Hammett analysis of a C-C hydrolase-catalysed reaction using synthetic 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acid substrates

Article abstract of DOI:10.1039/b208168g

A Hammett plot (ρ = 20.71) has been measured for C-C hydrolase enzyme BphD from Pseudomonas LB400, using six 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acids synthesised by a Heck coupling strategy.

Full text of DOI:10.1039/b208168g

Hammett analysis of a C–C hydrolase-catalysed reaction using synthetic
6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acid substrates
Damian M. Speare, Petra Olf and Timothy D. H. Bugg*
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
E-mail: T.D.Bugg@warwick.ac.uk; Fax: 02476-524112; Tel: 02476-573018
Received (in Cambridge, UK) 20th August 2002, Accepted 6th September 2002
First published as an Advance Article on the web 20th September 2002
A Hammett plot (r = 20.71) has been measured for C–C
hydrolase enzyme BphD from Pseudomonas LB400, using
six 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acids synthes-
ised by a Heck coupling strategy.
Aryl ketones containing electron-withdrawing substituents
gave low yields of the corresponding ketal 2. In these cases,
Heck coupling was carried out using the corresponding allylic
alcohols 5a (X = H), 5d (X = Cl), 5e (X = NO2) and 5f (X =
CN), synthesised by reaction of the p-substituted benzaldehyde
with allyl magnesium bromide. Reaction of 5a with bromide 1,
in the presence of Pd(OAc)2/AgOAc/PPh3, was found to give
the desired alcohol 6a as the major product, although other
alkene by-products were also visible in the crude reaction
The bacterial degradation of aromatic compounds by soil
bacteria such as Pseudomonas spp. commonly proceeds via the
oxidative cleavage of catechol intermediates.1 Extradiol cate-
chol cleavage of 3-substituted catechols, catalysed by non-haem
iron(II) dependent catechol 2,3-dioxygenase, yields 6-substi-
tuted 2-hydroxy-6-ketohexa-2,4-dienoic acids (see Fig. 1).
NMR spectroscopic analysis has shown that 2-hydroxy-
6-ketonona-2,4-dioic acid, the meta ring fission product on the
phenylpropionate catabolic pathway of Escherichia coli, exists
as the dienol tautomer in the trans,transoid conformation.2
This family of meta-ring fission products are substrates for a
hydrolytic C–C cleavage reaction, catalysed by C–C hydrolases
which belong to the a/b-hydrolase family (see Fig. 1).3
Crystallographic studies on C–C hydrolase BphD have revealed
that this family of enzymes contain a serine–histidine–aspartate
triad at their active site,4 yet mechanistic studies to date are not
consistent with a nucleophilic mechanism, but instead favour a
general base mechanism involving a gem-diol intermediate.5–7
Studies on this unusual family of enzymes are hampered by the
lack of a synthetic route for the dienol substrates. In this paper
we report the first total synthesis of these biological inter-
mediates, and the kinetic evaluation via a Hammett plot of a
series of synthetic substrates for C–C hydrolase BphD.
1
product by H NMR spectroscopy. The labile alcohol 6a was
oxidised to ketone 4a using CrO3/H2SO4, and isolated after
chromatography in 69% overall yield. Alcohols 5d–f were
similarly coupled by this method, yielding the ketones 4d–f in
70–80% yield.
Deprotection of di-esters 4a–f was carried out by alkaline
hydrolysis in aqueous sodium hydroxide for 2 h at room
temperature, followed by neutralisation to pH 7 to give the
1
products 7a–f as their sodium salts in 80–90% yields. The H
NMR spectrum of 7b showed signals for the diene portion at
7.07 (d, J 11 Hz, H-3), 7.47 (dd, J 15, 11 Hz, H-4) and 7.23 ppm,
indicating that 7 exists in the trans,transoid (2-Z, 4-E) diene
conformation, as found previously for an enzymatically-
generated ring fission intermediate.2
C–C hydrolase BphD from the biphenyl degradation pathway
of Pseudomonas spp. LB40010,11 was purified from an
overexpressing strain of E. coli, to specific activity 3.6 u mg21
.
Treatment of a solution of 7a in 50 mM potassium phosphate
buffer pH 7.0 with an aliquot of BphD gave a linear decrease in
absorbance at 434 nm, confirming the identity of 7a.
Steady-state kinetic parameters were measured for the
processing of synthetic substrates 7a–f by hydrolase BphD. A
plot of log(kcat) vs. substituent parameter s (Fig. 2) shows a
decreasing rate for electron-withdrawing substituents, with
reaction constant r = 20.71 ± 0.1.† The two larger substituents
Synthesis of the diene portion of the target molecule was
attempted using the palladium-catalysed Heck coupling8 of
bromo-enol acetate 1, prepared from ethyl 3-bromopyruvate.9
Palladium-catalysed couplings of 1 with phenyl vinyl ketone
gave < 10% yield of the coupled diene product. However,
reaction of 1 with the corresponding ketal 2a in the presence of
palladium(II) acetate (0.1 equiv.), silver( ) acetate and triphenyl-
I
phosphine at 80 °C cleanly gave the coupled ketal 3a in 78%
yield. Treatment with aqueous 2 M HCl gave the desired ketone
4a in 93% yield. No other regioisomer was detected by NMR
spectroscopy, thus reaction occurred selectively at the terminal
b-carbon of alkene 2a. Ketals 2b (X = CH3) and 2c (X =
OCH3) were also coupled to give 4b and 4c in 76 and 68% yield,
respectively, as shown in Scheme 1.
Scheme 1 Synthetic route for 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic
acids. X = H (a), CH3 (b), OCH3 (c), Cl (d), CN (e), CF3 (f). Reagents and
conditions: i. Pd(OAc)2 (0.1 equiv.), AgOAc, PPh3, toluene, 80 °C; ii. 2 M
HCl/H2O; iii. Pd(OAc)2 (0.1 equiv.), AgOAc, PPh3, toluene, 80 oC; iv.
CrO3, H2SO4; v. NaOH, H2O, 2 h. Yields described in text.
Fig. 1 Bacterial meta-cleavage pathway for degradation of 3-substituted
catechols (R
= H, CH3, CH2CH2CO2H, Ph), via extradiol catechol
oxidative cleavage, to give a 6-substituted 2-hydroxy-6-ketohexa-2,4-die-
noic acid, followed by C–C hydrolytic cleavage.
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This journal is © The Royal Society of Chemistry 2002

of +1.8 and +0.9 reported for serine proteases a-chymotrypsin12
and subtilisin13 respectively (using substituted phenyl acetates
as substrates), which proceed via a nucleophilic mechanism. For
comparison, r values for base-catalysed and acid-catalysed
ester hydrolysis are +2.55 and 20.57, respectively.14
The availability of a synthetic route to this class of biological
intermediates has made possible the synthesis of a range of
substrates for C–C hydrolase BphD, and could be used to
synthesise ring fission intermediates found on other aromatic
meta-cleavage pathways.
We wish to thank Dr B. Hofer (GBF, Braunschweig) for
provision of the BphD expression strain, and BBSRC (D. M. S.)
and the EU ERASMUS/SOCRATES exchange programme (P.
O.) for financial support.
Notes and references
† The kcat kinetic parameter was used, since values of Km for this enzyme
are extremely low ( < 1 mM),11 generating more experimental error in kcat
/
K
m data.
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Fig. 2 Hammett plot of log(kcat) vs. substituent coefficient s for processing
of substrates 7a–f by C–C hydrolase BphD. Gradient of least squares fit line
r = 20.71 ± 0.1. The scheme shows likely transition state for C–C cleavage
of a gem-diol reaction intermediate.
7 S. M. Fleming, T. A. Robertson, G. J. Langley and T. D. H. Bugg,
Biochemistry, 2000, 39, 1522.
8 R. F. Heck, Org. React., 1982, 27, 345.
OCH3 and CN both give relatively low values of kcat, probably
due to steric effects upon substrate binding, but comparison of
isosteric CH3 vs. CF3 and OCH3 vs. CN gives a consistent
pattern. These data give some insight into the transition state for
C–C bond cleavage during the enzymatic reaction, implying an
accumulation of d+ charge adjacent to the aryl ring. This is
consistent with heterolytic cleavage of the C–C bond, and
delocalisation of the departing d2 charge by the adjacent a,b-
unsaturated ketone. This value is opposite in sign to the r values
9 M. D. Fryzuk and B. Bosnich, J. Am. Chem. Soc., 1979, 101, 3043.
10 B. Hofer, S. Backhaus and K. N. Timmis, Gene, 1994, 144, 9.
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D. Eltis, J. Biol. Chem., 1998, 273, 22943.
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14 N. S. Isaacs, Physical Organic Chemistry, Longman Press, Harlow,
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