Elucidation of the catalytic mechanisms of the non-haem iron-dependent catechol dioxygenases: Synthesis of carba-analogues for hydroperoxide reaction intermediates

Article abstract of DOI:10.1039/b004265j

The catalytic mechanisms of the non-haem iron-dependent intradiol and extradiol catechol dioxygenases are thought to involve transient hydroperoxide reaction intermediates, formed by reaction of a catechol substrate with dioxygen. The synthesis of carba-analogues of these intermediates is described in which the hydroperoxide functional group (-OOH) is replaced by a hydroxymethyl group (-CH₂OH), and the cyclohexadienone skeleton simplified to a cyclohexanone. Analogues of the "proximal" hydroperoxide in which the hydroxymethyl group was positioned axially with respect to the ring were found to act as reversible competitive inhibitors (K_i 0.7-7.6 mM) for the extradiol enzyme 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) from Escherichia coli, whereas analogues in which the hydroxymethyl group was positioned equatorially showed no inhibition. In contrast, assays versus the intradiol-cleaving protocatechuate 3,4-dioxygenase from Pseudomonas sp. showed inhibition only by an analogue containing an equatorial hydroxymethyl group (IC₅₀ 9.5 mM). These data support the existence of a proximal hydroperoxide intermediate in the extradiol catechol dioxygenase mechanism, and suggest that the conformation adopted by the hydroperoxide reaction intermediate may be an important determinant in the reaction specificity of the extradiol and intradiol dioxygenases. The Royal Society of Chemistry 2000.

Full text of DOI:10.1039/b004265j

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Elucidation of the catalytic mechanisms of the non-haem iron-
dependent catechol dioxygenases: synthesis of carba-analogues for
hydroperoxide reaction intermediates
1
Christopher J. Winfield,a Zeyana Al-Mahrizy,a Michael Gravestockb and
Timothy D. H. Bugg*a†
a Department of Chemistry, University of Southampton, Highfield, Southampton,
UK SO17 1BJ
b AstraZeneca Pharmaceuticals, Alderley Park, Alderley Edge, Macclesfield, Cheshire,
UK SK10 4TG
Received (in Cambridge, UK) 30th May 2000, Accepted 10th August 2000
First published as an Advance Article on the web 18th September 2000
The catalytic mechanisms of the non-haem iron-dependent intradiol and extradiol catechol dioxygenases are
thought to involve transient hydroperoxide reaction intermediates, formed by reaction of a catechol substrate
with dioxygen. The synthesis of carba-analogues of these intermediates is described in which the hydroperoxide
functional group (–OOH) is replaced by a hydroxymethyl group (–CH2OH), and the cyclohexadienone skeleton
simplified to a cyclohexanone. Analogues of the “proximal” hydroperoxide in which the hydroxymethyl group was
positioned axially with respect to the ring were found to act as reversible competitive inhibitors (Ki 0.7–7.6 mM)
for the extradiol enzyme 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) from Escherichia coli, whereas
analogues in which the hydroxymethyl group was positioned equatorially showed no inhibition. In contrast,
assays versus the intradiol-cleaving protocatechuate 3,4-dioxygenase from Pseudomonas sp. showed inhibition only
by an analogue containing an equatorial hydroxymethyl group (IC50 9.5 mM). These data support the existence
of a proximal hydroperoxide intermediate in the extradiol catechol dioxygenase mechanism, and suggest that the
conformation adopted by the hydroperoxide reaction intermediate may be an important determinant in the reaction
specificity of the extradiol and intradiol dioxygenases.
the catechol and dioxygen by the iron() centre, the catalytic
Introduction
mechanism proceeds via single electron transfers to give a
The catechol dioxygenase family of enzymes catalyses the oxid-
semiquinone–iron()–superoxide intermediate.7 C–O bond
ative cleavage of catechol and substituted catechols which lie on
formation could then take place to give two possible hydro-
catabolic pathways for the bacterial degradation of aromatic
peroxide intermediates, either a C-1 hydroperoxide (distal to
compounds.1 The intradiol dioxygenases catalyse the cleavage
the catechol oxygens) or a C-2 hydroperoxide (proximal to the
of the bond situated between the two catechol hydroxy groups,
catechol oxygens). 18O-Labelling studies have established the
utilising non-haem iron() as cofactor, whereas the extradiol
dioxygenases catalyse the cleavage of the bond adjacent to the
existence of a seven-membered lactone intermediate, formed by
Criegee rearrangement of the hydroperoxide: either via acyl
two hydroxy groups, utilising non-haem iron() as cofactor.
migration of the distal hydroperoxide, or via alkenyl migration
The three-dimensional structures of three extradiol catechol
of the proximal hydroperoxide.8 The reaction is completed
dioxygenases, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)
by attack of iron() hydroxide upon the lactone to give the
from Pseudomonas LB400,2 catechol 2,3-dioxygenase (XylE)
extradiol ring fission product.8
from Pseudomonas putida mt-2,3 and protocatechuate 4,5-
The present study concerns the choice of proximal vs. distal
hydroperoxide in the catalytic mechanism of MhpB (Fig. 1).
The prior observation that substrate analogues containing
dioxygenase (LigAB) from Sphingomonas paucimobilis4 have
been determined, and in each case the mononuclear iron()
centre is ligated by two histidine ligands and one glutamic
either –OCH CO H or –CH᎐CHCO H sidechains at C-1 were
᎐
2
2
2
acid ligand. In contrast the mononuclear iron() centre of
the intradiol-cleaving protocatechuate 3,4-dioxygenase from
Pseudomonas aeruginosa is ligated by two histidine ligands and
two tyrosine ligands.5 The choice of reaction pathway, intradiol
versus extradiol, and its relationship to the oxidation state and
coordination state of the metal centre, is therefore an intriguing
mechanistic problem.
Previous studies in our laboratory have focussed on the reac-
tion mechanism of the extradiol enzyme 2,3-dihydroxyphenyl-
propionate 1,2-dioxygenase (MhpB) from Escherichia coli.6
Mechanistic studies using substrates containing cyclopropyl
radical traps have established that, following the ligation of
processed at comparable rates by MhpB disfavoured the distal
hydroperoxide,7 however since there are literature examples of
both acyl migration and alkenyl migration in hydroperoxide
rearrangements,1 a more direct probe for the hydroperoxide
intermediate was required.
The approach described in this paper is the synthesis of
carba-analogues of each hydroperoxide in which the peroxide
functional group (–OOH) is replaced by a hydroxymethyl group
(–CH2OH), and the cyclohexadienone skeleton simplified to a
cyclohexanone. Although the latter skeleton is more puckered
in conformation than a cyclohexadienone, the incorporation of
suitable substituents on the cyclohexane ring would allow an
investigation of the preferred conformation of the hydro-
peroxide in the active site: axial with respect to the ring, or
equatorial. It was anticipated that the optimal conformation of
† Present address: Department of Chemistry, University of Warwick,
Coventry, UK CV4 7AL.
DOI: 10.1039/b004265j
J. Chem. Soc., Perkin Trans. 1, 2000, 3277–3289
This journal is © The Royal Society of Chemistry 2000
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Scheme 1 Synthetic route for distal carba-analogue (5). Reagents and
yields: a, t-BuOK–MeI, Aliquat 336, 97%; b, HO(CH2)2OH, Hϩ, 63%;
c, LiAlH4, THF, 90%; d, NaH, BnBr, THF, 61%; e, Hϩ–acetone–H2O,
87%; f, KHMDS, THF, Ϫ78 ЊC; g, PhCH(O)NTs, 62% overall; h, H2–
Pd–C, 85%.
Synthesis of carba-analogues of proximal hydroperoxide
A series of carba-analogues for the proximal hydroperoxide,
containing methyl, tert-butyl or phenyl sidechains, were syn-
thesised using the route shown in Scheme 2. The commercially
available 2-substituted cyclohexanones were converted to the
corresponding tosylhydrazones (6a–c), in 90–99% yield. The
tosylhydrazones were then subjected to a Shapiro lithiation
procedure:12 treatment with n-butyllithium at Ϫ78 ЊC generated
in each case the intermediate vinyllithium species, which was
reacted with paraformaldehyde to give the allylic alcohols
(7a–c) regioselectively in 31–55% yield. The modest yields are
attributed to the sluggish reaction of paraformaldehyde, since
other carbonyl reagents were found to react in higher yield
(data not shown).
The primary alcohol functional group was then protected,
either with a tert-butyldimethylsilyl group (to give 8a in 93%
yield) or with a methoxymethyl protecting group (to give 8b,c in
81–85% yield). When bulky sidechains were present the MOM
group was found to be superior in this sequence to the TBDMS
group, the latter giving low yields in the subsequent dihydroxy-
lation step, presumably due to increased steric hindrance.
Dihydroxylation of the alkene functional group was then
carried out using the conditions of Minato et al.,13 employing
1–5 mol% K2OsO4 in the presence of K3Fe(CN)6 and Me-
SO2NH2. Reaction occurred selectively on the face opposite the
R substituent, although as mentioned above the reaction of this
alkene was sluggish and highly sensitive to steric bulk. The
methyl analogue (9a) was isolated in 96% yield as a 7:3
diastereomeric mixture after 24 hours at room temperature,
whereas only a 40% yield of the tert-butyl analogue (9b) was
obtained after 7 days, and a 25% yield of the phenyl analogue
(9c) was obtained, in each case as a single diastereoisomer.
Synthesis of the desired analogues (10a–c) was completed by
oxidation of the secondary alcohol, using either the Dess–
Martin oxidation14 or the Swern oxidation15 conditions, fol-
lowed by deprotection of the primary hydroxy group (yields
given in Scheme 2).
Fig.
1
Proposed catalytic mechanisms for 2,3-dihydroxyphenyl-
propionate 1,2-dioxygenase (MhpB), illustrating the proximal and dis-
tal hydroperoxide intermediates and their respective carba-analogues.
the proximal hydroperoxide for alkenyl migration might be one
in which the hydroperoxide group was positioned axially with
respect to the ring, thus aligning the migrating C–C bond anti-
periplanar to the O–O bond being broken, a stereoelectronic
effect which has recently been supported experimentally.9 We
describe the synthesis of carba-analogues of proximal and
distal hydroperoxides, an analysis of their conformational
preference, and their interaction with extradiol and intradiol
dioxygenase enzymes.
Results
Synthesis of carba-analogue of distal hydroperoxide
A carba-analogue for the distal hydroperoxide, containing a
methyl sidechain, was synthesised as shown in Scheme 1.
2-Ethoxycarbonylcyclohexanone was alkylated at C-2 by
methyl iodide, using potassium tert-butoxide and phase transfer
conditions,10 to give (1) in 97% yield. The ketone group was
protected as its ethylene ketal, in 63% yield, and the ester group
reduced to alcohol (2) using lithium aluminium hydride, in 90%
yield. Protection of the primary alcohol as its benzyl ether and
deprotection of the ketal proceeded in 53% overall yield to give
ketone (3). The Davis oxaziridine method11 was used to carry
out the α-hydroxylation of ketone (3), giving the α-hydroxy
ketone (4) as a 2:1 mixture of diastereoisomers, in 62% yield.
Attempts to separate the diastereoisomers by chromatography
were unsuccessful. Deprotection of the benzyl ether proceeded
in 85% yield to give analogue (5) as a 2:1 mixture of
diastereoisomers.
Analysis of analogue 10a (R = Me) by NMR spectroscopy
in CDCl3 revealed an unexpected conformational preference.
The methyl sidechain was found to adopt an axial position, as
shown by analysis of coupling constants and by measurement
of NOE’s from the –CH3 group to the –CH2OH sidechain (5%)
Analogue (5) was assayed against dioxygenase MhpB, as
described in the Experimental section, but showed no observ-
able enzyme inhibition at 10 mM concentration.
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Scheme 2 Synthetic route for proximal carba-analogues 10a (R =
CH3), 10b (R = t-Bu), and 10c (R = Ph). P = protecting group: either
TBDMS or MOM. Reagents and yields shown below:
R = CH3 R = t-Bu R = Ph
a, TsNHNH2, MeOH, Hϩ
b, n-BuLi, THF, Ϫ78 ЊC; c, CH2O
P = TBDMS: d, TBDMSCl,
DMAP, DMF
6a 99%
7a 53%
8a 93%
6b 92%
7b 31%
—
6c 73%
7c 43%
—
P = MOM: d, CH2(OMe)2, LiBr–
—
8b 85%
9b 40%
—
8c 81%
9c 25%
—
TsOH
e, K2OsO4–K3Fe(CN)6, MeSO2-
NH2, t-BuOH–H2O
P = TBDMS: f, Swern ox.; g,
TBAF, THF
P = MOM: f, Dess–Martin ox.;
g, HCl–MeOH
9a 96%
10a 30%
—
Fig. 2 X-Ray crystal structures of carba-analogues 10b (A) and
10c (B).
10b 35%
10c 43%
and to a C-5 axial ring hydrogen (4%). This conformation is
stabilised by the formation of an intramolecular hydrogen bond
between the primary alcohol of the sidechain and the C-3
ketone group. In contrast, analogues 10b (R = t-Bu) and 10c
(R = Ph) were found by NMR spectroscopy to adopt the
expected conformation in which the R sidechain is situated
equatorially, and the hydroxymethyl group axially. This assign-
ment was confirmed by determination of X-ray crystal struc-
tures for single crystals of 10b and 10c, which are shown in
Fig. 2.
Assay of analogues 10a–c versus dioxygenase MhpB revealed
that 10a showed no inhibition at 10 mM concentration, whereas
10b and 10c both acted as reversible inhibitors, with Ki values
of 4.9 mM and 0.7 mM respectively (see Table 1). Kinetic
analysis using a Dixon plot16 showed in each case that inhib-
ition was competitive with respect to the natural substrate 2,3-
dihydroxyphenylpropionic acid (see Fig. 3). Although the
Ki values are significantly higher than the Km for its natural
substrate (Km 26 µM for 2,3-dihydroxyphenylpropionic acid6),
the MhpB active site does appear to recognise the carba-
analogues of the proximal hydroperoxide in which the hydroxy-
methyl group is positioned axially with respect to the ring.
Fig. 3 Dixon plot16 showing inhibition of E. coli 2,3-dihydroxyphenyl-
propionate 1,2-dioxygenase (MhpB) by carba-analogue 10c. Assays
were carried out at 50, 100, and 150 µM concentrations of 2,3-
dihydroxyphenylpropionic acid (DHP), as indicated. The point of
intersection of the lines gives a Ki value of 0.7 mM.
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Analogues 10a–c were incubated for extended periods of
time with MhpB, in order to examine whether any enzyme-
catalysed reactions might take place (e.g. pinacol rearrange-
ment reactions), however no new characterisable peaks were
found by HPLC analysis.
The effect of the steric bulk of the tert-butyl and phenyl
sidechains on substrate binding was investigated by synthesis of
the corresponding catechol substrates, 3-tert-butylcatechol and
3-phenylcatechol. This was carried out by an ortho-lithiation
strategy reported concurrently to this work by Snieckus et al.,17
as shown in Scheme 3. Ortho-lithiation of MOM-protected
Scheme 3 Synthetic route of 3-substituted catechols 13a (R = Ph) and
13b (R = t-Bu). Reagents and yields (for 13b): a, CH3OCH2Cl, NaH,
THF, 89%; b, n-BuLi, THF, Ϫ78 ЊC; c, B(OMe)3; d, Oxone, 65%
overall; e, Hϩ–H2O, 82%.
Scheme 4 Synthetic route for carba-analogues 17 and 18. Reagents
and yields: a, TsNHNH2, MeOH, Hϩ, 99%; b, n-BuLi, THF, Ϫ78 ЊC;
c, CH2O, 26% overall; d, CH2(OMe)2, LiBr–TsOH, 81%; e, K2OsO4–
K3Fe(CN)6, MeSO2NH2, t-BuOH–H2O, 97%; f, Dess–Martin ox.; g,
HCl–MeOH, 74–79% overall.
phenols (11a/b) was found to proceed smoothly using n-butyl-
lithium as base, and the resulting alkyllithiums were trapped by
trimethyl borate. Oxidation of the boronic acids to phenols
(12a/b) was achieved best using Oxone in aqueous acetone–
NaHCO3, a reagent which in our hands was superior to aque-
ous hydrogen peroxide. The catechols (13a/b) were assayed
against MhpB, and 3-phenylcatechol (13a) was found to be a
good substrate (Km 78 µM), indicating that a phenyl substituent
can be accommodated at the enzyme active site. The 3-tert-
butylcatechol (13b) was found to be very unstable towards air
oxidation at pH 7.5–8.0 in aqueous solution, thus a reliable Km
determination was not possible.
effective bridge to the non-haem iron centre at the dioxygenase
active site. In order to investigate this hypothesis, a further
analogue was synthesised, containing an extended sidechain
(–CH2CH2OH).
The synthetic strategy used for this compound was a
Horner–Wadsworth–Emmons reaction on the 2-substituted
cyclohexanone, as shown in Scheme 5. This reaction failed
with 2-tert-butylcyclohexanone, but proceeded in 75% yield
Analogues were also synthesised in which the alkyl substitu-
ent was situated para with respect to the hydroxymethyl sub-
stituent, instead of ortho. The synthetic strategy was the same
as that used for synthesis of analogues 10a–c, but the starting
material in this case was 4-tert-butylcyclohexanone. Synthetic
steps and yields are shown in Scheme 4. Shapiro reaction with
formaldehyde proceeded in 26% yield to give alcohol 14, and
after MOM protection the dihydroxylation procedure as above
gave a mixture of two diastereoisomers 15 and 16, in a 1:1
ratio, which were separated by careful silica column chromato-
graphy. Oxidation separately gave the syn (17) and anti (18)
carba-analogues with the hydroxymethyl group axial and
equatorial respectively. When assayed versus MhpB, analogue
17 showed competitive reversible inhibition (Ki 7.6 mM),
whereas analogue 18 showed no enzyme inhibition at 10 mM
concentration (see Table 1). These data are entirely consistent
with the results obtained for 10a–c, in that only those analogues
containing an axial hydroxymethyl group showed inhibition of
MhpB.
Synthesis and assay of a hydroxyethyl carba-analogue
One possible explanation for the relatively low affinity of the
above carba-analogues is that the hydroxymethyl substituent
(–CH2OH) is shorter in length than the putative hydroperoxide
intermediate (–OOH). Consequently, it is conceivable that the
hydroxymethyl group is not of sufficient length to form an
Scheme 5 Synthetic route for extended carba-analogue 22. Reagents
and yields: a, (EtO)2POCH2CO2Et, NaH, THF, 75%; b, LDA, THF,
Ϫ78 ЊC; c, NH4Cl–H2O, 85% overall; d, LiAlH4, 96%; e, MOMCl,
i-Pr2NEt, CH2Cl2, 94%; f, K2OsO4–K3Fe(CN)6, MeSO2NH2, t-BuOH–
H2O, 98%; f, Dess–Martin ox.; g, HCl–MeOH, 42% overall.
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Table 1 Inhibition of E. coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) and Pseudomonas sp. protocatechuate 3,4-dioxygenase (3,4-
PCD) by carba-analogues. Assays were carried out as described in the Experimental section. Ki values were determined using a Dixon plot. Km
values: MhpB 26 µM (2,3-dihydroxyphenylpropionic acid); 3,4-PCD 20 µM (protocatechuic acid). NA = not assayed
Compound
Hydroperoxide mimic
Sidechain
MhpB Ki (mM)
3,4-PCD IC50 (mM)
5
Distal –CH2OH
2-CH3
2-CH3
2-t-Bu
2-Ph
4-t-Bu
4-t-Bu
2-Ph
No inhibition
No inhibition
4.9
0.7
NA
10a
10b
10c
17
Proximal –CH2OHeq
Proximal –CH2OHax
Proximal –CH2OHax
Proximal –CH2OHax
Proximal –CH2OHeq
Proximal –CH2CH2OHax
No inhibition
No inhibition
NA
No inhibition
9.5
7.6
18
22
No inhibition
1.4
NA
with 2-phenylcyclohexanone to give the unsaturated ester 19.
Treatment with LDA at Ϫ78 ЊC effected the isomerisation to
endocyclic 2,3-alkene (20). Only a small amount of the 1,2-
alkene was observed in the reaction product (3–4% by NMR
spectroscopy) when this reaction was carried out at Ϫ78 ЊC,
which could be removed by careful column chromatography.
Reduction of the ester by lithium aluminium hydride, followed
by MOM protection of the primary alcohol, gave the MOM
ether 21. Dihydroxylation as before, followed by Dess–Martin
oxidation and deprotection, gave the extended analogue 22.
Assays versus MhpB revealed that 22 was a reversible, com-
petitive inhibitor with Ki 1.4 mM, of comparable affinity to the
earlier series (see Table 1).
Assay of carba-analogues against an intradiol dioxygenase
Having observed some selectivity for binding by extradiol
dioxygenase MhpB, it was of interest to examine whether the
same, or different, selectivity was exhibited by an intradiol
dioxygenase enzyme. Accordingly, a selection of analogues
were tested as inhibitors of commercially available Pseu-
domonas sp. protocatechuate 3,4-dioxygenase. No inhibition
was observed by the 2-methyl analogue 10a, the 2-tert-butyl
analogue 10b, or the syn 4-tert-butyl analogue 17, however
inhibition was observed by the anti tert-butyl analogue 18 (IC50
9.5 mM). Again the observed IC50 value is higher than the sub-
strate Km (Km 20 µM determined for protocatechuic acid), how-
ever the selectivity for these analogues is markedly different to
the selectivity found for MhpB (see Table 1).
Discussion
A series of carba-analogues for the putative proximal and distal
hydroperoxide reaction intermediates have been synthesised
and tested as inhibitors for extradiol and intradiol catechol
dioxygenases. The observation of a selective interaction of
these analogues with the respective enzymes gives experimental
support and insight into the existence of hydroperoxide inter-
mediates which to date have been presumed but unverified.
Although the binding affinity of these analogues is approxi-
mately 100-fold weaker than the observed Km values for their
natural substrates (see below), a clear pattern of enzyme inhib-
ition is observed. Carba-analogues 10b, 10c and 17 of the prox-
imal hydroperoxide in which the hydroxymethyl group was
positioned axially with respect to the cyclohexane ring acted as
reversible competitive inhibitors for MhpB, whereas analogue 5
of the distal hydroperoxide, and analogues 10a and 18 of the
proximal hydroperoxide in which the hydroxymethyl group is
positioned equatorially show no inhibition. These observations
provide experimental support for a proximal hydroperoxide
intermediate in the MhpB catalytic mechanism, rather than a
distal hydroperoxide, a conclusion which is consistent with
earlier observations of the substrate specificity of MhpB.7 The
proximal hydroperoxide structure has also been observed in a
model transition metal catechol–dioxygen adduct.18
Fig. 4 Illustration of the convergence of the reaction mechanisms of
the extradiol and intradiol catechol dioxygenases onto a similar prox-
imal hydroperoxide intermediate, followed by divergence via alkenyl vs.
acyl migration.
intermediate, but catalyse intradiol C–C cleavage to give an
anhydride species.1 This work therefore leads to the conclusion
that the catalytic mechanisms of these two families of dioxygen-
ase enzymes converge on a similar hydroperoxide intermediate,
and subsequently diverge to give regiospecific oxidative cleavage
(see Fig. 4). How then is the site of bond cleavage dictated in
intradiol vs. extradiol enzymes?
The observed inhibition data suggest that there is a confor-
mational preference of the MhpB active site, since 10b, 10c and
17 are recognised by the enzymes whereas 10a and 18 are not. If
one considers the Criegee rearrangement of the presumed prox-
imal hydroperoxide intermediate on stereoelectronic grounds
(Fig. 5), then one might predict that this species would be
optimally aligned for O–O bond cleavage if the hydroperoxide
functional group is positioned axially with respect to the
cyclohexadienone ring. In this conformation the O–O bond is
The iron()-dependent intradiol dioxygenases also presum-
ably access the same type of proximal hydroperoxide reaction
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leading to extradiol cleavage, and an equatorial hydroperoxide
leading to intradiol cleavage.
Preliminary computational studies indicate that for
a
6,6-disubstituted cyclohexa-2,4-dienone the energy barrier
between these two extreme conformations is small (<2 kJ
molϪ1), however if bound to an active site iron cofactor then the
choice of conformation could be dictated by the coordination
state of the metal centre. Thus, the extradiol iron() centre
bound facially by three protein ligands would have three vacant
coordination sites with which it could stabilise an axial
hydroperoxide conformation. The intradiol iron() centre
bound initially by four protein ligands might have only two
vacant coordination sites, which might favour an equatorial
hydroperoxide. Nearby active site amino acid sidechains also
presumably contribute to the choice of extradiol vs. intradiol
reaction pathways.
There are several possible explanations for the relatively weak
binding observed for the carba-analogues. First is that the
cyclohexane ring of the analogues is not as planar as the
cyclohexadienone ring of the presumed reaction intermediate.
The axial substituents on the cyclohexane ring may give rise to
unfavourable binding interactions, although the observation
that the 4-tert-butyl analogue 17 also inhibits MhpB suggests
that the enzyme active site tolerates considerable substitution in
this position at least. Second is that the length of the hydroxy-
methyl substituent may not effectively mimic the length of the
hydroperoxide functional group. However, analogue 22 contain-
ing a hydroxyethyl group exhibited a very similar Ki value to
hydroxymethyl analogue 10c containing a hydroxymethyl
group, suggesting that the length of the sidechain is not a major
factor. Third (and perhaps most likely) is that the acidity of the
alcohol functional groups of the carba-analogues (pKa 15–16)
does not match the acidity of the phenolic hydroxy groups of
the natural substrate (pKa 9–10) and the acidity of the
hydroperoxide group (pKa 12.8 for t-BuOOH21). In order to
effectively ligate the active site iron() centre the ligand atoms
should be deprotonated, for which active site histidine bases
have been identified in the X-ray crystal structure of BphC.2
The lower acidity of the alcohol functional groups in the ana-
logues may dictate their relatively weak binding efficacy. Never-
theless, we note that these analogues are the first non-aromatic
compounds found to inhibit the catechol dioxygenases.
In conclusion, these data support the existence of a proximal
hydroperoxide reaction intermediate in the reaction mechan-
isms of both the extradiol and intradiol dioxygenases, raising
the intriguing question of how the extradiol vs. intradiol select-
ivity is controlled by the two families of enzyme. This work
provides experimental evidence that the MhpB active site shows
a conformational preference for the binding of reaction inter-
mediate analogues, hence providing a clue that an important
factor in the reaction specificity of the extradiol vs. intradiol
dioxygenases may be the conformation in which the hydro-
peroxide intermediate is bound at the respective enzyme active
sites.
Fig. 5 Mechanisms for alkenyl migration of an axial proximal
hydroperoxide intermediate, either via Criegee rearrangement (σ bond
migration) or participation of diene π system. Literature precedent for
the latter mechanism (see ref. 19) is illustrated.
aligned antiperiplanar with respect to the migrating C–C bond,
an arrangement which is known to be favoured experimentally
for the Criegee rearrangement.9 It is therefore significant that
only the carba-analogues containing an axial hydroxymethyl
group inhibit MhpB, which provides some experimental sup-
port for the existence of this active conformation for extradiol
cleavage.
In this conformation an alternative mechanism is also pos-
sible: the participation of the π electrons of the diene system,
which would overlap with the σ* orbital of the O–O bond, to
give a transient 1,2-epoxide containing an allylic carbocation,
which could fragment with C–C cleavage to give the desired
lactone (see Fig. 5). The latter π participation mechanism has
some precedent in the rearrangement of a known cyclohexa-
dienyl hydroperoxide,19 and the rearrangements of lipid
hydroperoxides,20 to give epoxide products. Analysis of the
X-ray crystal structure of the iridium() catechol model com-
plex mentioned above, which contains a proximal hydroperox-
ide species, reveals that in this case the hydroperoxide func-
tional group is also positioned axially with respect to the ring
system.18
Experimental
General
Assays of the carba-analogues versus the intradiol enzyme
protocatechuate 3,4-dioxygenase from Pseudomonas sp. reveal
a different selectivity, this enzyme being inhibited only by
analogue 18 containing an equatorial hydroxymethyl group.
Assuming that both families of enzyme access a proximal
hydroperoxide intermediate, mimicked by the carba-analogues,
these data suggest that the conformation adopted by the
hydroperoxide intermediate may be an important determinant
in the choice of reaction pathway. We speculate that the reac-
tion specificity of the intradiol vs. extradiol dioxygenases may
be controlled by the precise conformation in which the
hydroperoxide intermediate is bound: an axial hydroperoxide
Nuclear magnetic resonance spectra were recorded on a Bruker
AM300 Fourier transform spectrometer (300 MHz). Ultra-
violet–visible spectra were recorded on a Cary-1 UV–visible
spectrophotometer. Infrared spectra were recorded on a 1600
series Perkin-Elmer FTIR spectrometer. Mass spectra were
recorded on a VG-70-250 mass spectrometer in electron impact
(EI) or chemical ionisation (CI) mode, or a VG Platform
Quadrupole Electrospray Ionisation mass spectrometer (ES).
3-(2,3-Dihydroxyphenyl)propionic acid was prepared from
2,3-dimethoxycinnamic acid using a published procedure.22
2-(p-Tolylsulfonyl)-3-phenyl-1,2-oxaziridine was prepared by
3282
J. Chem. Soc., Perkin Trans. 1, 2000, 3277–3289

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the method of Davis.11 Dess–Martin periodinane was prepared
according to reference 14. All other chemicals were supplied by
Sigma-Aldrich Chemical Co.
the evolution of H2 had ceased, benzyl bromide (4.65 g, 27.19
mmol, 2.0 eq.) and tetra-n-butylammonium iodide (0.15 g, 0.41
mmol, 3 mol%) in THF (10 ml) was added dropwise over
5 minutes. The solution was stirred for a further hour under
N2 then quenched with saturated NH4Cl solution (30 ml),
extracted with ethyl acetate (3 × 20 ml), washed with brine,
dried (Na2SO4) and concentrated in vacuo. The resultant oil was
purified by flash silica chromatography (10% EtOAc–hexane) to
afford the benzyl ether as a colourless oil (2.30 g, 8.34 mmol,
61%). Rf (10% EtOAc–hexane) 0.24; IR (liquid film) 2936 (s),
2868 (s), 1601 (w), 1583 cmϪ1; δH (300 MHz, CDCl3) 7.20–7.40
(5H, m, Ar), 4.52 (2 H, 2 × d, J = 12 Hz, OCH2Ph), 3.90 (4H,
m, OCH2CH2O), 3.55 (1H, d, J = 9 Hz, CHHOBn), 3.40 (1H,
d, J = 9 Hz, CHHOBn), 1.40–1.77 (8H, m), 1.09 (3H, s, CH3)
ppm; δC (75 MHz, CDCl3) 139.3, 128.4, 127.6, 127.4, 111.9,
74.1, 73.6, 65.1, 64.8, 43.4, 33.0, 31.2, 23.8, 20.9, 18.5 ppm; m/z
(EI) 276.2 (8%, Mϩ), 185.0 (96%, [M Ϫ C7H7]ϩ).
To a solution of (6-methyl-1,4-dioxaspiro[4.5]decan-6-yl)-
(phenylmethoxy)methane (2.15 g, 7.80 mmol) in acetone–water
(90:10, 50 ml) was added a catalytic amount of toluene-p-
sulfonic acid (10 mg, 0.04 mmol) and the mixture heated under
reflux for 15 hours. The mixture was then diluted with diethyl
ether (50 ml), washed with brine (20 ml), dried (MgSO4), then
filtered through a short pad of silica and the solvent removed
in vacuo to afford the product ketone 3 as a colourless oil (1.57
g, 6.77 mmol, 87%). Rf (10% EtOAc–hexane) 0.31; IR (liquid
film) 2938 (m), 2867 (m), 1701 (s), 1602 (w) cmϪ1; δH (300 MHz,
CDCl3) 7.35 (5H, m, Ar), 4.55 (2H, 2 × d, J = 12 Hz, OCH2Ph),
3.52 (2H, s, CH2OBn), 2.40 (2H, m, CH2CO), 1.65–1.95 (6H,
m), 1.18 (3H, s, CH3) ppm; δC (75 MHz, CDCl3) 214.6, 138.6,
128.5, 127.6, 75.5, 73.5, 49.8, 39.2, 36.5, 27.2, 21.3, 21.2 ppm;
m/z (CI) 250 ([M ϩ NH4]ϩ); HRMS (CI) found [M ϩ NH4]ϩ,
250.1816. C15H24O2N requires 250.1807.
Ethyl 1-methyl-2-oxocyclohexan-1-carboxylate 1
To ethyl 2-oxocyclohexane-1-carboxylate (5.00 g, 29.38 mmol)
was added Aliquat 336 (0.72 g, 1.76 mmol, 6 mol%). The mix-
ture was then cooled (ice bath) and solid potassium tert-
butoxide (3.30 g, 29.40 mmol, 1 eq.) added portionwise over
10 minutes with magnetic stirring. Methyl iodide (4.17 g, 29.40
mmol, 1 eq.) was then added slowly, and stirring was continued
for a further 30 minutes. The mixture was then diluted with
ethyl acetate (50 ml), filtered through a pad of Celite and the
solvent removed in vacuo. The resultant yellow oil was further
purified by short path distillation to afford 1 as a colourless oil
(5.15 g, 28.0 mmol, 97%). Rf (40% EtOAc–petroleum ether)
0.57; IR (liquid film) 2938 (m), 2867 (m), 1711 (s) cmϪ1; δH (300
MHz, CDCl3) 4.20 (2H, q, J = 7 Hz, OCH2CH3), 2.46 (3H, m),
2.00 (1H, m), 1.70 (3H, m), 1.45 (1H, m), 1.29 (3H, s, CH3), 1.26
(3H, t, J = 7 Hz, OCH2CH3) ppm; δC (75 MHz, CDCl3) 208.5,
173.2, 61.4, 57.3, 40.8, 38.4, 27.7, 22.8, 21.4, 14.2 ppm; m/z (EI)
184.0 (62%, Mϩ), 155.9 (82%, [M Ϫ C2H5]ϩ).
(6-Methyl-1,4-dioxaspiro[4.5]decan-6-yl)methanol 2
Ethylene glycol (158 g, 25.50 mmol) and p-TsOH (0.22 g, 1.16
mmol) were added to a solution of 1 (4.27 g, 23.20 mmol) in
toluene (150 ml). The mixture was heated under reflux with
azeotropic removal of water (Dean–Stark apparatus) for 16
hours. The mixture was washed with sat. sodium bicarbonate
solution (20 ml), brine (20 ml), dried over anhydrous K2CO3
and the solvent removed in vacuo. The resultant oil was then
purified by flash silica chromatography (EtOAc–hexane, 20:80)
to afford the ethylene ketal as a colourless oil (3.36 g, 14.70
mmol, 63%). Rf (20% EtOAc–hexane) 0.32; IR (liquid film)
2935 (s), 2867 (s), 1721 (s) cmϪ1; δH (300 MHz, CDCl3) 4.18
(2H, q, J = 7 Hz, OCH2CH3), 3.95 (4H, m, OCH2CH2O), 1.35–
2.15 (8H, m), 1.25 (3H, s, CH3 and 3H, t, J = 7 Hz, OCH2CH3)
ppm; δC (75 MHz, CDCl3) 175.0, 110.8, 65.5, 64.7, 60.4, 51.2,
34.8, 32.0, 23.5, 21.7, 19.2, 14.3 ppm; m/z (EI) 228.0 (38%,
Mϩ), 182.9 (23%, [M Ϫ C2H5O]ϩ).
To a cooled (Ϫ60 ЊC acetone–dry ice) solution of LiAlH4
(1.0 M in THF, 30 ml) in dry THF (50 ml) was added ethyl
6-methyl-1,4-dioxaspiro[4.5]decane-6-carboxylate (3.66 g,
16.09 mmol) in dry THF (50 ml) dropwise over 20 minutes with
stirring. The solution was then stirred for a further 3 hours at
Ϫ30 ЊC then diluted with ethyl acetate (50 ml). Saturated
NH4Cl solution (150 ml) was added and the resultant viscous
solution warmed to room temperature. The solution was fil-
tered through Celite and the filter cake washed with ethyl acet-
ate (50 ml). The aqueous phase was then extracted with ethyl
acetate (3 × 20 ml), and the combined organic phase washed
with sat. NaHCO3(aq) (50 ml), brine (50 ml), dried (Na2SO4) and
the solvent removed in vacuo. The resultant oil was then puri-
fied by flash silica chromatography (40% EtOAc–hexane) to
afford the product alcohol 2 as a colourless oil (2.68 g, 14.43
mmol, 90%). Rf (40% EtOAc–hexane) 0.31; IR (liquid film)
3495 (m, br), 2935 (s), 2864 (s) cmϪ1; δH (300 MHz, CDCl3) 3.98
(4H, m, OCH2CH2O), 3.62 (1H, d, J = 11 Hz, CHHOH), 3.48
(1H, d, J = 11 Hz, CHHOH), 2.80 (1H, s, OH), 1.30–1.80 (8H,
m), 1.02 (3H, s, CH3) ppm; δC (75 MHz, CDCl3) 113.9, 68.9,
64.7, 64.4, 41.9, 33.3, 30.6, 23.6, 20.6, 18.8 ppm; m/z (EI)
186.1 (4%, Mϩ), 159.9 (20%, [M Ϫ CH2O]ϩ), 112.8 (82%,
[M Ϫ C3H5O2]ϩ).
6-Hydroxy-2-(hydroxymethyl)-2-methylcyclohexan-1-one 5
Potassium hexamethyldisilazanide (0.5 M in THF, 12.93 ml,
6.46 mmol) was diluted with dry THF (100 ml) and purged with
N2. The solution was cooled to Ϫ78 ЊC (acetone–dry ice) and a
solution of 3 (1.00 g, 4.31 mmol) in dry THF (10 ml) was added
slowly. The reaction mixture was stirred for 30 minutes followed
by dropwise addition of 2-(p-tolylsulfonyl)-3-phenyl-1,2-oxazir-
idine (1.77 g, 6.46 mmol, prepared by the method of Davis11) in
dry THF (10 ml) over 10 minutes. The mixture was stirred for a
further 2 hours then quenched with sat. NH4Cl(aq) and allowed
to warm to room temperature. The mixture was then extracted
with ethyl acetate (50 ml), washed with sat. NaHCO3 solution
(50 ml), brine (50 ml), dried (MgSO4) and concentrated to
ca. 10 ml in vacuo. Addition of hexane (10 ml) precipitated
by-product as a white crystalline solid which was removed by
filtration. Removal of the remaining solvent and purification by
flash silica chromatography (EtOAc–hexane, 10:90) afforded
the crude product 4 as a pale yellow oil (0.66 g, 2.67 mmol,
62%). Rf 0.17 (EtOAc–petroleum ether, 10:90), which was used
directly in the next experiment.
To a solution of 5% palladium on charcoal (20 mg) in
degassed ethyl acetate (10 ml) under N2 was added a solution of
4 (0.50 g, 2.01 mmol) in ethyl acetate (5 ml), and the solution
was thoroughly degassed. The solution was then placed under a
hydrogen atmosphere and stirred for 15 hours. The solution was
then filtered through a short pad of silica and the solvent
removed in vacuo. The resultant oil was then purified by flash
silica chromatography (EtOAc) to afford 5 as a colourless oil
(0.27 g, 1.71 mmol, 85%, 3:1 mixture of diastereomers). Rf
(EtOAc) 0.46; IR (liquid film) 3391 (s, br), 2939 (s), 2873 (s),
1703 (s) cmϪ1; δH (300 MHz, d6-DMSO) 4.82 (0.25H, t, J = 6
Hz, minor CH2OH), 4.75 (0.75H, d, J = 5 Hz, major CHOH),
4.61 (0.25H, d, J = 5 Hz, minor CHOH), 4.42 (0.75H, t, J = 6
Hz, major CH2OH), 4.31 (1H, m, CHOH both diastereomers),
3.68 (0.25H, dd, J = 6, 11 Hz, minor CHHOH), 3.52 (0.75H,
2-Methyl-2-[(phenylmethoxy)methyl]cyclohexan-1-one 3
To a solution of NaH (0.78 g, 16.31 mmol, 1.2 eq.) in THF (20
ml) cooled to 0 ЊC was added a solution of 2 (2.52 g, 13.59
mmol) in THF (5 ml) dropwise over 10 minutes under N2. Once
J. Chem. Soc., Perkin Trans. 1, 2000, 3277–3289
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dd, J = 6, 11 Hz, major CHHOH), 3.33 (0.25H, dd, J = 6,
11 Hz, minor CHHOH), 3.21 (0.75H, dd, J = 6, 11 Hz, major
CHHOH), 1.30–2.15 (6H, m, CH2, both diastereomers), 1.08
(2.25H, s, major CH3), 0.95 (0.75H, s, minor CH3) ppm;
δC (75 MHz, d6-DMSO) 212.7, 211.9, 70.9, 70.3, 65.1, 64.6,
49.6, 48.0, 35.2, 35.0, 34.6, 33.5, 19.2, 18.5, 17.7, 17.2 ppm; m/z
(CI) 176 (80%, [M ϩ NH4]ϩ), 159 (21%, [M ϩ H]ϩ). HRMS
(CI) found [M ϩ NH4]ϩ, 176.1290. C8H18O3N requires
176.1287.
HRMS (CI) [M ϩ NH4]ϩ found 186.1860. C11H24ON requires
186.1858.
Data for 7c: Rf (20% EtOAc–hexane) 0.17; IR (liquid film)
3316 (br), 2927 (s), 2861 (s), 1599 (m) cmϪ1; δH (300 MHz,
CDCl ) 7.15–7.48 (5H, m, Ar), 6.04 (1H, m, CH᎐C), 3.82 (1H,
᎐
d, J =39 Hz, CHHOH), 3.67 (1H, d, J = 9 Hz, CHHOH), 3.54
(1H, m, benzylic H), 1.40–2.53 (6H, m) ppm; δC (75 MHz,
CDCl3), 145.2, 138.5, 130.7, 128.6, 127.5, 126.3, 71.1, 41.7,
32.7, 25.6, 18.6 ppm; m/z (EI) 188 (5%, Mϩ), 170 (30%,
[M Ϫ H2O]ϩ); HRMS (CI) [M ϩ NH4]ϩ found 206.1554.
C13H20ON requires 206.1545.
Preparation of tosylhydrazones 6a–c
Procedure for 6a. To a stirred suspension of tosylhydrazine
(5.0 g, 26.85 mmol) in MeOH (20 ml) was added 2-methyl-
cyclohexanone (3.0 g, 26.75 mmol) in MeOH (10 ml). Addition
of HCl (0.5 ml) caused the mixture to clear rapidly. After stir-
ring overnight crystals had formed which were filtered off,
washed with cold MeOH and dried yielding tosylhydrazone 6a
as a white crystalline solid, 6.14 g (26.44 mmol, 99%). Mp
122 ЊC [lit. 121–122 ЊC].23 The same procedure was used to
prepare the tosylhydrazones of 2-tert-butylcyclohexanone (to
give 6b in 92% yield), 2-phenylcyclohexanone (to give 6c in
73% yield), and 4-tert-butylcyclohexanone (in 99% yield).
Data for 6a: IR (Nujol mull) 3201 (m), 2956 (s), 2853 (s),
1632 (w), 1596 (w) cmϪ1; δH (300 MHz, CDCl3) 7.85 (2H, d,
J = 7.9 Hz, Ar), 7.30 (2H, d, J = 7.9 Hz, Ar), 2.57 (1H, m,
CHMe), 2.42 (3 H, s, CH3Ph), 2.24 (1H, m), 1.87 (2H, m), 1.72
(2H, m), 1.42 (2H, m), 1.22 (1H, m), 1.02 (3H, d, J = 6.6 Hz,
CHCH3) ppm; δC (75 MHz, CDCl3) 165.4, 143.9, 135.4, 129.4,
128.4, 39.3, 35.5, 26.6, 26.3, 24.6, 21.8, 17.1 ppm; m/z (ESϩ)
281.2 (100%, [M ϩ H]ϩ).
tert-Butyl(1,1-dimethyl)silyl [(6-methylcyclohex-1-enyl)methyl]
ether 8a
To a solution of 7a (0.578 g, 4.58 mmol) in DMF (5 ml) were
added imidazole (0.469 g, 6.88 mmol, 1.5 eq.) and DMAP
(0.005 g, catalytic amount). The solution was cooled to Ϫ10 ЊC
and TBDMSCl (0.761 g, 5.05 mmol, 1.1 eq.) was added and the
reaction mixture stirred overnight. The mixture was then
extracted with diethyl ether (3 × 10 ml), washed with water
(5 × 10 ml) and washed with brine (2 × 10 ml). The resulting
pale yellow oil was purified by silica column chromatography
(CH2Cl2) to yield 8a as a colourless oil (1.02 g, 4.25 mmol,
93%). Rf (CH2Cl2) 0.80; IR (liquid film) 2928 (s), 2856 (s), 1640
(w) cmϪ1; δH (300 MHz, CDCl ) 5.65 (m, 1H, CH᎐C), 4.12 (d,
᎐
3
1H, J = 13 Hz, CHHOSi), 4.03 (d, 1H, J = 13 Hz, CHHOSi),
2.36 (m, 1H), 2.00 (m, 2H), 1.50–1.80 (m, 3H), 1.41 (m, 1H),
1.05 (d, 3H, J = 7 Hz, CH3CH), 0.92 (s, 9H, t-Bu), 0.08 (s, 6H,
CH3Si) ppm; δC (75 MHz, CDCl3) 141.2, 121.6, 65.7, 31.1, 29.2,
26.0, 25.3, 19.5, 19.4, 18.4, Ϫ5.2 ppm; m/z (EI) 240 (100%, Mϩ).
Preparation of 6-substituted cyclohex-1-ene-1-methanols 7a–c
6-tert-Butyl-1-[(methoxymethoxy)methyl]cyclohex-1-ene 8b
Procedure for 7a. 2-Methylcyclohexanone tosylhydrazone 6a
(2.0 g, 7.14 mmol) was placed in a flame dried 100 ml 3-necked
RBQF flask fitted with a solids addition adapter, charged with
paraformaldehyde (1.5 g, 50 mmol). THF (50 ml) and TMEDA
(10 ml) were added and the solvent degassed with dry nitrogen.
The solution was then cooled to Ϫ78 ЊC (acetone–dry ice) and
n-BuLi (10 ml, 2.5 M in hexane) added dropwise via syringe.
The solution was then stirred at Ϫ78 ЊC for 30 minutes until a
bright orange colour was observed. The solution was then
warmed to RT slowly with the evolution of N2 gas, until a
yellow solution was obtained. After the evolution of gas had
ceased the solution was cooled once more to Ϫ78 ЊC and the
paraformaldehyde was added, giving rise to an exothermic reac-
tion. After stirring for 1 hour the solution was poured onto
crushed ice (20 g) and acidified to ~ pH 6.0 (c. HCl) and the
water layer extracted with diethyl ether (4 × 50 ml). The com-
bined organic layers were washed with 10% aqueous citric acid
(50 ml), saturated brine (2 × 50 ml), dried (MgSO4), and evap-
orated in vacuo. The resulting oil was purified by flash silica
chromatography (20% EtOAc–hexane) to give alcohol 7a as a
colourless oil (476 mg, 3.78 mmol, 53%). The same procedure
was used for the preparation of the 6-tert-butyl alcohol 7b (31%
yield) and 6-phenyl alcohol 7c (43% yield).
To a stirred solution of 7b (0.601 g, 3.58 mmol) in anhydrous
dimethoxymethane (10 ml) were added LiBr (62 mg, 0.72
mmol) and p-TsOHؒH2O (68 mg, 0.36 mmol) and the solution
stirred for 2 hours at which time a further portion of LiBr (62
mg, 0.72 mmol) was added and stirring continued for 4 hours at
room temperature. The mixture was then treated with saturated
NaCl(aq) and the mixture extracted with diethyl ether (2 ×
20 ml). The combined organic layers were dried (Na2SO4),
and concentrated in vacuo. The resulting oil was purified by
silica column chromatography (EtOAc–petroleum ether, 10:90)
to give the product as a colourless oil (0.642 g, 3.03 mmol,
85%). Rf (EtOAc–petroleum ether, 10:90) 0.55; IR (liquid film)
2942 (s), 2875 (s), 1061 (w) cmϪ1; δH (300 MHz, CDCl3) 5.90
(m, 1H, CH᎐C), 4.64 (2 × d, 2H, J = 6 Hz, CH OCH ), 4.15
᎐
2
(d, 1H, J = 12 Hz, CHHOMOM), 4.01 (d, 1H, J = 312 Hz,
CHHOMOM), 3.40 (s, 3H, CH3O), 2.10 (m, 2H), 1.78 (m,
2H), 1.55 (m, 3H), 1.02 (s, 9H, t-Bu) ppm; δC (75 MHz,
CDCl3) 136.7, 129.0, 95.4, 72.9, 55.4, 43.6, 34.2, 30.2, 26.3,
24.9, 19.9 ppm; m/z (CI) 230 (13%, [M ϩ NH4]ϩ), 151 (100%,
[M Ϫ OCH2OCH3]ϩ).
6-Phenyl-1-[(methoxymethoxy)methyl]cyclohex-1-ene 8c
Data for 7a: Rf (CH2Cl2) 0.23; IR (liquid film) 3331 (s, br),
To a solution of 7c (1.34 g, 7.10 mmol) in CH2Cl2 (20 ml) was
added diisopropylethylamine (1.86 ml, 1.38 g, 10.65 mmol, 1.5
eq.) and chloromethyl methyl ether (0.81 m, 0.86 g, 10.65 mmol,
1.5 eq.) under nitrogen at 0 ЊC and the solution stirred over-
night at room temperature. Aqueous KOH (2 M, 20 ml) was
then added and stirring continued for 30 minutes. The aqueous
phase was then extracted with diethyl ether, and the organic
phase dried (Na2SO4) and concentrated in vacuo. The resulting
oil was purified by silica column chromatography (5% EtOAc–
hexane) to afford 8c as a colourless oil (1.33 g, 5.74 mmol,
81%). Rf (5% EtOAc–hexane) 0.25; IR (liquid film) 2929 (s),
2865 (s), 1599 (m) cmϪ1; δH (300 MHz, CDCl3) 7.30 (5H, m,
2926 (s), 2870 (s), 1635 (m) cmϪ1; δH (300 MHz, CDCl3) 5.67
(1H, s, CH᎐C), 4.11 (1H, d, J = 12.5 Hz, CH OH), 3.98 (1H, d,
᎐
2
J = 12.5 Hz, CH2OH), 2.31 (1H, m), 2.00 (2H, m), 1.34–1.71
(4H, m), 1.04 (3H, d, J = 6.9 Hz, CHMe) ppm; δC (75 MHz,
CDCl3) 141.8, 123.7, 65.9, 31.3, 29.6, 25.5, 19.7, 19.5 ppm;
m/z (APCI) 126 ([M]ϩ, 70%). HRMS (CI) found [M ϩ NH4 Ϫ
H2O]ϩ, 126.1287. C8H16N requires 126.1283.
Data for 7b: Rf (CH2Cl2) 0.38; IR (liquid film) 3387 (br), 2953
(s), 2867 (s) 1582 (w) cmϪ1; δH (300 MHz, CDCl3) 5.90 (m, 1H,
CH᎐C), 4.15 (2 × d, 2H, J = 12 Hz, CH OH), 2.10 (m, 2H),
᎐
2
1.75 (m, 2H), 1.52 (m, 3H), 1.00 (s, 9H, t-Bu) ppm; δH (75
MHz, CDCl3) 140.5, 126.8, 68.5, 43.8, 34.2, 30.0, 26.4, 24.7,
20.2 ppm; m/z (EI) 168 (5%, Mϩ), 150 (12%, [M Ϫ H2O]ϩ);
Ar), 6.02 (1H, CH᎐C), 4.60 (2H, m, OCH O), 3.79 (1H, d, J = 9
᎐
2
Hz, CHHOMOM), 3.42 (1H, d, J = 9 Hz, CHHOMOM), 3.54
3284
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(1H, m, benzylic H), 3.28 (3H, s, OCH3), 1.50–2.30 (6H, M)
ppm; δC (75 MHz, CDCl3) 146.0, 135.6, 131.3, 128.5, 128.1,
125.9, 96.6, 75.7, 55.2, 41.6, 33.1, 25.3, 18.3 ppm; m/z (CI) 250
(60%, [M ϩ NH4]ϩ). HRMS (CI) [M ϩ NH4]ϩ found 250.1816.
C15H24O2N requires 250.1807.
(3 × 10 ml). The combined organic layers were dried (MgSO4)
and concentrated in vacuo. Flash column chromatography
afforded the product ketone as a colourless oil (0.610 g, 2.24
mmol, 74%). Rf (CH2Cl2) 0.51.
To a solution of 2-{tert-butyl(dimethyl)silyloxymethyl}-2-
hydroxy-3-methylcyclohexan-1-one (0.5668 g, 2.09 mmol)
in anhydrous THF (5 ml) was added excess tetra-n-butyl-
ammonium fluoride (1.0 M in 95% THF–H2O, 4.0 ml) and
glacial acetic acid (0.25 ml, 0.263 g, 4.38 mmol), and the reac-
tion mixture stirred for 12 hours at room temperature. Water
(5 ml) was then added and the mixture extracted with ethyl
acetate (3 × 10 ml). The combined organic layers were washed
with 10% citric acid (10 ml) and brine (10 ml), dried (Na2SO4)
and concentrated in vacuo. Silica column chromatography (60%
EtOAc–hexane) afforded 10a as a colourless oil (0.130 g, 0.83
mmol, 40%). Rf (60% EtOAc–hexane) 0.27; IR (liquid film)
3426 (br), 2980 (s), 2871 (s), 1713 (s) cmϪ1; δH (300 MHz,
CDCl3) 4.05 (d, 0.3H, J = 11 Hz, minor CHH2OH), 4.00 (d,
0.7H, J = 12 Hz, major CHH2OH), 3.87 (d, 0.7H, J = 12 Hz,
major CHH2OH), 3.74 (d, 0.3H, J = 11 Hz, minor CHH2OH),
3.21–3.45 (br s, 2H, OH), 1.50–2.75 (m, 7H), 1.09 (d, 0.7H,
J = 7 Hz, major CH3), 0.81 (d, 0.3H, J = 7 Hz, minor CH3)
ppm; δC (75 MHz, CDCl3) 213.2, 212.8, 82.9, 82.7, 67.1, 63.4,
44.5, 38.9, 37.7, 30.7, 28.8, 26.3, 22.7, 15.2, 13.2 ppm; m/z (CI)
176 (15%, [M ϩ NH4]ϩ); HRMS (CI) found [M ϩ NH4]ϩ
176.1295. C8H18O3N requires 176.1287.
Dihydroxylation of protected 6-substituted hydroxymethylcyclo-
hex-1-ene (preparation of 9a–c)
Preparation of 9a. To a 50% aqueous solution of t-BuOH (20
ml) were added K2CO3 (1.65 g, 11.94 mmol, 3.7 eq.), K3Fe(CN)6
(3.92 g, 11.94 mmol, 3.7 eq.), MeSO2NH2 (0.340 g, 3.57 mmol,
1.1 eq.) and K2OsO4ؒ2H2O (11 mg, 0.03 mmol, 1 mol%). The
solution was cooled to 0 ЊC (ice bath) and a solution of 8a (0.78
g, 3.24 mmol) in t-BuOH (2 ml) was added dropwise, and the
solution stirred vigorously for 24 hours at 0 ЊC. After the reac-
tion was judged to be complete by thin layer chromatography,
excess Na2SO3 (4.0 g) was added with ice cooling and the
mixture was stirred for 1 hour. The mixture was then extracted
with CH2Cl2 (20 ml). The aqueous portion was then further
extracted with CH2Cl2 (3 × 10 ml) and the combined organic
fractions washed with aqueous KOH (1 M), then dried
(MgSO4) and evaporated in vacuo. Silica column chromato-
graphy (3% EtOAc–CH2Cl2) afforded the product diol 9a as a
colourless oil (0.754 g, 3.11 mmol, 96%), as a mixture of two
diastereoisomers (7:3 ratio). Rf (CH2Cl2) 0.21; IR (liquid film)
3456 (br), 2926 (s), 2856 (s) cmϪ1; δH (300 MHz, CDCl3) 3.84
(2 × d, 1H, J = 10 Hz, major and minor CHHOH), 3.64 (2 × d,
1H, J = 10 Hz, major and minor CHHOH), 1.10–2.05 (m, 8H),
0.9–1.0 (m, 12H, CHCH3 and t-Bu), 0.10 (s, 6H, CH3Si) ppm;
δC (75 MHz, CDCl3) 74.5, 74.3, 73.2, 70.4, 69.2, 65.7, 35.9, 34.3,
29.7, 29.5, 25.8, 23.4, 18.9, 18.2, 14.9, Ϫ5.5 ppm; m/z (ESϪ) 273
(100%, [M Ϫ H]).
Diol 9b was prepared from 6-tert-butyl MOM ether 8b in
40% yield by the same procedure, except that the reaction was
increased to 7 days at room temperature. Data for 9b: Rf
(EtOAc–petroleum ether, 10:90) 0.55; IR (liquid film) 3446
(br), 2940 (s), 2875 (s) cmϪ1; δH (300 MHz, CDCl3) 4.66 (2H,
2 × d, J = 7 Hz, OCH2OCH3), 3.97 (1H, m, CHOH), 3.84 (2H,
s, CH2OMOM), 3.39 (3H, s, OCH3), 2.53 (2H, br s, OH), 1.10–
1.90 (7H, m), 1.02 (9H, s, t-Bu) ppm; δC (75 MHz, CDCl3) 97.3,
78.1, 71.2, 68.7, 55.7, 47.2, 33.9, 30.7, 29.0, 25.9, 19.9 ppm; m/z
(CI) 246 (8%, Mϩ).
3-(tert-Butyl)-2-hydroxy-2-(hydroxymethyl)cyclohexan-1-one 10b
To a solution of Dess–Martin periodinane14 (0.68 g, 1.62 mmol)
in CH2Cl2 (5 ml) was added a solution of 9b (0.20 g, 0.81 mmol)
in CH2Cl2 (5 ml) with stirring which was continued overnight
at room temperature. Once complete (15 hours) the reaction
mixture was diluted with diethyl ether (30 ml). Aqueous
1.0 M NaOH (10 ml) was added, and stirring was continued for
a further 20 minutes. The organic phase was then separated,
and the aqueous phase extracted with Et2O (3 × 10 ml). The
combined organic phases were dried (Na2SO4) and concen-
trated in vacuo. The resulting oil was then purified by flash silica
chromatography (40% EtOAc–hexane) to afford the product
ketone as a colourless oil (0.170 g, 0.70 mmol, 86%). Rf (40%
EtOAc–hexane) 0.53; IR (liquid film) 3446 (w), 2950 (s), 2869
(s), 1712 (s) cmϪ1; δH (300 MHz, CDCl3) 4.60 (2H, 2 × d, J = 7
Hz, OCH2OCH3), 4.52 (1H, s, OH), 4.16 (1H, d, J = 10 Hz,
CHH2ORMOM), 3.86 (1H, d, J = 10 Hz, CHH2ORMOM),
3.32 (3H, s, OMe), 2.59 (2H, m, CH2CO), 2.18 (1H, m), 1.95
(1H, m), 1.30–1.80 (3H, m), 1.05 (9H, s, t-Bu) ppm; δC (75
MHz, CDCl3) 211.6, 97.1, 84.4, 70.1, 58.5, 55.8, 38.4, 35.2, 30.6,
26.5, 25.7 ppm; m/z (CI) 244 (100%, Mϩ) 213 (24%,
[M Ϫ OCH3]ϩ), 183 (63%, [M Ϫ OCH2OCH3]ϩ).
To a stirred solution of 3-tert-butyl-2-hydroxy-2-[(methoxy-
methoxy)methyl]cyclohexan-1-one (94 mg, 0.30 mmol) in meth-
anol (5 ml) was added 40% c. HCl in methanol (5 ml) to give a
solution containing 20% HCl. Stirring was continued for 1 hour
and the reaction monitored by thin layer chromatography. Once
complete, water (5 ml) was added and the mixture extracted
with ethyl acetate (5 × 10 ml). The organic phase was washed
once with saturated NaHCO3 solution (5 ml) and dried
(MgSO4). Flash silica chromatography (40% EtOAc–petroleum
ether) afforded 10b as a colourless oil (48 mg, 0.18 mmol, 60%).
Rf (40% EtOAC–petroleum ether) 0.36; IR (liquid film) 3416
(s), 3371 (s), 2951 (s), 2865 (s), 1712 (s) cmϪ1; δH (300 MHz,
CDCl3) 4.60 (1H, s, OH), 4.05 (2H, two d, J = 10 Hz, CH2OH),
2.60 (2H, m), 2.20 (2H, m), 1.95 (1H, m), 1.35–1.75 (3H, m),
1.04 (s, 9H, t-Bu) ppm; δC (75 MHz, CDCl3) 212.7, 85.8,
64.3, 58.3, 38.1, 35.1, 30.5, 26.5, 25.6 ppm; m/z (EI) 200 (25%,
Mϩ), 182 (20%, [M Ϫ H2O]ϩ); HRMS (CI) [M ϩ NH4]ϩ found
218.1760. C11H24O3N requires 218.1756.
Diol 9c was prepared from 6-phenyl MOM ether 8c in 25%
yield by the same procedure, using a reaction time of 4 days at
room temperature. Data for 9c: Rf (30% EtOAc–hexane) 0.13;
IR (liquid film) 3415 (br), 2932 (s), 1600 (m) cmϪ1; δH (300
MHz, CDCl3) 7.20 (5H, m, Ar), 4.51 (2H, two d, J = 6 Hz,
OCH2OCH3), 4.14 (1H, m, CH(OH)), 3.93 (1H, d, J = 11 Hz,
OCHHOMOM), 3.34 (1H, m, benzylic H), 3.30 (3H, s, OMe),
3.23 (1H, d, J = 11 Hz, OCHHOMOM), 2.50 (2H, br s, OH),
1.94 (1H, m), 1.75 (3H, m), 1.60 (2H, m) ppm; δC (75 MHz,
CDCl3), 141.0, 129.6, 128.0, 126.7, 97.2, 75.1, 69.8, 68.9, 55.7,
45.3, 28.8, 28.3, 19.6 ppm; m/z (CI) 284 (24%, [M ϩ NH4]ϩ),
252 (82%, [M ϩ NH4 Ϫ MeOH]ϩ), 207 [58%, [M ϩ NH4 Ϫ
Ph]ϩ).
2-(Hydroxymethyl)-2-hydroxy-3-methylcyclohexan-1-one 10a
A solution of oxalyl chloride (0.32 ml, 0.425 g, 3.35 mmol,
1.1 eq.) in anhydrous CH2Cl2 (20 ml) was cooled to Ϫ78 ЊC
(acetone–dry ice bath) and anhydrous (DMSO (0.57 ml, 0.571
g, 7.31 mmol, 2.4 eq.) in CH2Cl2 (10 ml) added dropwise. The
solution was stirred for 5 minutes until gas evolution ceased.
A solution of 9a (0.836 g, 3.05 mmol) in CH2Cl2 (10 ml) was
added dropwise over 5 minutes. Stirring was continued for 15
minutes at Ϫ60 ЊC after which time anhydrous triethlamine
(1.541 g, 15.23 mmol, 5.0 eq.) was added, and stirring was con-
tinued for a further 10 minutes. The solution was then warmed
to room temperature and quenched with water (20 ml). The
aqueous layer was then separated and extracted with CH2Cl2
The same procedures were used to oxidise 9c to give 3-
phenyl-2-hydroxy-2-[(methoxymethoxy)methyl]cyclohexan-1-
J. Chem. Soc., Perkin Trans. 1, 2000, 3277–3289
3285

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one in 52% yield, which was deprotected in 83% yield to give
10c. Data for 10c: Rf (30% EtOAc–hexane) 0.22; IR (liquid
film) 3481 (br), 2930 (m), 2886 (m), 1718 (s), 1595 (w) cmϪ1; δH
(300 MHz, CDCl3) 7.30 (5H, m, Ar-H), 4.50 (2H, two d, J = 7
Hz, OCH2OCH3), 4.20 (1H, d, J = 10 Hz, CHHOMOM), 3.29
(3H, s, OCH3), 3.24 (1H, d, J = 10 Hz, CHHOMOM), 2.94
(1H, dd, J = 4, 13 Hz, CHPh), 2.72 (2H, m), 2.38 (2H, m), 2.02
(1H, m), 1.78 (1H, m) ppm; δC (75 MHz, CDCl3), 211.1, 138.8,
128.9, 128.3, 127.3, 97.1, 81.5, 70.4, 55.7, 54.6, 38.4, 28.3, 26.3
ppm; m/z (CI) 282 (29%, [M ϩ NH4]ϩ), 250 (32%, [M ϩ NH4 Ϫ
MeOH]ϩ), 205 (74%, [M ϩ NH4 Ϫ Ph]ϩ). Data for 10c: Rf (30%
EtOAc–hexane) 0.12; IR (liquid film) 3453 (br), 2925 (w), 2870
(w), 1703 (s), 1593 (w) cmϪ1; δH (300 MHz, CDCl3) 7.16–7.28
(5H, m, Ar), 4.26 (1H, br s, OH), 4.05 (1H, d, J = 12 Hz,
CHHOH), 3.37 (1H, d, J = 12 Hz, CHHOH), 2.85 (1H, dd,
J = 4, 13 Hz, benzylic H), 2.73 (1H, dt, J = 6, 14 Hz, CHax-
stirred at room temperature for 1.5 h. The solution was then
neutralised with solid NaHCO3 portionwise until effervescence
ceased, followed by addition of solid MgSO4. The solution was
filtered and the residue washed twice with methanol. The
material was then purified by flash silica chromatography (30%
EtOAc–hexane) to afford 13b as a yellow oil (0.97 g, 5.86 mmol,
82%). IR (liquid film) 3466 (br), 2954 (s), 2870 (m), 1617 (w),
1587 (m) cmϪ1; δH (300 MHz, CDCl3) 6.89 (1H, m), 6.73 (2H,
m), 5.73 (1H, s, OH), 5.41 (1H, s, OH), 1.45 (9H, s, t-Bu) ppm;
δC (75 MHz, CDCl3) 143.4, 142.9, 136.6, 119.2, 119.1, 112.9,
34.6, 29.5 ppm; m/z (EI) 166 (50%, Mϩ), 151 (100%, [M Ϫ
CH3]ϩ); HRMS (CI) [M ϩ NH4]ϩ found 184.1345, C10H18O2N
requires 184.1338. The same method was used to prepare
3-phenylcatechol (13a) from 12a, in 44% yield. Data: Rf (30%
EtOAc–petroleum ether) 0.05; IR (liquid film) 3542 (s) cmϪ1
;
δH (300 MHz, CDCl3) 7.50 (5H, s), 7.42 (1H, m), 6.93 (1H, t,
J = 7 Hz), 6.84 (1H, dd, J = 2.7 Hz), 5.48 (1H, br s), 5.33 (1H, br
s) ppm.
H C᎐O), 2.59 (1H, m, CH H C᎐O), 2.25 (2H, m), 1.94
᎐
᎐
ax eq
eq
(1H, m), 1.71 (1H, m) ppm; δC (75 MHz, CDCl3) 211.9, 138.5,
128.8, 128.1, 127.2, 82.4, 64.3, 54.5, 37.7, 27.8, 26.2 ppm; m/z
(EI) 220 (20%, Mϩ), 190 (12%, [M Ϫ CH2O]ϩ); HRMS (CI)
[M ϩ NH4]ϩ found 238.1448. C13H20O3N requires 238.1443.
(4-tert-Butylcyclohex-1-enyl)methanol 14
NЈ-(4-tert-Butylcyclohexylidene)-4-methylbenzene-1-sulfono-
hydrazide (prepared from 4-tert-butylcyclohexanone by the
above method in 99% yield, 2.00 g, 6.20 mmol) was placed in a
flame dried 100 ml 3-necked flash fitted with a solids addition
adapter charged with paraformaldehyde (0.65 g, 21.7 mmol).
THF (30 ml) and TMEDA (10 ml) were added, the solution was
then cooled to Ϫ78 ЊC (acetone–dry ice) and n-butyllithium (2.2
ml, 10.0 M in hexane, 3.5 eq., 21.71 mmol) added dropwise
via syringe. The solution was stirred at Ϫ78 ЊC for 30 minutes,
giving an orange precipitate. The solution was warmed to room
temperature slowly, with the evolution of N2 gas, giving a dark
green solution. After the evolution of gas had ceased the solu-
tion was cooled to Ϫ78 ЊC and paraformaldehyde added (exo-
thermic). After stirring for 1 hour, the colourless solution was
poured onto crushed ice and acidified to ~ pH 6.0 (c. HCl), and
the product extracted with diethyl ether (4 × 20 ml). The com-
bined organic layers were washed with 10% aqueous citric acid
(20 ml), saturated brine (2 × 20 ml), dried (MgSO4), then con-
centrated in vacuo. The resulting yellow oil was purified by flash
silica chromatography (EtOAc–hexane, 15:85) to give the
product 14 as a colourless oil (0.269 g, 1.60 mmol, 26%). Rf
(EtOAc–hexane, 15:85) 0.19; IR (liquid film) 3330 (br), 2943
(s), 2865 (s), 1632 (w) cmϪ1; δH (300 MHz, CDCl3) 5.68 (m, 1H,
1-tert-Butyl-2-(methoxymethoxy)benzene 11b
NaH (1.69 g, 35.2 mmol, 2 eq., dispersion in mineral oil) was
washed twice with dry hexane 5 ml) and re-suspended in dry
THF (50 ml ). 2-tert-Butylphenol (2.65 g, 17.6 mmol, 2.7 ml)
was added dropwise with ice cooling and stirring continued for
30 minutes until H2 evolution had cased. Chloromethyl methyl
ether (CARE! known carcinogen, 4.38 g, 54.4 mmol, 3.1 eq.)
was then added dropwise and the solution stirred for a further
12 hours. Aqueous KOH (2 M, 50 ml) was then added and
stirring continued for 30 minutes. The aqueous phase was then
extracted with diethyl ether, and the organic phase dried
(Na2SO4) and concentrated in vacuo. The resulting oil was puri-
fied by column chromatography (hexane) to afford 11b as a
colourless oil (3.03 g, 15.6 mmol, 89%). Rf (hexane) 0.40; IR
(liquid film) 2954 (s), 1598 (w), 1580 (m) cmϪ1; δH (300 MHz,
CDCl3) 6.90–7.35 (4H, m, Ar-H), 5.26 (2H, s, OCH2OCH3),
3.53 (3H, s, OCH2OCH3), 1.43 (9H, s, t-Bu) ppm; m/z (CI) 212
(20%, [M ϩ NH4]ϩ), 195 (20%, [M ϩ H]ϩ), 194 (18%, Mϩ).
The same method was used to prepare 1-phenyl-2-(methoxy-
methoxy)benzene (11a) from 2-phenylphenol, in 65% yield.
CH᎐C), 4.00 (2 × d, 2H, J = 12 Hz, CH OH), 2.11 (m, 2H),
᎐
2
1-tert-Butyl-2-(methoxymethoxy)phenol 12b
1.84 (m, 2H), 1.55 (m, 1H), 1.24 (2H, m), 0.89 (s, 9H, t-Bu)
ppm; δC (75 MHz, CDCl3) 137.4, 123.3, 67.3, 44.2, 32.2, 27.2,
27.0, 26.5, 23.8 ppm; m/z (EI) 168 (11%, Mϩ), 150 (19%,
[M Ϫ H2O]ϩ); HRMS (CI) [M ϩ NH4]ϩ found 186.1865.
C11H24ON requires 186.1858.
To a solution of 11b (3.01 g, 15.5 mmol) in dry THF (50 ml)
was added n-butyllithium (10.1 ml, 1.7 M solution in hexane,
17.1 mmol, 1.1 eq.) with ice cooling under N2. The solution was
warmed to room temperature and stirred for 1 hour (yellow
colour observed), then cooled to 0 ЊC (ice bath) and quenched
with trimethyl borate (1.94 g, 18.1 mmol, 1.2 eq.). Stirring was
continued for a further 30 minutes, then the solution was con-
centrated in vacuo to afford a solid. The solid was resuspended
in aqueous acetone (20%, 50 ml) containing NaHCO3 (5 g).
Oxone (9.54 g, 15.5 mmol, 1 eq.) was added, and stirring con-
tinued. After 5 minutes solid NaHSO3 (2 g) was added. The
solution was then extracted with ethyl acetate (3 × 50 ml), dried
(Na2SO4) and concentrated in vacuo. The resulting oil was puri-
fied by flash silica chromatography (EtOAc–hexane, 1:19) to
afford 12b as a yellow oil (2.12 g, 10.1 mmol, 65%). Rf (EtOAc–
hexane, 1:19) 0.39; δH (300 MHz, CDCl3) 6.70–7.10 (3H, m,
Ar-H), 6.12 (1H, s, ArOH), 5.73 (2H, s, ArOCH2OMe), 3.53
(3H, s, OCH2CH3), 1.43 (9H, s, t-Bu) ppm. The same method
was used to prepare 1-phenyl-2-(methoxymethoxy)phenol (12a)
from 11a, in 81% yield.
4-tert-Butyl-1-[(methoxymethoxy)methyl]cyclohexane-1,2-diol
15, 16
Alcohol 14 was converted to its MOM ether using the same
procedure employed for preparation of 8b above, in 81% yield.
Data: Rf (EtOAc–hexane, 10:90) 0.27; IR (liquid film) 2945 (s),
2867 (s) cmϪ1; δH (300 MHz, CDCl ) 5.72 (m, 1H, CH᎐C), 4.64
᎐
3
(s, 2H, OCH2OCH3), 3.96 (s, 2H, CH2OMOM), 3.39 (s, 3H,
OCH2OCH3), 2.10 (m, 3H), 1.85 (m, 2H), 1.29 (m, 2H), 0.90 (s,
9H, t-Bu) ppm; δC (75 MHz, CDCl3) 134.2, 125.7, 95.3, 71.6,
55.1, 44.0, 32.2, 27.5, 27.2, 26.6, 23.8 ppm; m/z (CI) 230 (44%,
[M ϩ NH4]ϩ), 151 (100%, [M Ϫ OCH2OMe]ϩ).
To a 50% aqueous solution of t-BuOH (10 ml) were added
K2CO3 (589 mg, 4.26 mmol, 3.7 eq.), K3Fe(CN)6 (1.41 g, 4.26
mmol, 3.7 eq.), MeSO2NH2 (123 mg, 1.29 mmol, 1.1 eq.) and
K2OsO4ؒ2H2O (5 mg, 0.01 mmol, 1 mol%). The solution was
cooled to 0 ЊC (ice bath) and a solution of 4-tert-butyl-1-
[(methoxymethoxy)methyl]cyclohex-1-ene (247 mg, 1.17 mmol)
in t-BuOH (2 ml) added dropwise, then the solution was stirred
vigorously for 24 hours. Solid Na2SO3 (3 g) was added with ice
3-tert-Butylcatechol 13b
To a solution of 12b (1.50 g, 7.14 mmol) in methanol (20 ml)
was added 20% c. HCl in methanol (10 ml), and the reaction
3286
J. Chem. Soc., Perkin Trans. 1, 2000, 3277–3289

View Article Online
cooling and the mixture was stirred for 1 hour. The mixture was
then extracted with CH2Cl2 (10 ml). The aqueous portion was
then further extracted with CH2Cl2 (3 × 10 ml). The combined
organic fractions were washed with aqueous KOH (1 M, 10 ml),
dried (MgSO4) and evaporated in vacuo to give an oil, found to
contain 1:1 mixture of diastereomers. Careful silica column
chromatography (petroleum ether–EtOAc, 40:60) allowed
separation of the products to give compounds 15 and 16.
Compound 15 (axial–CH2OMOM) as a colourless oil (0.13
g, 0.53 mmol, 45%). Rf (EtOAc–petroleum ether, 60:40) 0.28;
IR (liquid film) 3419 (br), 2951 (s), 2867 (s) cmϪ1; δH (300 MHz,
CDCl3) 4.68 (s, 2H, OCH2OCH3), 3.89 (t, 1H, J = 3 Hz,
equatorial CH(OH)), 3.66 (d, 1H, J = 11 Hz, CH2OMOM),
3.56 (d, 1H, J = 11 Hz, CH2OMOM), 3.40 (s, 3H, OCH2OCH3),
2.97 (s, 1H, OH), 2.65 (br s, 1H, OH), 1.97 (m, 1H), 1.50–1.85
(m, 4H), 1.25 (m, 1H), 1.02 (m, 1H), 0.87 (s, 9H, t-Bu) ppm;
δC (75 MHz, CDCl3) 97.2, 74.2, 71.4, 55.5, 46.1, 39.5, 32.5, 30.9,
27.5, 21.0 ppm; m/z (CI) 264 (8%, [M ϩ NH4]ϩ), 232 (58%,
[M ϩ NH4 Ϫ MeOH]ϩ).
Compound 16 (equatorial–CH2OMOM) as a white crystal-
line solid (0.12 g, 0.50 mmol, 42%). Mp 75–76 ЊC; Rf (EtOAc–
petroleum ether, 60:40) 0.34; IR (liquid film) 3459 (br), 2953
(s), 2865 (s) cmϪ1; δH (300 MHz, CDCl3) 4.67 (2 × d, 2H, J = 7
Hz, OCH2OCH3), 3.66 (dd, 1H, J = 4 and 11 Hz, axial
CH(OH)), 3.58 (2 × d, 2H, J = 10 Hz, CH2OMOM), 3.40 (s,
3H, OCH2OCH3), 2.78 (br s, 2H, OH), 1.80 (m, 2H), 1.52 (m,
1H), 1.31 (m, 4H), 0.90 (s, 9H, t-Bu) ppm; δC (75 MHz, CDCl3)
97.1, 74.2, 71.2, 55.5, 46.1, 32.5, 32.3, 30.9, 27.5, 21.0 ppm;
m/z (CI) 264 (14%, [M ϩ NH4]ϩ), 232 (100%, [M ϩ NH4 Ϫ
MeOH]ϩ).
m), 1.45 (2H, m), 0.91 (9H, s, t-Bu) ppm; δH (75 MHz, CDCl3)
215.5, 75.8, 66.7, 50.2, 39.9, 34.5, 33.0, 27.6, 21.2 ppm; m/z (CI)
218 (17%, [M ϩ NH4]ϩ); HRMS (CI) [M ϩ NH4]ϩ found
218.1760. C11H24O3N requires 218.1756.
The same procedures were used to oxidise diol 16 in 83%
yield to the corresponding ketone, which was deprotected in
79% yield to give the (2S,5S)-isomer 18. Data for protected
ketone: Rf (EtOAc–hexane, 30:70) 0.30; IR (liquid film) 3461
(br), 2949 (s), 2869 (s), 1715 (s) cmϪ1; δH (300 MHz, CDCl) 4.62
(2H, 2 × d, J = 6, 7 Hz, OCH2OCH3), 4.03 (1H, d, J = 10 Hz,
CHHOR), 3.62 (1H, d, J = 10 Hz, CHHOR), 3.40 (1H, s, OH),
3.35 (3H, s, OCH3), 2.60 (1H, m), 2.18–2.40 (2H, m), 1.89 (1H,
m), 1.63–1.40 (3H, m), 0.93 (9H, s, t-Bu) ppm; δC (75 MHz,
CDCl3), 212.1, 96.8, 78.7, 72.1, 55.5, 50.4, 39.9, 36.6, 32.8, 27.2,
24.0 ppm; m/z (CI) 262 (19%, [M ϩ NH4]ϩ), 230 (18%, [M ϩ
NH4 Ϫ MeOH]ϩ). Data for 18: Rf (40% EtOAc–petroleum
ether) 0.18; IR (liquid film) 3326 (br), 2954 (s), 2865 (m), 1716 (s)
cmϪ1; δH (300 MHz, CDCl3) 3.94 (1H, d, J = 12 Hz, CH2OH),
3.70 (1H, d, J = 12 Hz, CH2OH), 2.60 (1H, m), 2.35 (1H, t,
J = 12 Hz), 2.25 (1H, m), 1.85 (1H, m), 1.40–1.60 (3H, m), 0.91
(9H, s, t-Bu) ppm; δC (75 MHz, CDCl3) 213.0, 80.0, 66.9, 50.8,
39.8, 36.5, 33.0, 27.3, 23.9 ppm; m/z (CI) 218 (15%, [M ϩ
NH4]ϩ); HRMS (CI) [M ϩ NH4]ϩ found 218.1762. C11H24O3N
requires 218.1756.
Ethyl 2-(2-phenylcyclohexylidene)acetate 19
To a suspension of NaH (50% dispersion in mineral oil, 2.19 g,
45.69 mmol) in THF (50 ml) was added triethyl phosphono-
acetate (9.0 ml, 10.24 g, 45.69 mmol) dropwise at 0 ЊC (ice
bath). Once effervescence had ceased, a solution of 2-phenyl-
cyclohexanone (5.31 g, 30.46 mmol) in dry THF (10 ml) was
added dropwise over 10 minutes, and the reaction stirred for
1 hour at room temperature. Water (50 ml) and ethyl acetate (50
ml) were added, and the phases were separated. The aqueous
phase was extracted with ethyl acetate (3 × 20 ml), and the
combined organic phase dried (Na2SO4). The crude product
was then purified by flash silica chromatography (5% EtOAc–
hexane) to afford 19 as a colourless oil (5.55 g, 22.74 mmol,
75%). Rf (5% EtOAc–hexane) 0.34; IR (liquid film) 2930 (s),
2857 (s), 1711 (s), 1640 (s) cmϪ1; δH (300 MHz, CDCl3) 7.17–
(2R,5S)-5-tert-Butyl-2-hydroxy-2-(hydroxymethyl)cyclohexan-
1-one 17
To a solution of Dess–Martin periodinane (3.24 g, 7.65 mmol)
in CH2Cl2 (10 ml) was added a solution of diol 15 (0.62 g, 2.55
mmol) in CH2Cl2 (10 ml), and the reaction was stirred overnight
at room temperature. The reaction mixture was diluted with
diethyl ether (30 ml) and aqueous 1.0 M NaOH (30 ml) was
added, and the mixture was stirred for a further 20 minutes. The
organic phase was then separated, and the aqueous phase
extracted with diethyl ether (3 × 20 ml). The combined organic
phase was then dried (Na2SO4), and concentrated in vacuo. The
resulting oil was then purified by flash silica chromatography
(EtOAc–hexane, 30:70) to afford the product as a colourless oil
(0.55 g, 2.24 mmol, 88%). Rf (EtOAc–hexane, 30:70) 0.36; IR
(liquid film) 3406 (br), 2952 (s), 2870 (s), 1712 (s) cmϪ1; δH (300
MHz, CDCl3) 4.68 (2H, 2 × d, J = 6, 7 Hz, OCH2OCH3), 3.90
(1H, d, J = 10 Hz, CHHOR), 3.45 (1H, d, J = 10 Hz, CHHOR),
3.40 (3H, s, OCH2OCH3), 2.75 (1H, t, J = 12 Hz, OH), 2.32
(1H, m), 2.05 (1H, m), 1.40–1.80 (5H, m), 0.92 (9H, s, t-Bu)
ppm; δC (75 MHz, CDCl3) 212.7, 97.2, 76.1, 71.5, 55.4, 50.5,
39.7, 35.7, 33.0, 27.2, 21.3 ppm; m/z (CI) 262 (18%,
[M ϩ NH4]ϩ), 230 (19%, [M ϩ NH4 Ϫ MeOH]ϩ), 213 (100%,
[M Ϫ OCH3]ϩ).
7.40 (5H, m, Ar-H), 5.13 (1H, s, CH᎐C), 4.10 (2H, q, J = 7 Hz,
᎐
OCH2CH3), 3.75 (1H, m), 3.43 (1H, dd, J = 4, 11 Hz, CHPh),
2.25 (1H, m), 2.00 (4H, m), 1.65 (2H, m), 1.25 (3H, t, J = 7 Hz,
OCH2CH3) ppm; δC (75 MHz, CDCl3) 167.1, 165.4, 141.7,
128.5, 126.6, 114.4, 59.6, 51.8, 34.3, 30.1, 28.1, 25.9, 14.2 ppm;
m/z (EI) 244 (84%, Mϩ), 199 (51%, [M Ϫ C2H5O]ϩ).
Ethyl 2-(6-phenylcyclohex-1-enyl)acetate 20
To a solution of diisopropylamine (3.16 ml, 2.28 g, 22.55 mmol)
in dry THF (50 ml) was added n-butyllithium (2.38 M solution
in hexane, 9.47 ml, 22.55 mmol) dropwise via syringe at Ϫ78 ЊC
under nitrogen. The solution was warmed to room temperature
for 10 minutes, then cooled to Ϫ78 ЊC prior to the addition of a
solution of ethyl 2-(2-phenylcyclohexylidene)acetate (5.00 g,
20.50 mmol) in dry THF (10 ml) dropwise over 5 minutes. The
solution was maintained at Ϫ78 ЊC for a further 30 minutes
before the addition of aqueous NH4Cl (1 M, 50 ml). The solu-
tion was warmed to room temperature, diluted with ethyl acet-
ate (50 ml), and the phases separated. The aqueous phase was
extracted with ethyl acetate (3 × 20 ml) and the combined
organic phase dried (Na2SO4) and evaporated in vacuo. The
resulting oil was then purified by careful flash silica chromato-
graphy (2% EtOAc–hexane) to afford 20 as a colourless oil (4.27
g, 17.51 mmol, 85%). Rf (2% EtOAc–hexane) 0.11; IR (liquid
film) 2931 (m), 2861 (m), 1731 (s), 1559 (m) cmϪ1; δH (300 MHz,
To
a
solution of 5-tert-butyl-2-hydroxy-2-[(methoxy-
methoxy)methyl]cyclohexan-1-one (330 mg, 1.35 mmol) in
methanol (5 ml) was added 40% c. HCl in methanol (5 ml) to
give a solution containing 20% HCl. Stirring was continued for
1 hour and the reaction monitored by thin layer chrom-
atography. Once complete, water (5 ml) was added and the mix-
ture extracted with ethyl acetate (5 × 10 ml). The organic phase
was washed with saturated NaHCO3 solution (5 ml), dried
(MgSO4) and evaporated in vacuo. Flash silica chromatography
(40% EtOAC–petroleum ether) afforded 17 as a colourless oil
(199 mg, 1.00 mmol, 74%). Rf (40% EtOAc–petroleum ether)
0.20; IR (liquid film) 3448 (s), 3238 (s), 2953 (s), 2865 (s), 1705
(s) cmϪ1; δH (300 MHz, CDCl3) 3.85 (1H, d, J = 12 Hz,
CH2OH), 3.30 (1H, d, J = 12 Hz, CH2OH), 2.90 (2H, br s, OH),
2.72 (1H, t, J = 12 Hz), 2.30 (1H, m), 1.89 (1H, m), 1.73 (2H,
CDCl ) 7.15–7.35 (5H, m, Ar-H), 5.88 (1H, m, CH᎐C),
᎐
3
4.05 (2H, 2 × q, J = 7 Hz, OCH2CH3), 3.54 (1H, br m, CHPh),
2.88 (1H, d, J = 15 Hz, CHHCO2Et), 2.75 (1H, d, J = 15 Hz,
CHHCO2Et), 2.18 (2H, m), 2.02 (1H, m), 1.50–1.75 (3H, m),
J. Chem. Soc., Perkin Trans. 1, 2000, 3277–3289
3287

View Article Online
1.22 (3H, t, J = 7 Hz, OCH2CH3) ppm; δC (75 MHz, CDCl3)
172.1, 144.6, 132.3, 128.5, 128.4, 128.3, 126.1, 60.3, 44.1, 41.6,
32.5, 25.5, 18.6, 14.1 ppm; m/z (EI) 244 (40%, Mϩ), 198 (100%,
[M Ϫ EtOH]ϩ).
3366 (br), 2937 (m), 2859 (m), 1702 (s), 1598 (w) cmϪ1; δH (300
MHz, CDCl3) 7.15–7.30 (5H, m, Ar-H), 4.34 (1H, br s,
OH), 3.96 (2H, q, J = 8 Hz, CH2CH2OH), 3.80 (1H, t, J = 8 Hz,
CH2CH2OH), 3.48 (1H, m, CHPh), 2.80 (2H, t, J = 8 Hz,
CH2CH2OH), 1.80–2.60 (4H, m), 1.36 (2H, m) ppm; δC (75
MHz, CDCl3) 206.4, 140.9, 129.4, 128.5, 127.9, 127.2, 104.7,
62.8, 49.7, 34.9, 32.6, 29.0, 22.7 ppm; m/z (CI) 234 (60%, Mϩ),
216 (30%, [M Ϫ H2O]ϩ); HRMS (CI) [M ϩ NH4]ϩ found
252.1605. C14H22O3N requires 252.1605.
[2-(6-Phenylcyclohex-1-enyl)ethoxy]methoxymethane 21
To a suspension of lithium aluminium hydride (1.21 g, 31.98
mmol) in dry THF (50 ml) was added 20 (3.12 g, 12.79 mmol)
under nitrogen. The solution was then heated under gentle
reflux for 12 hours, then cooled to 0 ЊC (ice bath), then carefully
quenched by the slow addition of water (50 ml). The solution
was then acidified to ~ pH 7 (1 M HCl), then diluted with ethyl
acetate (50 ml), and the phases separated. The aqueous phase
was extracted with ethyl acetate (3 × 20 ml). The combined
organic phases were washed with aqueous NaHCO3 (30 ml),
brine (30 ml), dried (Na2SO4), and evaporated in vacuo. The
resulting oil was then purified by flash silica chromatography
(20% EtOAc–hexane) to afford the product alcohol as a colour-
less oil (2.49 g, 12.35 mmol, 96%). Rf (20% EtOAc–hexane)
0.20; IR (liquid film) 3301 (br), 2928 (s), 2858 (s), 1599 (m)
cmϪ1; δH (300 MHz, CDCl3) 7.15–7.35 (5H, m, Ar-H), 5.85
Inhibition of 2,3-dihydroxyphenylpropionate 1,2-dioxygenase
(MhpB)
Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxy-
genase was purified to near homogeneity by the method
described previously.6,7 Enzyme was re-activated by treatment
with iron() ammonium sulfate and sodium ascorbate prior to
assay.6 Stock solutions of inhibitors were made in water at 50–
100 mM concentration. MhpB was assayed by UV spec-
troscopy, monitoring the appearance of the extradiol ring
fission product at 394 nm (ε = 15600 MϪ1 cmϪ1) at pH 8.0 in 50
mM potassium phosphate buffer. Assays (1.0 ml) contained
50 mM potassium phosphate buffer, 50–150 µM 2,3-dihydroxy-
phenylpropionic acid, 1–10 mM inhibitor, and 0.1 unit of
re-activated enzyme. Assays were carried out at 20 ЊC, in dupli-
cate, at a fixed concentration of substrate, and variable concen-
trations of inhibitor. Ki values and type of inhibition were then
determined by Dixon plot (1/v versus [I]).16 In each case linear
plots were obtained, and competitive inhibition was observed.
Data are shown in Table 1.
(1H, s, CH᎐C), 3.58 (2H, m, CH OH), 3.39 (1H, m, CHPh),
᎐
2
1.90–2.25 (4H, m), 1.40–1.74 (4H, m) ppm; δC (75 MHz,
CDCl3) 145.2, 135.2, 128.6, 128.4, 127.0, 126.2, 60.6, 43.9,
39.3, 32.9, 25.6, 18.9 ppm; m/z (EI) 202 (35%, Mϩ), 184
(36%, [M Ϫ H2O]ϩ), 170 (30%, [M Ϫ MeOH]ϩ); HRMS (CI)
[M ϩ NH4]ϩ found 220.1701. C14H22ON requires 220.1701.
The alcohol was then converted to the corresponding MOM
ether 21 in 94% yield, using the same procedure used above for
preparation of 8c. Data for 21: Rf (5% EtOAc–hexane) 0.25; IR
(liquid film) 2928 (s), 2879 (s), 1599 (w) cmϪ1; δH (300 MHz,
Inhibition of protocatechuate 3,4-dioxygenase
CDCl ) 7.17–7.34 (5H, m, Ar-H), 5.80 (1H, m, CH᎐C), 4.56 (2H,
᎐
3
Pseudomonas sp. protocatechuate 3,4-dioxygenase was pur-
chased from Sigma Chemical Co. The enzyme was assayed by
UV spectroscopy, monitoring the appearance of the intradiol
ring fission product at 290 nm (ε = 3890 MϪ1 cmϪ1).24 Assays
(1.0 ml) contained 50 mM Tris buffer pH 8.5, 50 µM proto-
catechuic acid, 1–20 mM inhibitor, and 0.05 unit enzyme.
Assays were carried out at 20 ЊC, in duplicate, at a fixed concen-
tration of substrate, and variable concentrations of inhibitor.
The IC50 value for 18 was determined by pot of v vs. [I] (see
Table 1). Under these conditions a Km value of 20 µM was
determined for protocatechuic acid.
s, OCH2OCH3), 3.54 (2H, t, J = 7 Hz, CH2CH2OMOM), 3.38
(1H, m, CHPh), 3.33 (3H, s, OCH3), 1.93–2.24 (5H, m), 1.45–
1.73 (3H, m) ppm; δC (75 MHz, CDCl3) 145.2, 135.5, 128.5,
128.1, 125.9, 125.5, 96.2, 66.2, 55.1, 44.2, 35.9, 32.7, 25.5, 18.8
ppm; m/z (CI) 264 (35%, [M ϩ NH4]ϩ).
2-Hydroxy-2-(2-hydroxyethyl)-3-phenylcyclohexan-1-one 22
Dihydroxylation of 21 was carried out using the procedure
given above for preparation of 9a, to give after chromatography
a 4:1 mixture of diastereomeric diols as a colourless oil (1.37 g,
4.92 mmol, 98%). Rf (30% EtOAc–hexane) 0.15; IR (liquid film)
3456 (br), 2931 (s), 2875 (s), 1660 (w) cmϪ1; δH (300 MHz,
CDCl3) 7.14–7.31 (5H, m, Ar-H), 4.57 (0.2H, 2 × d, J = 8 Hz,
minor OCH2OCH3), 4.49 (0.8H, 2 × d, J = 7 Hz, major
OCH2OCH3), 4.00 (1H, m, CHPh), 3.43–3.69 (2H, m,
CH2OR), 3.35 (0.6H, s, minor OCH3), 3.28 (2.4H, s, major
OCH3), 3.22 (0.8H, dd, J = 5, 9 Hz, major CHOH), 2.60–3.05
(2H, br s, OH), 2.50 (0.2H, dd, J = 4, 14 Hz, minor CHOH),
1.45–2.00 (6H, m, CH2) ppm; δC (75 MHz, CDCl3) 142.6, 141.2,
129.9, 129.5, 128.3, 127.9, 126.6, 96.6, 75.5, 74.9, 74.2, 71.0,
64.3, 64.0, 55.8, 55.7, 51.1, 46.8, 37.5, 30.9, 29.5, 29.1, 28.8,
28.4, 24.1, 19.7 ppm; m/z (CI) 298 (5%, [M ϩ NH4]ϩ), 266
(25%, [M ϩ NH4 Ϫ MeOH]ϩ).
Using the methods given above for preparation of 10b, the
mixture of diols was then oxidised using the Dess–Martin
periodinane in 52% yield to give the corresponding ketone,
which was deprotected under acidic conditions to give 22 in
80% yield. Data for ketone: Rf (30% EtOAc–hexane) 0.35; IR
(liquid film) 3448 (br), 2934 (s), 2871 (s), 1703 (s), 1599 (w)
cmϪ1; δH (300 MHz, CDCl3), 7.20–7.40 (5H, m, Ar-H), 4.41
(2H, 2 × d, J = 7 Hz, OCH2OCH3), 3.43 (2H, m, CH2OMOM),
3.28 (3H, s, OCH3), 2.92 (2H, m), 2.61 (1H, m), 2.40 (2H, m),
2.25 (1H, m), 1.97 (1H, m), 1.65 (1H, m), 1.55 (1H, m) ppm;
δC (75 MHz, CDCl3) 213.7, 139.2, 129.5, 128.0, 127.2, 96.8,
79.9, 62.8, 56.9, 55.5, 38.3, 33.5, 28.1, 26.9 ppm; m/z (CI) 296
(5%, [M ϩ NH4]ϩ), 264 (15%, [M ϩ NH4 Ϫ MeOH]ϩ). Data
for 22: Rf (30% EtOAc–hexane) 0.14; IR (CH2Cl2 solution)
Crystal structure determinations
Crystal data for 10b. C11H20O3, M = 200.27, triclinic, a =
6.5434(13), b = 6.8746(14), c = 13.017(3) Å, U = 562.2(2) Å3,
T = 150(2) K, space group P1, Z = 2, µ = 0.084 mmϪ1, 5176
¯
reflections measured, 1954 unique reflections (Rint = 0.0659), R
indices (all data) R1 = 0.0851, wR2 = 0.2018. CCDC reference
number 207/470.
Crystal data for 10c. C13H16O3, M = 220.26, monoclinic, a =
14.783(3), b = 5.9121(12), c = 12.150(2) Å, U = 1059.6(4) Å3,
T = 150(2) K, space group P21/c, Z = 4, µ = 0.097 mmϪ1, 9544
reflections measured, 2400 unique reflections (Rint = 0.0496), R
indices (all data) R1 = 0.0599, wR2 = 0.1586. CCDC reference
number 207/470.
Acknowledgements
We thank EPSRC and Zeneca Pharmaceuticals for provision of
a CASE studentship (C. J. W.), and Professor M. Hursthouse
(University of Southampton) for solution of the X-ray crystal
structures for 10b and 10c.
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