 3.0, 1.5 Hz, allyl C-3),
4.35 (2H, q, J = 7.0 Hz, COOCH2CH3), 4.00 (2H, dt, J = 5.5,
1.5 Hz, allyl C-1), 3.44 (1H, qui, J = 6.0 Hz, H-6), 2.51 (2H,
ddd, J = 7.5, 6.0, 1.5 Hz, H-5), 1.55 (2H, m, H-7), 1.38 (3H, t,
J = 7.0 Hz, COOCH2CH3), 0.92 (3H, t, J = 7.5 Hz, H-8) ppm; dC
(75 MHz, CDCl3) 183.3, 162.4, 151.6, 135.0, 127.0, 117.1, 78.8,
70.4, 62.5, 37.5, 27.0, 14.2, 9.6 ppm; m/z (EI) 240 (M+, 1%), 167
(M − CO2Et, 5%); HRMS (CI) calc. (MH+, C13H21O4) 241.1440,
obs. 241.1441.
2-p-Methoxyphenyl-2-vinyl-[1,3]dioxolane (9c, X = OCH3).
Yield 0.84 g (68%). dH (300 MHz, CDCl3) 7.38 (2H, d,
J = 8.0 Hz), 6.83 (2H, d, J = 8.0 Hz), 6.01 (1H, dd, J = 16.0,
11.0 Hz), 5.32 (1H, dd, J = 16.0, 1.5 Hz), 5.23 (1H, dd, J = 11.0,
1.5 Hz), 3.99 (4H, m), 3.78 (3H, s); dC (75 MHz, CDCl3) 162.8,
138.7, 138.5, 129.7, 116.8, 113.5, 108.0, 65.2, 64.0; m/z (EI) 206
(M+); HRMS calc. (C12H14O3) 206.0939, obs. 206.0943.
General procedures for ethyl 2-acetoxy-6-oxo-6-phenylhexa-2,4-
dienoates (8a–f)
Method A (for 8a–c, by Heck reaction of vinyl ketal 9)
Preparation of ethyl 2,2-dihydroxy-3-vinyl-4-ketohexanoate (5)
To ethyl 2-acetoxy-3-bromo-2-propenonate (7) (50 mg,
0.25 mmol) under nitrogen was added dropwise a solution
containing palladium acetate (10 mg, 0.2 equiv.) and silver(I)
To a stirred solution of silyl dienol ether 2 (242 mg, 1.13 mmol)
and propionic anhydride (0.15 mL, 1.13 mmol) in anhydrous
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 4 2 – 2 9 5 0
2 9 4 7

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carbonate (78 mg, 1 equiv.) in freshly distilled THF (10 mL). To
this was then added the vinyl ketal (9a–c) (0.2 mmol) in THF
(5 mL). The reaction was stirred at room temperature for 1 h, be-
fore refluxing overnight. The solution was then cooled to room
temperature, the solution diluted with diethyl ether and filtered
through Celite. The organic layer was washed with brine (20 mL)
and the products extracted into ethyl acetate (3 × 20 mL). The
combined extracts were dried (MgSO4), and concentrated under
reduced pressure, to give 10 as a dark orange oil. Data for 10a: dH
(300 MHz, CDCl3) 7.49 (2H, dd, J = 8.5, 1.3 Hz), 7.35 (3H, m),
6.95 (1H, d, J = 11.0 Hz, H-5), 6.54 (1H, dd, J = 16.5, 11.0 Hz,
H-4), 6.20 (1H, d, J = 16.5 Hz, H-3), 4.22 (2H, q, J = 8.2 Hz),
4.01 (4H, m), 2.26 (3H, s), 1.28 (3H, t, J = 8.2 Hz); m/z (EI)
332 (M+); HRMS calc. (C18H20O6) 332.1254, obs. 332.1260.
To the ketal (10a–c) (0.1 mmol) in acetone (5 mL) was added
pyridinium tosylate (0.01 g, cat) and conc. sulfuric acid (0.1 mL)
and the reaction stirred vigorously for 1 h. The reaction was
quenched by the addition of water (15 mL). The aqueous layer
was separated, extracted with diethyl ether (2 × 5 mL) and the
organic layers combined, further washed with water (2 × 15 mL),
dried (MgSO4) and the solvent removed under reduced pres-
sure. Purification by silica column chromatography (eluent 20%
EtOAc/petroleum ether) gave 8a–c as dark orange oils.
129.3, 128.7, 125.0, 61.4, 20.3, 19.8, 14.0; m/z (CI) 303 (MH+);
HRMS calc. (C17H19O5) 303.1227, obs. 303.1233.
Ethyl 2-acetoxy-6-oxo-6-(p-methoxyphenyl)hexa-2,4-dienoate
(8c, X = OCH3). Yield 26.6 mg (68%, method A). dH (300 MHz,
CDCl3) 7.98 (2H, d, J = 7.2 Hz), 7.56 (1H, dd, J = 15.2,
11.5 Hz, H-4), 7.31 (2H, d, J = 15.2 Hz, H-5), 7.28 (1H, d,
J = 11.5 Hz, H-3), 7.02 (2H, d, J = 7.2 Hz), 4.21 (2H, q, 2H,
J = 7.2 Hz), 3.84 (3H, s), 2.29 (3H, s), 1.28 (3H, t, J = 7.2 Hz); dC
(75 MHz, CDCl3) 189.4, 168.4, 162.9, 160.9, 142.4, 137.6, 132.0,
130.7, 129.7, 124.8, 107.8, 65.2, 61.5, 20.2, 14.0; m/z (EI) 318
(M+); HRMS calc. (C17H18O6) 318.1098, obs. 318.1105.
Ethyl 2-acetoxy-6-oxo-6-(p-chlorophenyl)hexa-2,4-dienoate
(8d, X = Cl). Yield 29.2 mg (74%, method B). dH (300 MHz,
CDCl3) 7.68 (2H, d, J = 7.2 Hz), 7.52 (3H, m), 7.28 (2H, d,
J = 15.1 Hz, H-5), 7.14 (1H, d, J = 11.5 Hz, H-3), 4.20 (2H, q,
J = 7.2 Hz), 2.28 (3H, s), 1.27 (3H, t, J = 7.2 Hz); dC (75 MHz,
CDCl3) 189.3, 168.5, 161.3, 143.6, 142.5, 135.8, 133.8, 130.6,
129.7, 128.9, 125.0, 61.5, 20.2, 14.0; m/z (EI) 322 (M+); HRMS
calc. (C16H15ClO5) 322.0603, obs. 322.0621.
Ethyl2-acetoxy-6-oxo-6-(p-cyanophenyl)hexa-2,4-dienoate(8e,
X = CN). Yield 25.6 mg (65%, method B). dH (300 MHz, CDCl3)
7.98 (2H, d, J = 7.2 Hz), 7.64 (1H, dd, J = 15.0, 11.5 Hz, H-4),
7.53 (2H, d, J = 7.2 Hz), 7.36 (1H, d, J = 15.0 Hz, H-5), 7.23
(1H, d, J = 11.5 Hz, H-3), 4.22 (2H, q, J = 7.2 Hz), 2.26 (3H, s),
1.28 (3H, t, J = 7.2 Hz); dC (75 MHz, CDCl3) 189.5, 168.6, 161.4,
143.5, 142.3, 136.5, 132.2, 130.5, 129.4, 128.5, 117.1, 61.5, 20.2,
14.0; m/z (EI) 313 (M+); HRMS calc. (C17H15NO5) 313.0946,
obs. 313.0972.
Method B (for 8a,d–f, by Heck reaction of allylic alcohol 12)
Ethyl 2-acetoxy-3-bromo-2-propenonate (7) (50 mg, 0.25 mmol)
was dissolved in THF (5 mL) to which was added palladium
acetate (22 mg, 0.06 mmol), allylic alcohol (12) (0.2 mmol)
and silver(I) carbonate (276 mg, 1 mmol). The reaction was
then stirred for 1 h at room temperature and then at reflux
overnight. The reaction was then diluted with ether (10 mL)
and filtered through a Celite pad. The products were washed
with water (2 × 20 mL) and dried over MgSO4. The solvent
was removed under reduced pressure to give ethyl 2-acetoxy-6-
hydroxy-6-phenylhexa-2,4-dienoate 13 as a dark yellow/orange
oil. Data for 13a: dH (300 MHz, CDCl3) 7.32 (5H, s), 6.95 (1H,
d, J = 11.1 Hz, H-3), 6.53 (1H, ddd, J = 15.3, 11.3, 1.5 Hz, H-
4), 6.22 (1H, dd, J = 15.2, 5.6 Hz, H-5), 5.27 (1H, dd, J = 5.5,
1.5 Hz, H-6), 4.21 (2H, q, J = 7.1 Hz), 2.25 (3H, s), 1.27 (3H,
t, J = 7.1 Hz); dC (75 MHz, CDCl3) 168.8, 161.9, 143.6, 141.5,
136.9, 128.5, 127.9, 127.3, 126.3, 120.9, 74.0, 61.4, 20.1, 13.9; m/z
(EI) 290 (M+); HRMS calc. (C16H18O5) 290.1150, obs. 290.1155.
To the alcohol (13a,d–f) (0.1 mmol) dissolved in diethyl ether/
acetone (1:1, 5 mL) was added water (5 mL) then chromium
trioxide (0.2 g, excess) and conc. sulfuric acid (0.1 mL). The re-
action was then stirred at room temperature for 1 h. The reaction
was then extracted into ether (20 mL) and washed with brine
(2 × 20 mL). The organic layers were combined, dried (MgSO4),
and concentrated under reduced pressure. Purification by silica
column chromatography (eluent 20% EtOAc/petroleum ether)
gave 8a,d–f as dark orange oils.
Ethyl
2-acetoxy-6-oxo-6-(p-trifluoromethylphenyl)hexa-
2,4-dienoate (8f, X = CF3). Yield 30.6 mg (69%, method B).
dH (300 MHz, CDCl3) 8.12 (2H, d, J = 7.2 Hz), 7.61 (2H, d,
J = 7.2 Hz), 7.41 (1H, dd, J = 15.0, 11.5 Hz, H-4), 7.31 (1H, d,
J = 15.0 Hz, H-5), 7.08 (1H, d, J = 11.5 Hz, H-3), 4.22 (2H, q,
J = 7.2 Hz), 2.25 (3H, s), 1.26 (3H, t, J = 7.2 Hz); dC (75 MHz,
CDCl3) 189.5, 168.6, 161.3, 142.5, 137.2, 135.8, 132.5, 130.5,
129.5, 129.3, 128.7, 119.3, 61.5, 20.3, 14.0; m/z (EI) 356 (M+);
HRMS calc. (C17H15F3O5) 356.0867, obs. 356.0874.
Ethyl 2-acetoxy-6-oxoocta-2,4-dienoate (18). This was
prepared from 3-ethylenedioxypent-1-ene18 using method
A, in 34% overall yield. dH (300 MHz, CDCl3) 7.28 (1H, dd,
J = 15.0, 13.5 Hz, H-4), 7.02 (1H, d, J = 13.8 Hz, H-3), 6.48
(1H, d, J = 15.4 Hz, H-5), 4.31 (2H, q, J = 7.2 Hz), 2.66 (2H, t,
J = 7.2 Hz), 2.35 (3H, s), 1.35 (3H, t, J = 7.2 Hz), 1.15 (3H, t,
J = 7.2 Hz); dC (75 MHz, CDCl3) 142.1, 134.4, 131.8, 124.7, 61.6,
30.0, 20.2, 13.9, 7.6 (carbonyls not seen); m/z (CI) 214 (MH+);
HRMS calc. (C12H17O5) 214.1076, obs. 214.1074.
Ethyl 2-acetoxy-6-oxo-6-phenylhexa-2,4-dienoate (8a, X = H).
Yield 26.8 mg (73%, method A). dH (300 MHz, CDCl3) 7.97 (2H,
d, J = 7.2 Hz, Ar ortho), 7.62 (1H, t, J = 7.2 Hz, Ar para), 7.56
(1H,dd, J = 15.2,11.5Hz,H-4),7.49(2H,t, J = 7.2Hz,Armeta),
7.30 (1H, d, J = 15.2 Hz, H-5), 7.14 (1H, d, J = 11.5 Hz, H-3),
4.20 (2H, q, J = 7.2 Hz), 2.25 (3H, s), 1.23 (3H, t, J = 7.2 Hz); dC
(75 MHz, CDCl3) 189.2, 168.4, 161.2, 142.5, 137.1, 134.2, 133.2,
130.8, 128.6, 128.3, 124.9, 61.4, 20.1, 13.9; m/z (CI) 289 (MH+);
HRMS calc. (C16H17O5) 289.1071, obs. 289.1078.
2-Hydroxy-6-oxo-6-arylhexa-2,4-dienoic acids (16a–f)
To the ethyl 2-acetoxy-6-oxo-6-(p-substituted phenyl)hexa-2,4-
dienoate (8a–f) (0.05 mmol) dissolved in methanol (2 mL) was
added 2 M sodium hydroxide (2 mL). The solution immediately
turned orange and was rapidly stirred for 2 h. The organic layer
was washed with sat. sodium bicarbonate (10 mL) and the aque-
ous layers acidified to pH 3 with 2 M HCl. The product were
then extracted into ethyl acetate (2 × 5 mL), dried (MgSO4) and
concentrated under reduced pressure to give the acid 16a–f as a
yellow or orange solid.
Ethyl
2-acetoxy-6-oxo-6-(p-tolyl)hexa-2,4-dienoate
(8b,
X = CH3). Yield 27.6 mg (72%, method A). dH (300 MHz,
CDCl3) 7.81 (2H, d, J = 7.2 Hz), 7.47 (1H, dd, J = 15.2, 11.5 Hz,
H-4), 7.24 (2H, d, J = 7.2 Hz), 7.22 (1H, d, J = 15.2 Hz, H-5),
7.07 (1H, d, J = 11.5 Hz, H-3), 4.21 (2H, q, J = 7.2 Hz), 2.56
(3H, s), 2.26 (3H, s), 1.27 (3H, t, J = 7.2 Hz); dC (75 MHz,
CDCl3) 188.4, 168.4, 161.2, 144.0, 142.5, 137.1, 134.7, 130.8,
2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (16a, X = H).
Yield 26.8 mg (93%). dH (300 MHz, d6-acetone) 8.05 (2H, d,
J = 7.1 Hz), 7.90 (1H, dd, J = 15.2, 12.0 Hz, H-4), 7.65 (1H, t,
J = 7.1 Hz), 7.55 (2H, t, J = 7.1 Hz), 7.40 (1H, d, J = 15.2 Hz,
H-5), 6.55 (1H, d, J = 12.0 Hz, H-3); dC (75 MHz, d6-acetone)
2 9 4 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 9 4 2 – 2 9 5 0

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206.6, 172.5, 139.5, 138.3, 135.6, 134.0, 129.9, 129.8, 126.9,
110.2; m/z (CI) 218 (M+); HRMS calc. (C12H10O4) 218.0576,
obs. 218.0572.
determined by conversion with excess BphD to the correspond-
ing para-substituted benzoic acid (see Table 1). Km and kcat values
were determined using Eadie–Hofstee plots (see Table 2).
HPLC analysis of benzoic acid products
2-Hydroxy-6-oxo-6-(p-tolyl)hexa-2,4-dienoic
acid
(16b,
X = CH3). Yield 27.6 mg (91%). dH (300 MHz, d6-acetone) 7.89
(2H, d, J = 7.0 Hz), 7.84 (1H, dd, J = 15.0, 12.0 Hz, H-4), 7.40
(1H, d, J = 15.0 Hz, H-5), 7.25 (2H, d, J = 7.0 Hz), 6.53 (1H, d,
J = 12.0 Hz, H-3), 2.43 (3H, s); m/z (CI) 232 (M+); HRMS calc.
(C13H12O4) 232.0736, obs. 232.0721.
Benzoic acid (2.46 mg) was dissolved in methanol (10 mL) and
analysed on a Phenomenex ODS semi-prep C18 reverse-phase
HPLC column eluted with a gradient of 100% methanol to
100% water over 30 min (flow rate 0.5 mL min−1). The retention
time was 3.045 min. A standard curve for peak height vs. con-
centration was determined. 3.98 mg of substrate (16a) was then
added to a solution containing BhpD (1 unit) and the reaction
allowed to go to completion, as observed by the total loss of UV
absorbance at 434 nm. The products of the enzyme assay were
passed through a 0.2 l filter. A 50 mL aliquot was analysed on
the reverse-phase HPLC column under the same elution condi-
tions as for the standard.
2-Hydroxy-6-oxo-6-(p-methoxyphenyl)hexa-2,4-dienoic acid
(16c, X = OCH3). Yield 26.6 mg (84%). dH (300 MHz, d6-acetone)
7.92 (2H, d, J = 7.0 Hz), 7.83 (1H, dd, J = 15.0, 12.0 Hz, H-4),
7.34 (1H, d, J = 15.0 Hz, H-5), 7.12 (2H, d, J = 7.0 Hz), 6.62
(1H, d, J = 12.0 Hz, H-3), 3.73 (3H, s); m/z (EI) 248 (M+);
HRMS calc. (C13H12O5) 248.0681, obs. 248.0679.
BphD-catalysed conversion of reduced substrate 19
2-Hydroxy-6-oxo-6-(p-chlorophenyl)hexa-2,4-dienoic
acid (16d, X = Cl). Yield 29.2 mg (91%). dH (300 MHz, d6-
acetone) 7.83 (2H, d, J = 7.0 Hz), 7.74 (1H, dd, J = 15.0,
12.0 Hz, H-4), 7.49 (2H, d, J = 7.0 Hz), 7.34 (1H, d, J = 15.0 Hz,
H-5), 6.91 (1H, d, J = 12.0 Hz, H-3); m/z (EI) 252/254 (M+);
HRMS calc. (C12H9ClO4) 252.0186, obs. 252.0201.
A solution of allylic alcohol 13a (5 mg) in methanol (2 mL) was
added to 50 mM potassium phosphate buffer (pH 8.0; 8 mL),
to which was added pig liver esterase (20 units). The solution
was incubated for 2 h at 4 °C, generating the reduced substrate
19 (kmax 340 nm). BphD (5 units) was added, and the solution
incubated at 4 °C. Aliquots (1.0 mL) were withdrawn at 1 h
intervals. Each aliquot was acidified to pH 3.0, and the product
extracted with chloroform. A control experiment was performed
in parallel, with no added BphD. The samples were analysed
on a GC-17A gas chromatograph, linked to a QP-5000 mass
spectrometer, using a temperature gradient of 50 °C to 250 °C
over 25 min.
2-Hydroxy-6-oxo-6-(p-cyanophenyl)hexa-2,4-dienoic acid (16e,
X = CN). Yield 25.6 mg (82%). dH (300 MHz, d6-acetone) 8.11
(2H, d, J = 7.0 Hz), 7.89 (1H, dd, J = 15.0, 12.0 Hz, H-4), 7.65
(2H, d, J = 7.0 Hz), 7.42 (1H, d, J = 15.0 Hz, H-5), 6.58 (1H, d,
J = 12.0 Hz, H-3); m/z (EI) 243 (M+).
2-Hydroxy-6-oxo-6-(p-trifluoromethylphenyl)hexa-2,4-
dienoic acid (16f, X = CF3). Yield 30.6 mg (86%). dH (300 MHz,
d6-acetone) 7.92 (2H, d, J = 7.0 Hz), 7.79 (1H, dd, J = 15.0,
12.0 Hz, H-4), 7.68 (2H, d, J = 7.0 Hz), 7.38 (1H, d, J = 15.0 Hz,
H-5), 6.71 (1H, d, J = 12.0 Hz, H-3); m/z (EI) 286 (M+); HRMS
calc. (C13H9F3O4) 286.0450, obs. 286.0458.
Acknowledgements
We are grateful to BBSRC for the award of a studentship
(D.M.S.) and research grant B20467, and to EPSRC for the
award of a studentship (to S.M.F.). We are grateful to Petra Olf
for preliminary synthetic work, and to Dr B. Hofer (GBF, Braun-
schweig) for the gift of strain BL21(DE3)[pLysS]/pAIA51.
Partial purification of BphD
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pooled and stored at 4 °C. The purified enzyme (total 89 units,
14 mg protein) had specific activity 6.4 units mg−1.
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