Toluene dioxygenase-catalyzed synthesis and reactions of cis -diol metabolites derived from 2- and 3-methoxyphenols

Article abstract of DOI:10.1021/jo5028968

Using toluene dioxygenase as biocatalyst, enantiopure cis-dihydrodiol and cis-tetrahydrodiol metabolites, isolated as their ketone tautomers, were obtained from meta and ortho methoxyphenols. Although these isomeric phenol substrates are structurally similar, the major bioproducts from each of these biotransformations were found at different oxidation levels. The relatively stable cyclohexenone cis-diol metabolite from meta methoxyphenol was isolated, while the corresponding metabolite from ortho methoxyphenol was rapidly bioreduced to a cyclohexanone cis-diol. The chemistry of the 3-methoxycyclohexenone cis-diol product was investigated and elimination, aromatization, hydrogenation, regioselective O-exchange, Stork-Danheiser transposition and O-methylation reactions were observed. An offshoot of this technology provided a two-step chemoenzymatic synthesis, from meta methoxyphenol, of a recently reported chiral fungal metabolite; this synthesis also established the previously unassigned absolute configuration.

Full text of DOI:10.1021/jo5028968

Article
pubs.acs.org/joc
Toluene Dioxygenase-Catalyzed Synthesis and Reactions of cis-Diol
Metabolites Derived from 2- and 3‑Methoxyphenols
Derek R. Boyd,*,† Narain D. Sharma,† John F. Malone,† Peter B. A. McIntyre,† Colin McRoberts,§
Stewart Floyd,§ Christopher C. R. Allen,‡ Amit Gohil,‡ Simon J. Coles,∥ Peter N. Horton,∥
and Paul J. Stevenson*,†
†School of Chemistry and Chemical Engineering, Queen’s University of Belfast, Belfast BT9 5AG, U.K.
‡School of Biological Sciences, Queen’s University of Belfast, Belfast, BT9 5AG, U.K.
§Agri-food and Biosciences Institute for Northern Ireland, Belfast, BT9 5PX, U.K.
∥National Crystallography Service, School of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K.
S
* Supporting Information
ABSTRACT: Using toluene dioxygenase as biocatalyst, enantiopure cis-
dihydrodiol and cis-tetrahydrodiol metabolites, isolated as their ketone
tautomers, were obtained from meta and ortho methoxyphenols. Although
these isomeric phenol substrates are structurally similar, the major
bioproducts from each of these biotransformations were found at different
oxidation levels. The relatively stable cyclohexenone cis-diol metabolite from
meta methoxyphenol was isolated, while the corresponding metabolite from
ortho methoxyphenol was rapidly bioreduced to a cyclohexanone cis-diol.
The chemistry of the 3-methoxycyclohexenone cis-diol product was
investigated and elimination, aromatization, hydrogenation, regioselective
O-exchange, Stork−Danheiser transposition and O-methylation reactions were observed. An offshoot of this technology provided
a two-step chemoenzymatic synthesis, from meta methoxyphenol, of a recently reported chiral fungal metabolite; this synthesis
also established the previously unassigned absolute configuration.
Scheme 1. TDO-Catalyzed Oxidations of Monosubstituted
Phenols to Yield Catechols, Hydroquinones and cis-
Dihydrodiols/Cyclohexenone cis-Diols
INTRODUCTION
■
The formation of enantiopure cis-dihydrodiol metabolites, from
the biotransformation of monocyclic arene substrates, using
mutant and recombinant bacterial strains as sources of toluene
dioxygenase (TDO) and other arene dioxygenases, has been
extensively reviewed in recent years.1−17 A wide range of
substituted benzenes has been used as substrates for TDO,
resulting in cis-dihydrodiols with substituents including
halogens, alkyl, aryl, heteroaryl, alkene, alkynyl, carboxylic
acid, ester, ether, thioether, sulfoxide and nitrile groups.
Continuing interest in the application of the resulting
monocyclic cis-dihydrodiol bioproducts, as synthetic precursors
of chiral natural products, chiral ligands and compounds of
value in medicinal chemistry, is evident from the many recent
publications on this topic by the groups of Banwell,18
Hudlicky,19 Lewis20 and Stevenson.21
Until relatively recently, phenols were among the few types
of substituted benzene substrates not found to yield the
corresponding cis-dihydrodiol metabolites following TDO-
catalyzed oxidation. Catechols and hydroquinones were,
previously, identified as the major types of phenol derivatives
isolated from bacterial arene dioxygenase-catalyzed oxidations
(Scheme 1).22−32 cis-Dihydrodiols were, however, postulated as
possible intermediates during the formation of hydroquinones
from phenols, but were not detected under the reaction
conditions.32 Following the first reports of cis-dihydrodiol
metabolite formation from phenols, characterized as the more
stable keto tautomers (cyclohexenone cis-diols),33,34 a number
of new family members of enantiopure cyclohexenone cis-diol
metabolites (>20 examples) has recently been reported,33−37 as
a result of TDO-catalyzed cis-dihydroxylation or chemo-
enzymatic synthesis.
The difficulty in detecting cyclohexenone cis-diol metabolites
from phenols may have been due to (i) competing formation of
catechols, whose presence was known to inhibit TDO
Received: December 21, 2014
Published: March 10, 2015
© 2015 American Chemical Society
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Figure 1. Structures of naturally occurring mono- and disubstituted guaiacols.
activity,38 (ii) their instability, resulting in dehydration to yield
hydroquinones and (iii) further metabolism including enzy-
matic alkene and/or ketone reductions to yield cis-triols and
other bioproducts.35,36 The majority of isolated cyclohexenone
cis-diol metabolites have been derived from 3-substituted and
2,5-disubstituted phenols.33−38 To date, only one example of a
cis-diol metabolite has been reported from a 2-substituted
phenol (ortho cresol)34 and none from 4-substituted phenols.
Methoxyphenols are widely distributed in the environment,
due to (i) their role as monomers in the biosynthesis of lignin
in biomass, e.g., plants and trees, and (ii) their formation as a
result of lignin biodegradation, pyrolysis and pretreatment in
biorefinery operations.39−41 Many of these naturally occurring
phenolic products contain the ortho methoxyphenol moiety
(guaiacol) with either one, e.g., vanillin, vanillic acid, eugenol,
coniferyl alcohol, ferulic acid, or two, e.g., syringyl acid, sinapyl
alcohol, additional substituents (Figure 1). Anthropogenic
sources of substituted guaiacols, e.g., chloroguaiacols, are also
prevalent in the environment as a result of the chlorine
bleaching of wood pulp.41,42
Guaiacol, is itself a naturally occurring product, also formed
from the pyrolysis and biodegradation of wood or other plant
material and is responsible for the flavor of many foods in our
diet. It was also found to act both as a carbon and an energy
source during bacterial metabolism of chloroguaiacols.41,42
There has been considerable interest in a recent compound
derived from a combination of guaiacol and vanillyl alcohol to
yield bisguaiacol F (BGF) as a potential alternative to the
endocrine disrupting compound bisphenol A (BPA).43
Guaiacol is among the most abundant phenols in nature.
Investigation of its metabolic breakdown pathways in bacteria,
identification and reactions of the resulting metabolites, were
major priorities of the current program.
Scheme 2. TDO-Catalyzed Oxidation of 3-Methoxyphenol 1
to Yield Cyclohexenone cis-Diol 3, via cis-Dihydrodiol 2,
with Catechol 4 and Hydroquinone 5 as Minor Products
and column chromatography, yielded, mainly, 3-methoxycyclo-
hexenone cis-diol 3 (45 g, 38% yield), via the undetected enol
tautomer 2. Catechol 4 and hydroquinone 5 were also detected,
by GC−MS analysis of their silylated derivatives, among a
mixture of unidentified minor metabolites.
Phenols are enol forms of cyclohexadienones. The kinetics
and equilibrium constants for this tautomerism have been
evaluated.44 Because of the large gain in resonance energy, the
enol form is by far the most stable tautomer, with an
equilibrium constant K > 1011. However, chemoselective
removal of a non enolic CC double bond removes the
aromatic resonance energy and provides a novel entry to a new
enol, 2, with very different properties to the parent phenol.
Although several methods for preparing enols have been
reported, including hydration of alkynes,45 hydrolysis of enol
ethers,46,47 decarboxylation of β-keto acids,48 isomerization of
allyl alcohols,49 1,4-addition of thioacetic acid to enals50 and
photochemical fragmentations,51 the present enzymatic ap-
proach is novel, as it is the first method of asymmetric synthesis
for preparing a chiral enol by a dearomatisation of a phenol.
There is normally a large thermodynamic driving force for
enols to tautomerise, to the corresponding carbonyl com-
pounds, but since the activation energy for intramolecular
migration of hydrogen is high, enols can be regarded as being
metastable. Ethenol47 and prop-1-en-2-ol49 are sufficiently long-
lived in a dilute solution of an aprotic solvent, at room
temperature, to allow the NMR spectra of these species to be
recorded. An O-deuterated enol with two fluorine atoms on the
α-carbon has a half-life greater than 2 weeks in neutral
methanol-d4.52 Cyclohexa-1,3-dienol has a half-life of 5 s,53
further demonstrating the longevity of enols in aqueous media.
However, the proton transfer is greatly facilitated in protic
solvents, with most studies undertaken in water as solvent,
catalyzed both by acid and base.54
A recent small-scale biotransformation of 3-methoxyphenol,
using Pseudomonas putida UV4 whole cells, gave mainly a cis-
dihydrodiol metabolite as its preferred keto tautomer (cyclo-
hexenone cis-diol).37 This observation prompted the current
comprehensive biotransformation studies of 2- and 3-
methoxyphenol substrates with emphasis on effective scale
up, identification of the resulting metabolites, metabolic
pathways and chemical reactions.
RESULTS AND DISCUSSION
■
Biotransformation of 3-Methoxyphenol 1 to Yield cis-
Diols 2 and 3, Catechol 4 and Hydroquinone 5. Earlier
small-scale biotransformations of 3-methoxyphenol 1, using
either the mutant P. putida UV4 or the recombinant Escherichia
coli CL-4t strain, resulted in TDO-catalyzed formation of cis-
dihydrodiol 2, a major metabolite isolated as the preferred
cyclohexenone cis-diol-tautomer 3 (Scheme 2).36 A larger
quantity of cyclohexenone cis-diol metabolite 3 was required,
for studies of its reactivity and evaluation of its potential, as a
new chiral pool compound. A large-scale biotransformation of
3-methoxyphenol 1 (96 g in 120 L) was therefore conducted
using P. putida UV4. Concentration of the aqueous
biotransformed material, followed by ethyl acetate extraction
DFT calculations (Table 1) indicated that the relative
difference in free energy between enol 2 and ketone 3 in the gas
phase was −76.5 kJ mol−1. As such there was a huge
thermodynamic driving force for forming the ketone tautomer
1
and hence k1 ≫ k2 (Scheme 2). H NMR analysis of the
vinylogous ester 3 failed to detect any of the enol tautomer 2,
when the signals for the 13C satellites were clearly visible
indicating that the equilibrium constant K for the tautomerism
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1
determined by H NMR spectroscopy. Proton H-1 showed a
coupling constant of 4.3 Hz, when coupled to H-6, suggesting
an axial−equatorial arrangement, and proton H-6 was coupled
to H-5 with a coupling constant of 8.4 Hz, indicating that H-6
was axial and H-1 was equatorial. The tendency for electro-
negative allylic substituents to adopt pseudo axial positions, in
six-membered rings, is well documented57−59 and can be
Table 1. DFT Calculated Gas Phase Differences in Free
Energy of Ketones (3 and 11) with Respective Enols (2 and
10) and Comparison of Free Energy between Two Isomeric
Ketones (3 and 26)
entry
ketone
enol
ΔG (kJ mol−1
)
1
2
3
3
2
−76.5
−48.5
−11.7
11
10
attributed to a combination of minimization of 1,2-allylic
strain60 and increased hyperconjugation π−σ*C−O
this case with an electron-deficient alkene.
,
even in
61
3 → 26
Biotransformation of Guaiacol 9 to Yield cis-Diols 10,
11, 13, 14, Methoxyhydroquinone 5 and Methoxyca-
techol 4. The bacterial biotransformation study of guaiacol
substrate 9, was conducted using P. putida UV4, a source of
TDO and glucose as a carbon source (Scheme 4). The initial
objective of this study was to compare the results with those
observed earlier, using ortho-cresol as substrate, where the
corresponding cyclohexenone cis-diol was obtained in very low
yield (1%).34 During an early small-scale biotransformation,
surprisingly, the only keto cis-diol products isolated from
guaiacol 9 were metabolites 13 and 14. Intermediate 11, the
preferred tautomeric form of the initial cis-dihydrodiol
metabolite 10, was presumed to be reduced to cis-diol 14 and
epimer 13.
Results obtained from a later time course study of this P.
putida UV4 biotransformation, using LC-TOFMS analysis of
the crude culture medium, confirmed the presence of the major
metabolites 11 and 14, detected after 2 h, along with traces of
partially separated metabolite 13 from diol 14. After 20 h,
metabolite 11 showed a 7-fold decrease, and the reduced
bioproducts 13/14 a 7-fold increase in concentration; the
epimeric ketone 12 was not detected. Only a very minor
proportion of cyclohexenone cis-diol 11, remained, when the
biotransformation was terminated at 20 h, leaving a 1:10
mixture of cis-diol epimers 13:14 as major metabolites. The
diastereoisomers 13 and 14, separated by column chromatog-
raphy, did not interconvert during extensive NMR experiments
in CD3OD solution.
reaction was less than 10−3. For comparison purposes, keto−
enol tautomerism in 2-cyclohexenone, with water as solvent,
has an equilibrium constant K of 2.0 × 10−8,53,55 which is well
outside the range for 1H NMR spectroscopy to detect the enol.
When the TDO-expressing recombinant strain E. coli (CL-
4t) was used with the phenol substrate 1, GC−MS analysis
showed that cis-diol 3 and catechol 4 were again formed,
consistent with TDO being the enzyme involved.
To provide further confirmation of the (4S,5S) absolute
configuration of cis-diol-3, earlier assigned by ECD spectros-
copy,37 a stable derivative was synthesized. Hydrogenation of
enone 3 using a palladium catalyst (Pd−C, MeOH) gave an oil
(85% yield) which was identified as cyclohexanone cis-diol 6
(Scheme 3). This reaction was remarkably selective and only
one diastereoisomer was detected. The observed relative
configuration was consistent with delivery of hydrogen from
the sterically less encumbered face of enone 3.56 The relative
configuration of compound 6 was determined primarily from
the magnitude of vicinal proton coupling constants associated
with proton H-4 (J 5.6, 2.9, 2.9). The 5.6 Hz coupling constant
was due to coupling of H-4 to the hydroxyl group, and the
other two small coupling constants of 2.9 Hz were consistent
with two axial−equatorial couplings, confirming a cis-relation-
ship among protons H-3, H-4 and H-5, and hence establishing
a cis-relationship between the OMe and OH groups. For
unequivocal confirmation of the relative and absolute
configuration of compound 6, a suitable crystal of mono-
camphanate derivative 8, for X-ray crystallographic analysis, was
obtained by treatment with an excess of (1S)-camphanic
chloride in pyridine solution. This procedure yielded the
crystalline derivative 8 (Scheme 3); it was presumably derived
from the dicamphanate 7, which was clearly prone to facile
elimination reactions under the basic reaction conditions. The
X-ray crystal structure, confirmed that camphanate 8 had an
equatorial OMe group and a pseudoaxial O-camphanate group
in a cis-relationship in the solid phase and therefore a (1S,6R)
absolute configuration. The (4S,5S) configuration for cyclo-
hexenone cis-diol 3 and a (3S,4S,5R) configuration for
cyclohexanone cis-diol 6, were established from the stereo-
chemical correlation sequence shown in Scheme 3. The same
conformation of compound 8 was also dominant in solution, as
The metabolic sequence, proposed in Scheme 4, involved an
initial TDO-catalyzed cis-dihydroxylation, to yield guaiacol cis-
dihydrodiol 10. Enol-keto tautomerisation gave cyclohexenone
cis-diol 11, and a rapid ene reductase (ERED)-catalyzed
reduction (hydrogenation) produced the cyclohexanone cis-
diol 14, followed by an epimerization to give isomer 13. A
similar type of enzymatic alkene reduction was observed, with
other α,β-unsaturated ketones, e.g., 2-cyclohexen-1-one62 and a
2-cyclohexen-1-one cis-diol,34 using P. putida strains (M10 and
UV4 respectively) both expressing ene reductase enzyme
activity.
A large-scale biotransformation of guaiacol 9, (96 g in 120
L), using glucose as carbon source and P. putida UV4, yielded
Scheme 3. Synthesis of (1S,6R) Monocamphanate 8 from (3S,4S,5R) Cyclohexanone cis-Diol 6
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Scheme 4. Metabolism of Guaiacol 9 to Yield cis-Diols 10, 11, 13, 14, Catechol 4 and Hydroquinone 5
cyclohexanone cis-diols 14 (13% isolated yield) and 13 (1.3%
isolated yield, Scheme 4), after separation by column
chromatography. The relative stereochemistry of each of the
diastereoisomers 13 and 14 was determined by the magnitude
of the vicinal proton coupling constants. The major cis-diol 14
exhibited a small coupling constant (J2,3) of 3.0 Hz, indicating a
cis-axial−equatorial relationship between H-2 and H-3 protons.
The minor isomer 13 showed a much larger value (J2,3 9.8 Hz),
consistent with a trans-diaxial arrangement for H-2 and H-3
protons, and a corresponding trans-diequatorial relationship
between the OMe and OH groups at C-2 and C-3 respectively.
In both diastereoisomers, the conformer in which the methoxy
group was equatorial predominated, and in each isomer two
groups were equatorial and one was axial (Figure 2).
camphanates 8 and 16, indicates how finely balanced the
stereoelectronic and steric factors are in these molecules.
Methoxycatechol 4 and methoxyhydroquinone 5 were later
identified as very minor guaiacol metabolites, by GC−MS
analysis of the freeze-dried residue after silylation with MSTFA.
Catechol 4 could be formed via TDO-catalyzed dihydroxylation
of guaiacol 9 at the C1C6 bond followed by loss of water as
proposed for the formation of catechols from other phenol
substrates.28 Hydroquinone 5 may be formed from the
dehydration of intermediate cyclohexenone cis-diol 11 during
the biotransformation.
In one of the experiments when a flask-scale biotransforma-
tion of guaiacol 9 was conducted, using P. putida UV4 and
pyruvate as carbon source, a slightly higher concentration of
cyclohexenone-cis-diol 11 than usual was observed, during the
early phase of metabolism. Extraction and purification (PLC)
resulted in partial decomposition and yielded mainly
methoxyhydroquinone 5. However, a very small sample of
the residual cyclohexenone cis-diol 11 (<5 mg) survived the
1
purification. The H NMR spectrum of this impure sample
Figure 2. Preferred conformations of cis-diols 14 and 13.
showed chemical shift values (δ) and coupling constants (J) for
the alkene protons, 6.76 (1 H, dt, J 10.3, 2.2, H-3), 6.04 (1 H,
dd, J 10.3, 2.3, H-2) that were very similar to those found for
the corresponding, but more stable, cyclohexenone-cis-diol
metabolite from ortho-cresol.34 The relative stereochemistry of
diastereoisomer 11 was established by analysis of vicinal proton
coupling constants. Coupling constants J-4,5 and J-5,6 were
small, 3.9 and 2.3 Hz respectively indicating that H-4, H-5 and
H-6 were cis to one another. The epimeric enone 12 remained
undetected in these experiments. The NMR sample of the
elusive metabolite 11 showed further evidence of instability in
CDCl3 solution and slowly decomposed forming methoxyhy-
droquinone 5 and other unidentified products, before full
structural and stereochemical characterization could be carried
out. Other researchers have noted the lability of structurally
similar compounds, where facile dimerization occurred.63
Further attempts to isolate the intermediate cyclohexenone-
cis-diol 11, by this method were unsuccessful and the absolute
configuration of metabolite 11 was presumed to be identical to
that of the major metabolite 14.
Although compound 11, was identified as the initial major
cyclohexenone cis-diol metabolite, in the aqueous culture
medium, it became evident that our efforts to extract and
isolate a sample by PLC, were being thwarted by (a) the low
conversions during the very early stages of the P. putida UV4
biotransformation, (b) it being an excellent substrate for
ERED-catalyzed reduction and (c) its chemical/thermal
instability. When an E. coli recombinant strain (CL4t),
expressing TDO, but not the ERED enzyme, was used with
The absolute configuration of cis-diol 14, was determined by
single crystal X-ray crystal structure analysis of camphanate
derivative 16, followed by stereochemical correlation (Scheme
5). The undetected dicamphanate 15 eliminated camphanic
Scheme 5. Synthesis of (1S) Monocamphanate 16 from
(2S,3S,4S) Cyclohexanone cis-Diol 14
acid (CamOH) under the reaction conditions, to give (1S)
camphanate 16. The absolute stereochemistry of diols 14 and
11 could be assigned as (2S,3S,4S) and (4S,5S,6S) respectively.
In the solid state, the camphanate ester group in derivative 16
1
occupied a pseudoequatorial position. In solution, H NMR
spectroscopy showed that proton H-1 had vicinal coupling
constants of 6.0 and 4.4 Hz to protons H-6 and H-6′, indicating
that H-1 was axial in the dominant conformer, but a substantial
amount of at least one other conformer was also present. This
subtle difference in conformational preference, between
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guaiacol 9 as substrate, LC-TOFMS analysis showed that
compound 11 was the only cis-diol metabolite formed. Since it
was produced in an extremely low yield, and due to
decomposition on attempted purification, it was not examined
further.
Stability and Aromatization Studies of Methoxycy-
clohexenone cis-Diols. On the basis of earlier studies, it
appeared that cis-dihydrodiols from nonphenolic monosub-
stituted arene substrates (e.g., 17, R = OMe, Figure 3) were
observed, in the presence of the major silylated (triTMS) keto
tautomer 22, by GC−MS analysis.
The TDO-catalyzed ipso cis-dihydroxylation/dehydration
route for the oxidation of phenols to catechols, proposed
earlier by Gibson et al., involved an undetected triol
intermediate, with the additional oxygen atom being derived
from 18O-labeled oxygen gas; this mechanism may also be
involved in the formation of catechol 4.26,28 The formation of
methoxyhydroquinone 5, as a dehydration product from
metabolites 3 and 11 (Schemes 2 and 4), provides strong
support for a general pathway to hydroquinone metabolites
from phenols, based on the instability of cyclohexenone cis-diol
intermediates. An alternative biosynthetic pathway, for the
naphthalene dioxygenase-catalyzed formation of hydroquinones
and catechols, from the corresponding ortho- and meta-phenols,
was, however, recently reported to involve monohydroxylation
rather than cis-dihydroxylation and dehydration.31 Thus, with
m-cresol as substrate, monohydroxylation occurred via
nucleophilic attack by 18O-labeled water at the para position,
to yield methylhydroquinone with incorporation of an 18O-
labeled oxygen atom and consumption of dioxygen. A similar
pathway with water attacking at the ortho position was used, to
explain the formation of catechols; the role of oxygen was to
reoxidise the Fe(III) back to Fe(IV).
Figure 3. cis-Diol metabolites from monosubstituted benzenes 17,
from meta-phenols 3, 18, 19, and their silylated derivatives 20−23
from the corresponding cyclohexenone cis-diols and cis-dihydrodiols.
generally much less stable,64−67 at ambient temperature,
compared with known members of the cyclohexenone cis-diol
family of metabolites derived from meta-phenols or 2,5-
disubstituted phenols.33−37 During our attempts to purify
cyclohexenone cis-diol 11, it became evident that it was very
labile and decomposed readily, when compared with some
other members of this cyclohexenone cis-diol family; many
having half-lives of several hours in 6 M perchloric acid,34 thus
indicating a much wider stability range for this type of phenol
metabolite than anticipated.
Thermal dehydration of substituted benzene cis-dihydrodiols
17, yielded both ortho- or meta-phenols, with preference being
dependent upon the nature of the substituent group R.
Furthermore, acid-catalyzed dehydration of cis-dihydrodiols 17
afforded mainly ortho rather than meta phenols,33−37 and the
trend reversed under base-catalyzed conditions.67 Cyclo-
hexenone cis-diols (e.g., 18 and 19) were found to aromatize
under both acidic and alkaline conditions, yielding the
corresponding substituted 1,4-dihydroxybenzenes (hydroqui-
nones) rather than 1,3-dihydroxybenzenes (resorcinols). In the
present study, treatment of the methoxycyclohexenone cis-diols
3 and 11 under acid conditions (CF3CO2H) yielded
methoxyhydroquinone 5 as the sole product.
As indicated in Table 1, the proportion of the less stable enol
tautomer 2, at equilibrium with cyclohexenone cis-diol
metabolite 3, would be extremely small. This could account
our earlier unsuccessful attempts to observe or trap the elusive
enol tautomers through formation of triazolinedione cyclo-
adducts of the diene moiety, or methylation of the enol OH
group with diazomethane.34 Silylation and GC−MS analysis of
a pure sample of cyclohexenone cis-diol 3 showed the expected
major diTMS derivative 20 (ca. 95%) but also a minor
proportion (ca. 5%) of a triTMS derivative 21 (Figure 3).
Compound 21, was a derivative of the less stable enol (cis-
dihydrodiol) tautomer 2, the initial metabolite of phenol 1
(Scheme 2). A very minor quantity of a diTMS derivative of
hydroquinone 5 was also observed possibly due to dehydration
of cis-diol 3 during the trimethylsilylation procedure.
The isolated yields of cis-dihydrodiols 17, obtained with
monosubstituted benzene substrates and TDO as biocatalyst,
were generally much higher than those obtained using
disubstituted benzene substrates, due to (i) the increased steric
requirements of the latter substrates at the active site, (ii)
competition from alternative types of oxidation, particularly
associated with meta disubstituted benzene substrates (meta
effect).68 It was expected that lower yields of cyclohexenone cis-
diols, e.g., metabolite 3, would be obtained and alternative
pathways would operate, as 3-substituted phenols, e.g., 1, are
also meta disubstituted benzenes. The alkene and carbonyl
groups associated with enones can both be reduced, under the
biotransformation conditions,36 leading to mixtures of bio-
products and hence decreased overall yields. Because
metabolite 3 is a vinylogous ester, it has very different
electronic properties to a typical enone and appeared to be
resistant to biological reduction of both the alkene and carbonyl
functional groups and to hydrolysis. This property, allied to its
relative stability, may account for the isolated moderate yield of
cyclohexenone cis-diol 3. 3-Methoxyphenol 1 was thus one of
the phenolic substrates selected for large scale (120 L)
fermentation to yield cyclohexenone cis-diols. The sufficient
quantity of metabolite 3 produced, made it possible to conduct
a comprehensive study of its reactions and synthetic potential.
Synthetic Applications of cis-Diol Metabolite 3
Derived from 3-Methoxyphenol 1. Numerous synthetic
applications of enantiopure cis-dihydrodiol metabolites, derived
from substituted benzene substrates, have been reported, but,
to date, relatively few reactions and applications of the new
family of cyclohexenone cis-diol metabolites from phenols have
appeared in the literature. These reactions include substitution
of an iodine atom in cyclohexenone cis-diol 19 (Figure 3) by
different atoms or groups (R = H, CN and CO2Me) to provide
a chemoenzymatic route to other family members.34
Hydrolysis of the vinylogous ester 3, using aqueous 1 M
sodium hydroxide, proceeded smoothly to give the enolate
sodium salt 25 (Scheme 6). This process involved nucleophilic
substitution of the methoxy group by a hydroxyl group, via an
addition−elimination pathway, followed by the formation of
Biphenyl dioxygenase-catalyzed cis-dihydroxylation of 3,3′-
dihydroxybiphenyl earlier, yielded a cyclohexenone cis-diol
metabolite of similar structure to compound 3, but with the
MeO group replaced by a 3-HO·C6H4 group.33 A minor
amount of the silylated (tetraTMS) product 23 was also
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the minimized structure there was evidence of a hydrogen bond
between the α-hydroxyl and ketone carbonyl groups. It was
challenging to distinguish between compounds 3 and 26 by 1H
NMR spectroscopy. In both isomers, NOE was only observed
between the methoxy group and the olefinic proton. Key
proton carbon couplings, detected in HMBC experiments
(Figure 4), confirmed the carbon connectivity of both isomers.
Scheme 6. Hydrolysis of Vinylogous Ester 3 and
Esterification to Give Predominantly the Transposed
Vinylogous Ester 26
Figure 4. Key HMBC correlations for enone cis-diols 3 and 26
confirming the relative position of the carbonyl and methoxy groups in
both isomers.
enolate 25, under the basic reaction conditions. Acidification (1
M HCl) and removal of water, under reduced pressure, yielded
a very polar crude product, which was assumed to consist of
diketone 24d and its tautomers 24a−c and 24e. Attempts to
obtain direct evidence of intermediates 24a−e by NMR
When the crude mixture of tautomers 24a−e was dissolved
1
1
spectroscopy in D2O as solvent, were unsuccessful. H NMR
in methanol-d4, analysis of the H NMR spectrum of the new
analysis of the product showed very broad signals suggesting
chemical exchange between these various species on the NMR
time scale or aggregation. LC-TOFMS analysis did however
show the presence of a single peak with the correct mass for
any of the tautomeric intermediates 24a−e. GC−MS analysis
and trimethylsilylation of the tautomeric mixture, showed three
peaks consistent with the presence of two triTMS keto
tautomers (24a, 24b) and one tetraTMS derivative (24c or
24e) or both isomers coeluting. A triTMS derivative of 1,2,4-
trihydroxybenzene 27 was also observed, which was assumed to
be a dehydration product of the cyclohexenone cis-diol 24a or
its tautomers 24b−e.
The crude product, 24a−e, was extracted with methanol and
the residue, obtained after concentration of the extract, was
separated by PLC into a major less polar and a minor more
polar compound (9:2). The major compound was identified as
the transposed vinylogous ester 26, by LC-TOFMS, NMR and
IR spectroscopic analyses. It was less polar than its isomeric
precursor 3, presumably due to intramolecular hydrogen
bonding between the transposed carbonyl group and the
proximate OH group (Scheme 7). The minor compound was
found to be the reformed vinylogous ester 3. Gas phase DFT
calculations, (Table 1), indicated that compound 26 was more
stable than the isomeric compound 3, by 11.7 kJ mol−1, and in
vinylogous ester 26 indicated that the methyl group had been
replaced, by CD3 (26D3) and the vinyl proton signal had
diminished in intensity. This result was consistent with CD3
being incorporated into the product, after partial exchange of
the vinyl proton for deuterium via diketone 24d (26D4), by an
addition−elimination reaction of methanol-d4 with enol 24a.
Transposition of vinylogous esters, under acidic conditions, is
well documented69 and O-benzylation of unsymmetrical 1,3-
dicarbonyl compounds, under basic conditions, gives trans-
position isomer mixtures.70 The regenerated precursor 3 was
also found to have incorporated a CD3 group (3D3) and a vinyl
deuterium atom (3D4). Notably, in both cases no deuterium
was incorporated at carbons 4 and 6 suggesting limited
contributions from enol tautomers 24c and 24e in methanol-d4.
Under the strongly basic conditions that ketodiol 26 had
been subjected to, there was a possibility of epimerization at C-
6. The relative stereochemistry of diol 26 was established by
analysis of vicinal coupling constants. The J6,5 value of 3.0 Hz
was consistent with an axial−equatorial vicinal coupling,
confirming that the cis stereochemistry had been preserved.
The signal for proton H-5 appeared as a ddd, J 3.1, 3.0, 2.8 Hz,
indicating that H-5 was equatorial in the dominant conformer.
Therefore, in diol 26 the proton on C-6 is axial and the
hydroxyl group equatorial. This was in sharp contrast to
Scheme 7. Stork−Danheiser Transpositions of Compound 30 and Synthesis of the Fungal Metabolite 34
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compound 3, where the hydroxyl group on C-4 was axial,
presumably due to 1,2-allylic strain and hyperconjugation
effects. Intramolecular hydrogen bonding, to the carbonyl
group of diol 26, was only possible, when the hydroxyl group
was equatorial. The TDO-catalyzed synthesis of the vinylogous
esters 3 and 11 and chemoenzymatic synthesis of diols 24a and
26, from metabolite 3, add further members to the growing
family of chiral cyclohexenone cis-diols derived from phenols
and demonstrate their wide range of associated stabilities.
To further explore the chemistry of vinylogous ester 3, it was
necessary to protect the diol group as an acetonide. Initial
efforts, using 2,2-dimethoxypropane, in the presence of p-
TsOH at ambient temperature, proved to be unsuccessful. The
only isolated product was 2,4-dimethoxyphenol 29 (Scheme 7).
The mechanism for conversion of cis-diol 3 into phenol 29 is
unclear; it may involve aromatization via formation and
decomposition of the unstable hemiacetal intermediate 28.
When the protection step was repeated at a lower temperature
(0 °C), cyclohexenone cis-diol 3 was successfully converted into
the required acetonide 30 in good yield (82%), with only traces
of dimethoxyphenol 29 present (Scheme 7).
Acetonide 30 underwent a Stork−Danheiser transposition,
on reaction with nucleophiles followed by acid treatment.71
Thus, reaction of acetonide 30 with DIBAL followed by
treatment with p-TsOH in diethyl ether yielded the transposed
cyclohexenone 31, in an overall yield of 70%. The synthetic
potential of this new route to enantiopure cyclohexenone 31
was evident from its earlier use as an intermediate in the total
synthesis of natural products, e.g., (+)-clivonone,72 (+)-tri-
anthine73 and the antibiotic (+)-palitantin.74 Similarly, employ-
ing Grignard reagents (MeMgBr and PhMgBr) as nucleophiles,
followed by acidic workup, resulted in hydrolysis of the enol
ether and yielded the transposed cyclohexenones 32 (72%
yield) and 33 (75% yield) respectively (Scheme 7). Recently, a
new secondary metabolite, isolated from the phytopathogenic
fungus Peronophythora litchi, was identified as the chiral cis-diol
34 but its absolute configuration was not determined.75 On the
basis of the structural similarities of the bacterial metabolite 3
and the fungal metabolite 34, an attempted monomethylation
(MeI, Ag2O, CH2Cl2) under mildly basic conditions was carried
out, which resulted in the formation of a mixture of
monomethyl ethers 34, 35 and dimethyl ether 36 (Scheme 7).
The monomethyl ether derivative 34, was separated by PLC
from the other products 35 and 36 and was obtained in modest
yield (37%). A direct comparison of its NMR and IR data with
that reported for the fungal metabolite, was in good agreement
and confirmed its structure. Comparison of the optical rotation
of the synthetic sample of (4S,5S) monomethyl ether 34 ([a]D
−48.0, MeOH), derived from the enantiopure cis-diol 3, with
the reported value for the fungal metabolite ([a]D +28.4,
MeOH),75 suggested that the natural metabolite was of lower
enantiopurity (ca. 60% ee) and had the opposite (4R,5R)
absolute configuration. It is noteworthy, that despite the
densely packed functionality of fungal metabolite 34, it was
synthesized in two steps from the commodity chemical 3-
methoxyphenol 1.
products. The alkene bond in the initially formed conjugated
enone cis-diol 11 was found to be rapidly reduced (hydro-
genated), via ene reductase catalysis, yielding cyclohexanone cis-
diol 14, the major isolated bioproduct. A similar enzymatic
reduction (hydrogenation) of the substituted alkene group in
enone cis-diol 3 was not observed. The structures and absolute
configurations of the chiral cis-diol bioproducts 3, 11, 14 and
their derivatives were assigned by spectroscopic, X-ray
crystallographic and stereochemical correlation methods. The
isolated enone cis-diols 3 and 11 readily dehydrated to
hydroquinone 5, providing strong evidence that hydroquinones
were formed from diols by an nonenzymatic pathway.
A large-scale biotransformation of phenol 1 provided
sufficient quantities of cyclohexenone cis-diol 3 for further
studies, viz. diol protection, aromatization, alkene hydro-
genation, hydrolysis and re-esterification with transposition,
O-methylation and Stork−Danheiser transposition with a range
of nucleophiles. The versatility of enone cis-diol 3 was
demonstrated in the synthesis and stereochemical assignment
of the new type of cyclohexenone cis-diol 26, and a fungal
secondary metabolite 34. The value of cis-diol 3 and its
acetonide 30, as useful additions to the chiral pool, was further
demonstrated by the synthesis of (3aS,7aS) ketoacetonide 31,
which has been employed as a key intermediate for the
synthesis of known alkaloids and an antibiotic.
EXPERIMENTAL SECTION
■
1H and 13C NMR spectra were recorded on 300, 400, and 500 MHz
NMR spectrometers using the specified solvent. Chemical shifts (δ)
are reported in ppm relative to SiMe4 and coupling constants (J) are
given in Hz. IR spectra were recorded either as KBr discs or as thin
films on KBr plates. Mass spectra (EI) were run at 70 eV, on a heated
inlet system. Accurate molecular weights were determined by the peak
matching method, using heptacosafluorotributylamine as the standard
reference and were accurate to 5 × 10−6 ppm. Separations for
TOFMS analyses were performed using a reverse phase column (C18,
5 mm, 150 × 2.1 mm) together with the corresponding guard column
(5 mm, 12.5 × 2.1 mm). The mobile phase consisted of 95% methanol
containing 0.1% formic acid in channel A, and 5% methanol containing
0.1% formic acid in channel B. The system was programmed to
perform an analysis cycle consisting of 100% B for 1 min, followed by
gradient elution from 100 to 5% B over a 14 min period, hold at 5% B
for 10 min, return to initial conditions over 2 min and then hold these
conditions for a further 8 min. The flow rate was 0.20 mL min−1 and
the injection volume was 5 μL. MS experiments were carried out using
ESI in positive ion mode with the capillary voltage set at 4.0 kV. The
desolvation gas was nitrogen set at a flow rate of 8 L min−1 and
maintained at a temperature of 350 °C. Optical rotation ([α]D) values
were carried out on a polarimeter, using a specified solvent and
concentration (g/100 mL) at the sodium D-line (589 nm) and
ambient temperature. The measurements are in units of 10−1 deg cm2
g−1.
TLC was carried out on analytical plates. Visualization of bands/
spots was at 254 nm with a UV lamp or by anisaldehyde- or
permanganate-based stains. PLC was carried out on glass plates (20
cm × 20 cm) coated with silica gel (21 g silica gel in 62 mL water).
Column chromatography was performed on type 60 (250−400 mesh).
Phenol substrates 1, 9, hydroquinone 5, catechol 4 and 1,2,4-
trihydroxybenzene 27 were commercially available.
Geometry optimization and vibrational frequencies were evaluated
in the gas phase by DFT calculations at the B3LYP/6-31G(d) level of
theory. No imaginary frequencies were detected indicating that the
structures were correctly optimized.
cis-Diols 3, 11, 13 and 14 were analyzed by NMR, LC-TOFMS and
GC−MS (silylated). Catechol 4, trihydroxybenzene 27, and hydro-
quinone 5 were analyzed by GC−MS after silylation using N-methyl-
N-(trimethylsilyl) trifluoroacetamide (MSTFA) and a gas chromato-
CONCLUSION
■
TDO-catalyzed cis-dihydroxylation of meta methoxyphenol 1
and ortho methoxyphenol 9, using whole cell cultures of P.
putida UV4, was found to yield the corresponding cis-diols 3
and 14 as the major metabolites respectively, along with
methoxycatechol 4 and methoxyhydroquinone 5 as very minor
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graph linked to a 5973 mass selective detector. The sample (1 μL) was
injected in the split mode (20:1). The column used had dimensions
(25 m × 0.2 mm × 0.33 μm). The GC oven used was maintained at
100 °C for 1 min and then ramped at 10 °C/min to 300 °C and held
at this temperature for 5 min.
Small-scale biotransformations of 2-methoxyphenol 9 and 3-
methoxyphenol 1 were carried out, using whole cell cultures of P.
putida UV4 and E. coli (CL-4t), with glucose or pyruvate as a carbon
source, under conditions reported earlier for other substituted phenol
substrates.34−37 For time course studies, the biotransformation was
performed over 20 h and samples, collected at 2, 6 and 20 h intervals,
were analyzed by LC-TOFMS.
Large-Scale Biotransformations of 2-Methoxyphenol 9 and
3-Methoxyphenol 1. 2-Methoxyphenol 9 and 3-methoxyphenol 1
(96 g, 0.77 mol) were each metabolized, using P. putida UV4, in a
fermenter (pH 7.0, 30 °C, 400 rpm). D-Glucose was used as carbon
source substrate (4.8 g/L) and air flow rate was maintained at 70%
oxygen. Sodium hydroxide (2 M) was added, automatically, into the
fermenter to maintain the pH. When the oxygen tension in the
fermenter exceeded 50%, an additional quantity of D-glucose was
added (1.64 g/min). Co-substrate addition was controlled, to maintain
the oxygen tension in the fermenter in excess of 50%. The crude
aqueous biomixture was centrifuged (30 000 rpm, 180 min); the
aqueous supernatant solution was decanted off and concentrated at
∼40 °C under reduced pressure. The viscous concentrate was
extracted with ethyl acetate (3 × 2.5 L) and the extract concentrated
under reduced pressure to yield the crude mixture of bioproducts.
(4S,5S)-4,5-Dihydroxy-3-methoxycyclohex-2-enone 3. Iso-
lated from the biotransformation of 3-methoxyphenol 1 by
crystallization (EtOAc) of the crude mixture of bioproducts as
colorless plates (45 g, 38%); the remaining mother liquor was retained
for further study. Metabolite 3 was found to be indistinguishable from
a sample reported earlier.36 GC−MS analysis of a small sample of
cyclohexenone cis-diol 3, after treatment with MSTFA, showed the
presence of disilylated keto derivative 20 as the major product (ca.
95%); m/z 302 (M+, 1%), 287 (10), 187(14), 186(100), 147 (27), 73
(25) and the trimethylsilylated enol derivative 21 as the minor product
(ca. 5%); m/z 375 (M+, 11%), 374 (33), 359 (10), 286 (87), 285 (87),
284 (26), 271 (30), 269 (18), 254 (65), 239 (10), 191 (57), 147 (340,
133 (12), 75 (20), 73 (100).
CDCl3) δH 0.98 (3 H, s, Me), 1.10 (3 H, s, Me), 1.13 (3 H, s, Me),
1.71 (1 H, ddd, J 13.2, 9.3, 4.3, H-5a), 1.94 (1 H, ddd, J 13.2, 10.7, 4.7,
H-5a′), 2.07 (1 H, ddd, J 13.6, 9.5, 4.7, H-6a), 2.43 (1 H, ddd, J 13.6,
10.7, 4.2, H-6a′), 2.69 (1 H, dd, J 16.7, 3.9, H-5), 2.84 (1 H, dd, J 16.7,
8.4, H-5′), 3.41 (3 H, s, OMe), 3.95 (1 H, m, H-6), 5.88 (1 H, ddd, J
4.3, 4.3, 0.9, H-1), 6.20 (1 H, dd 10.1, 0.9, H-3), 6.82 (1 H, dd 10.1,
4.3, H-2); 13C NMR (100 MHz, CDCl3) δC 9.8, 16.7, 16.8, 29.1, 30.8,
31.1, 54.6, 55.0, 57.4, 67.7, 76.1, 91.1, 133.1, 141.8, 167.0, 177.8, 196.1;
LR EI MS m/z 322 (M+, 1%), 290 (9), 264 (49), 181 (19), 137 (20),
125 (100), 109 (24), 97 (47), 83 (88).
Crystal Data for 8. C17H22O6, M = 322.4, monoclinic, a =
6.604(1), b = 19.939(3), c = 6.680(1) Å, β = 110.3(1), U = 1100.0(5)
Å3, T = 293(2) K, space group P21 (no. 4), Mo Kα radiation, λ =
0.71073 Å, Z = 2, F(000) = 344, Dx = 1.298 g cm−3, μ = 0.098 mm−1,
ω scans, 6.5° < 2θ < 50.0°, measured/independent reflections: 2005/
1570, Rint = 0.014, direct methods solution, full-matrix least-squares
refinement on Fo2, anisotropic displacement parameters for non-
hydrogen atoms; all hydrogen atoms located in a difference Fourier
synthesis but included at positions calculated from the geometry of the
molecule using the riding model, with isotropic vibration parameters.
R1 = 0.029 for 1455 data with Fo > 4σ(Fo), 213 parameters, ωR2 =
0.079 (all data), GoF = 1.06, CCDC 1025986. The absolute
configuration was established as (1S,6R) relative to the known
absolute configuration of the (1S)-camphanate group.
(4S,5S,6S)-4,5-Dihydroxy-6-methoxycyclohex-2-enone 11.
The biotransformation of guaiacol 9, with P. putida UV4 on a flask
scale was carried out using pyruvate as carbon source for 2 h under
5
previously reported conditions.4 LC-TOFMS analysis showed the
presence of the transient cyclohexenone cis-diol 11 as a major product
(>85%) and cyclohexanone cis-diol 14 as a very minor metabolite.
Concentration of the aq. biomixture under reduced pressure and
purification of the crude product by PLC (80% EtOAc in hexane)
b,
1
yielded hydroquinone 6 (12 mg) and metabolite 11 (ca. 5 mg); H
NMR (300 MHz, CDCl3) δH 6.76 (1 H, dt, J 10.3, 2.2, H-3), 6.04 (1
H, dd, J 10.3, 2.3, H-2), 4.54 (1 H, dt, J 3.9, 2.4, H-4), 4.50 (1 H, dt, J
3.9, 2.3, H-5), 3.83 (1 H, d, J 2.3 Hz, H-6), 3.59 (3 H, s, OMe);
HRMS (LC-TOFMS) [M + H]+ found 159.06517, C7H10O4 calcd.
159.06519+; [M + NH4]+ found 176.01965, C7H14O4N+ calcd.
176.01973; [M + K]+ found 197.02129, C7H10O4K+ calcd. 197.02107.
A 20 L aq. portion (from 120 L) of the biomixture, obtained using
guaiacol 9 as substrate, was worked up as described earlier. The crude
bioproduct mixture, on purification by column chromatography (20%
hexane in EtOAc → 100% EtOAc) followed by multiple elution PLC
(65% EtOAc in hexane), of the pooled major similar fractions,
furnished cis-diol metabolites 13 and 14. The minor fractions
containing other bioproducts are currently under investigation.
(2R,3S,4S)-3,4-Dihydroxy-2-methoxycyclohexanone 13. Bio-
transformation of guaiacol 9 yielded compound 13, a minor metabolite
as a colorless oil (250 mg, 1.3%); Rf 0.45 (65% EtOAc in hexane, 2
elutions); [α]D +92 (c 0.55, CHCl3); HRMS (LC-TOFMS) [M + H]+
(3S,4S,5R)-3,4-Dihydroxy-5-methoxycyclohexanone 6. To a
solution of enone diol 3 (200 mg, 1.26 mmol) in MeOH (10 mL) was
added 10% Pd/C (25 mg) and the mixture stirred overnight at room
temperature under 1 atm of hydrogen. The catalyst was filtered off, the
filtrate concentrated under reduced pressure, and the residue purified
by column chromatography (90% EtOAc in hexane) to give the
cyclohexanone 6 as a colorless oil (172 mg, 85%); Rf 0.21 (EtOAc);
[α]D −14.1 (c 0.7, CHCl3); HRMS (LC-TOFMS) [M + H]+ found
+
161.08091, C7H13O4 calcd. 161.08084; [M + Na]+ found 183.06273,
C7H12O4Na+ calcd. 183.06273; 1H NMR (400 MHz, CDCl3) δH
2.53−2.63 (2 H, m, H-2, H-6), 2.73−2.80 (2 H, m, H-2′, H-6′), 3.0(2
H, bm, 2 × OH), 3.41 (3 H, s, OMe), 3.69 (1 H, m, H-5), 4.02 (1 H,
m, H-3), 4.20 (1 H, ddd, J 5.6, 2.9, 2.9, H-4); 13C NMR (100 MHz,
CDCl3) δC 41.8, 46.2, 57.4, 69.8, 70.3, 79.8, 206.0; IR (film) νmax/cm−1
3411, 2922, 1714, 1264, 1063.
+
found 161.08136, C7H13O4 calcd. 161.08168; [M + K]+ found
199.03869, C7H12O4K+ calcd. 199.03727; 1H NMR (400 MHz,
CDCl3) δH 1.68 (1 H, tddd, J 14.4, 4.7, 2.4, 1.8, H-5), 2.18 (1 H, dddd,
J 14.4, 6.2, 3.6, 2.5, H-5′), 2.27 (1 H, dddd, J 13.7, 4.7, 2.5, 0.7, H-6),
2.80 (1 H, tddd, J 13.7, 6.2, 1.3, 0.7, H-6′), 2.91 (1 H, t, J 1.9, OH),
3.09 (1 H, d, J 2.0, OH), 3.53 (3 H, s, OMe), 3.71 (1 H, ddd, J 9.8, 2.8,
1.8, H-3), 4.07 (1 H, dd, J 9.8, 1.0, H-2), 4.26 (1 H, m, H-4); 13C
NMR (100 MHz, CDCl3) δC 27.5, 34.8, 59.7, 68.1, 76.4, 85.8, 207.2.
(2S,3S,4S)-3,4-Dihydroxy-2-methoxycyclohexanone 14.
Major metabolite from guaiacol 9, compound 14 was obtained as
colorless needles (2.5 g, 13%); mp 124−125 °C (EtOAc/hexane); Rf
0.2 (EtOAc); [α]D −54.2 (c 0.5, CHCl3); HRMS (LC-TOFMS) [M +
(1aS,4aR,1S,6R)-(6-Methoxy-4-oxocyclohex-2-enyl)-
4a,7a,7a-trimethyl-3a-oxo-2a oxabicyclo[2.2.1]heptane-1a-
carboxylate 8. A solution of cyclohexanone diol 6 (62 mg, 0.39
mmol) in dry pyridine (0.5 mL) was treated with (1S)-camphanic
chloride (208 mg, 0.96 mmol) and the mixture stirred at room
temperature for 12 h. The pyridine was removed in vacuo, the residue
dissolved in CH2Cl2 (10 mL) and the solution washed with brine (2 ×
10 mL). It was dried (Na2SO4) and concentrated under reduced
pressure to give a yellow oil, which was purified by PLC (40% EtOAc
in hexane) to give camphanate 8 as a colorless crystalline solid (68 mg,
55%); Rf 0.36 (40% EtOAc in hexane); mp 129−131 °C (acetone/
hexane); [α]D +129.3 (c 0.5, CHCl3); HRMS (LC-TOFMS) [M +
+
H]+ found 161.08136, C7H13O4 calcd. 161.08084; 1H NMR (400
MHz, CDCl3) δH 2.04 (1 H, m, H-5), 2.13 (1 H, dddd, J 12.5, 12.5,
10.2, 4.9, H-5′), 2.29 (1 H, dddd, J 14.1, 12.1, 6.5, 1.3, H-6), 2.45 (1 H,
dddd, J 14.1, 5.0, 4.3, 0.5, H-6′), 2.49 (1 H, br s, OH), 2.78 (1 H, br s,
OH), 3.51 (3 H, s, OMe), 3.80 (1 H, dd, J 3.0, 1.1, H-2), 4.10 (1 H,
ddd, J 10.2, 5.0, 2.6, H-4), 4.34 (1 H, ddd, J 3.0, 3.0, 1.5, H-3); 13C
NMR (100 MHz, CDCl3) δC 28.76, 35.31, 58.81, 69.70, 74.78, 84.51,
206.79; IR (film) νmax/cm−1 3418, 2926, 2856, 1731, 1494, 1452.
+
NH4]+ found 340.17538, C17H26NO6 calcd. 340.17546; [M + Na]+
found 345.13050, C17H22O6Na+ calcd. 345.13086; [M + K]+ found
361.10478, C17H22O6K+ calcd. 361.10480; 1H NMR (400 MHz,
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(1aS,4aR)-(S)-3-Methoxy-4-oxocyclohex-2-enyl)4a,7a,7a-tri-
methyl-3a-oxo-2a-oxabicyclo[2.2.1] heptane-1a-carboxylate
16. A solution of cyclohexanone diol 14 (100 mg, 0.62 mmol) in
dry pyridine (1 mL) was reacted with (1S)-camphanic chloride (342
mg, 1.58 mmol) as described for the synthesis of camphanate 8.
Camphanate 16 was obtained as a colorless crystalline solid (110 mg,
55%); mp 132−133 °C (acetone/hexane or EtOAc); [α]D −114.5 (c
0.75, CHCl3); HRMS (LC-TOFMS) [M + H]+ found 323.14797,
C17H23O6+calcd. 323.14891; [M+ NH3]+ found 340.17469,
C17H25O6N+ calcd. 340.17546; [M + Na]+ found 345.13014,
C17H22O6Na+ calcd. 345.13086; [M + K]+ found 361.1042,
Trimethylsilylated derivative of tetrahydroxy tautomers 24c or 24e:
(10.21 min); m/z 432 (M+, 9%), 344 (11), 343 (28), 342 (11), 329
(10), 239 (17), 191 (19), 147 (24), 133 (7), 75 (13), 74 (9), 73 (100).
Trimethylsilylated derivative of 1,2,4-trihydroxybenzene 27:(10.79
min); m/z 342 (M+, 71%), 254 (11), 240 (11), 239 (17), 73 (100).
Following similar treatment of enone cis-diol 3 and employing
CD3OD instead of methanol yielded deuteriated compounds 3D3, 3D4
and isomers 26D3, 26D4. On separation by PLC, using methanol in the
solvent mixture, the deuterium on the two hydroxyl groups was
replaced with hydrogen.
cis-Diol 3: HRMS (LC-TOFMS) [M + H]+ found159.06534,
1
C17H22O6K+ calcd. 361.1048; H NMR (400 MHz, CDCl3) δH 0.97
+
C7H10O4 calcd. 159.06519.
cis-Diol 3D3: HRMS (LC-TOFMS) [M + H]+ found 162.08391,
(3 H, s, Me), 1.06 (3 H, s, Me), 1.13 (3 H, s, Me), 1.71 (1 H, ddd, J
13.5, 9.5, 4.2, H-5a), 1.94 (1 H, ddd, J 13.3, 10.9, 4.6, H-5a), 2.06 (1 H,
ddd, J 13.8, 9.4, 4.6, H-6a), 2.17 (1 H, m, H-6), 2.37 (1 H, m, H-6),
2.44 (1 H, ddd, J 13.6, 10.8, 4.4, H-6a′), 2.55 (1 H, ddd, J 17.0, 7.8, 4.7,
H-5), 2.79 (1 H, ddd, J 17.0, 9.0, 4.8, H-5′), 3.65 (3 H, s, OMe), 5.76
(1 H, m, H-1), 5.81 (1 H, d, J 4.5, H-2); 13C NMR (100 MHz, CDCl3)
δC 9.8, 16.9, 17.0, 28.4, 29.0, 30.8, 34.5, 54.5, 54.9, 55.4, 69.5, 91.0,
111.3, 153.2, 167.4, 178.0, 192.6; LR EI MS m/z 322 (M+, 1%), 170
(23), 139 (36), 124 (91), 109 (100), 93 (35), 109 (24), 89 (39), 81
(77).
+
C7H8D3O4 calcd. 162.08402.
cis-Diol 3D4: HRMS (LC-TOFMS) [M + H]+ found 163.08999,
+
C7H7D4O4 calcd. 163.09029.
cis-Diol 26: HRMS (LC-TOFMS) [M + H]+ found 159.06544,
+
C7H10O4 calcd. 159.06519.
cis-Diol 26D3: HRMS (LC-TOFMS) [M + H]+ found 162.08372,
+
C7H8D3O4 calcd. 162. 08402.
cis-Diol 26D4: HRMS (LC-TOFMS) [M + H]+ found 163.09037.
+
C7H7D4O4 calcd. 163. 09029.
Crystal Data for 16. C17H22O6, M = 322.4, monoclinic, a =
6.071(3), b = 18.734(7), c = 7.024(3) Å, β = 98.10(1)o, U = 790.9(6)
Å3, T = 100(2) K, space group P21 (no. 4), Cu Kα radiation, λ =
1,54178 Å, Z = 2, F(000) = 344, Dx = 1.354 g cm−3, μ = 0.851 mm−1,
ω scans, 14.7° < 2θ < 133.1°, measured/independent reflections:
8710/2445, Rint = 0.032, direct methods solution, full-matrix least-
squares refinement on Fo2, anisotropic displacement parameters for
non-hydrogen atoms; hydrogen atoms included at positions calculated
from the geometry of the molecule using the riding model, with
isotropic vibration parameters. R1 = 0.036 for 2346 data with Fo >
4σ(Fo), 213 parameters, ωR2 = 0.096 (all data), GoF = 1.06, CCDC
1025987. The absolute configuration is established as (1S) relative to
the known absolute configuration of the (1S)-camphanate group and
independently from the anomalous scattering arising from the oxygen
atoms; Flack parameter x = −0.05(14).
2,4-Dimethoxyphenol 29. To a stirred solution of enone cis-diol
3 (100 mg, 0.63 mmol) in a mixture of acetone (2 mL) and 2,2-
dimethoxypropane (2 mL), was added p-TsOH monohydrate (6 mg,
0.03 mmol) at room temperature. After stirring the mixture for 2 h, the
solvent was removed under reduced pressure and the residue dissolved
in EtOAc (15 mL). The solution was successively washed with 5% aq.
NaHCO3 (10 mL), brine (10 mL), and dried (Na2SO4). Removal of
solvent under reduced pressure gave a light brown colored oil, which
was purified by column chromatography (66% EtOAc in hexane), to
yield phenol 29 as a light yellow viscous oil (81 mg, 83%); Rf 0.21
(66% EtOAc in hexane); 1H NMR (400 MHz, CDCl3) δH 3.76 (3H, s,
OMe), 3.85 (3H, s, OMe), 5.35 (1H, br s, OH), 6.39 (1H, dd, J 8.7,
2.7, H-5), 6.49 (1H, d, J 2.7, H-3),6.83 (1H, d, J 8.7, H-6); 13C NMR
(100 MHz, CDCl3) δC 55.87, 55.94, 99.5, 104.3, 114.2, 139.9, 147.2,
153.6. The spectroscopic data of phenol 29 was in agreement with the
literature.76
(2S,3S)-2,3-Dihydroxy-5-methoxycyclohex-6-enone 26.
Enone cis-diol 3 (50 mg, 0.32 mmol) was treated with an aqueous
solution of NaOH (1.5 mL, 1 M). The mixture was gently shaken, left
at room temperature for 24 h, neutralized with 1 M HCl and an
aliquot retained for LC-TOFMS analysis. The remaining reaction
mixture was concentrated under reduced pressure, the crude product
dried under a high vacuum. A small sample of the concentrate was
silylated for GC−MS analysis and the remainder taken up in methanol
(5 mL). The insoluble NaCl was filtered off, the filtrate concentrated,
and the crude product, on purification by PLC (EtOAc) separated into
two compounds. The major, less polar enone diol 26 was obtained as a
light yellow oil (12 mg, 24%); Rf 0.35 (EtOAc); [α]D +4.0 (c 1.0,
CHCl3); HRMS (LC-TOFMS) [M + H]+ found 159.06544,
(3aS,7aS)-7-Methoxy-2,2-dimethyl-3a,4-dihydrobenzo[d]-
[1,3]dioxol-5(7aH)-one 30. A solution of enone diol 3 (1.0 g, 6.33
mmol) in a mixture of acetone (4 mL) and 2,2-dimethoxypropane (8
mL), containing p-TsOH monohydrate (60 mg, 0.32 mmol), was
stirred at 0 °C for 45 min. The solvent was removed under reduced
pressure, the residue dissolved in EtOAc (35 mL) and the solution
successively washed with 5% aq. NaHCO3 (30 mL) and brine (30
mL). It was dried (Na2SO4) and concentrated under reduced pressure
to give a yellow oil, which was purified by column chromatography
(85% EtOAc in hexanes). Acetonide 30 was obtained as a white solid
(1.0 g, 80%); mp 99−101 °C (EtOAc); Rf 0.55 (85% EtOAc in
hexane); [α]D −58.4 (c 0.5, CHCl3); HRMS (ES): [M + H]+ found
+
C7H11O4 calcd. 159.06519; [M + Na]+ found 181.04713,
+
1
199.0975, C10H15O4 calcd. 199.0970; H NMR (400 MHz, CDCl3)
δH 1.37 (3 H, s, Me), 1.40 (3 H, s, Me), 2.64 (1 H, ddd, J 17.5, 2.7, 1.3,
H-4), 2.86 (1 H, dd, J 17.5, 1.6, H-4′), 3.75 (3 H, s, OMe), 4.64−4.66
(2 H, m, H-3a, H-7a), 5.42 (1 H, s, H-6); 13C NMR (100 MHz,
CDCl3) δC 25.4, 26.8, 37.6, 55.3, 71.3, 71.4, 101.8, 109.3, 170.9, 194.1;
LRMS (ES): 199 (6), 221 (20), 259 (48), 413 (80), 484 (42), 691
(35), 803 (100), 1082 (10), 1194 (24).
C7H10O4Na+ calcd. 181.04729; 1H NMR (400 MHz, CD3OD) δH
2.59 (1 H, dd, J 18.1, 2.8, H-4), 2.93 (1 H, ddd, J 18.1, 3.3, 1.7, H-4′),
3.76 (3 H, s, OMe), 4.21 (1 H, d, J 3.0, H-2), 4.34 (1 H, ddd, J 3.0, 3.0,
H-3), 5.43 (1 H, d, J 1.3, H-6); 13C NMR (100 MHz, CDCl3) δC 37.3,
57.1, 70.3, 76.0, 100.3, 177.3, 199.8; IR (film) νmax/cm−1 3397, 2659,
1658, 1603, 1386, 1227, 1199. The minor polar compound (3.1 mg,
6%); Rf 0.12 (EtOAc), was the starting enone cis-diol 3.
(3aS,7aS)-2,2-Dimethyl-7,7a-dihydrobenzo[d][1,3]dioxol-4-
(3aH)-one 31.72−74 A solution of DIBAL in toluene (1 M, 421 μL,
0.41 mmol) was added dropwise to a solution of acetonide 30 (59 mg,
0.30 mmol) in a mixture of THF and toluene (1:1, 5 mL) at 0 °C.
After stirring for 45 min, a saturated aq. solution of NH4Cl (0.3 mL)
was added and the mixture stirred for another 1 h. Magnesium sulfate
(200 mg) was added to the stirred reaction mixture and it was filtered
through diatomaceous earth. The filtrate was concentrated in vacuo to
yield a yellow oil, which was immediately dissolved in Et2O (10 mL)
and the solution treated with p-TsOH (5 mg). The reaction mixture
was washed successively with saturated aq. NaHCO3 (10 mL) and
brine (10 mL), dried (MgSO4) and concentrated in vacuo. The crude
product obtained was purified by PLC (50% EtOAc in hexane) to
LC-TOFMS analysis showed a strong peak consistent with the
+
presence of tautomers 24a−e; [M + H]+ found 145.04977, C6H9O4
calcd. 145.04954; [M + Na]+ found 167.03145, C6H8O4Na+ calcd.
167.03148; [M + K]+ found 183.00521, C6H8O4K+ calcd. 183.00542.
GC−MS analysis showed three peaks consistent with the presence
of trimethylsilylated derivatives of tautomers 24a or 24b (triTMS) and
24c or 24e (tetraTMS) and 1,2,4-trihydroxybenzene 27 (triTMS).
Trimethylsilylated derivatives of trihydroxy tautomers 24a and 24b:
Peak 1 (10.23 min); m/z 360 (M+, 8%), 345 (18), 242 (12), 204 (27),
147 (23), 133 (11), 75 (13), 74 (8), 73 (100); Peak 2 (10.87 min); m/
z 360 (M+, 2%), 345 (13), 246 (9), 245 (20), 244 (99), 147 (38), 133
(15), 75 (11), 74 (9), 73 (100).
3437
DOI: 10.1021/jo5028968
J. Org. Chem. 2015, 80, 3429−3439

The Journal of Organic Chemistry
Article
yield enone 31 as a colorless oil (32 mg, 70%); Rf 0.55 (50% EtOAc in
hexane); [α]D +83.9 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δH
1.34 (3 H, s, Me), 1.40 (3 H, s, Me), 2.79 (1 H, dddd, J 20.4, 5.7, 2.9,
2.8, H-7), 2.88 (1 H, dddd, J 20.4, 4.8, 1.7, 1.5, H-7′), 4.29 (1 H, d, J
5.2, H-3a), 4.63 (1 H, dddd, J 5.7, 5.2, 1.7, 1.7, H-7a), 6.12 (1 H, ddd, J
10.3, 2.8, 1.5, H-5), 6.83 (1 H, dddd, J 10.3, 4.8, 2.9, 1.7, H-6); 13C
NMR (100 MHz, CDCl3) δC 26.1, 27.6, 27.9, 73.0, 75.6, 109.4, 128.4,
146.4, 196.3.
(3aS,7aS)-2,2,6-Trimethyl-7,7a-dihydrobenzo[d][1,3]dioxol-
4(3aH)-one 32. To a solution of acetonide 30 (290 mg, 1.46 mmol)
in THF (6 mL) at 0 °C was added a solution of MeMgBr in Et2O (3
M, 1.46 mL, 4.0 mmol). The reaction mixture was stirred at 0 °C for 2
h, diluted with Et2O (20 mL), and then quenched with ice. It was
washed with 0.5 M HCl (2 × 15 mL), the aq. layers extracted with
Et2O (2 × 15 mL) and the combined organic layers washed with brine
(2 × 15 mL). The solution was dried (MgSO4) and concentrated in
vacuo to give a brown oil, which was purified by PLC (50% EtOAc in
hexane) to yield enone 32 as a light yellow oil (192 mg, 72%); Rf 0.25
(50% EtOAc in hexane); [α]D +42.8 (c 0.92, CHCl3); HRMS (ES):
[M + H]+ found 183.1020, C10H15O3+ calcd. 183.1021; 1H NMR (400
MHz, CDCl3) δH 1.35 (3 H, s, Me), 1.40 (3 H, s, Me), 2.00−2.02 (3
H, m, HCCMe), 2.76 (2 H, m, H-7, H-7′), 4.24 (1 H, d, J 5.1, H-
3a), 4.61 (1 H, ddd, J 6.8, 3.5, 3.5, H-7a), 5.98 (1 H, dq, J 2.8, 1.4, H-
5); 13C NMR (100 MHz, CDCl3) δC 24.6, 26.2, 27.6, 29.9, 32.8, 72.9,
74.8, 109.4, 125.3, 158.5, 196.1.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Department of Education and Learning,
Northern Ireland, for a Postgraduate Studentship (to PABMcI)
and to the EPSRC UK National Crystallography Service,
University of Southampton, for collection of the crystallo-
graphic data.
DEDICATION
■
This article is dedicated to the memory of the late Professor
David T. Gibson (University of Iowa), a good friend and
outstanding scientist.
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126.5 (2C), 129.0 (2C), 130.7, 138.0, 155.4, 196.5; LRMS (ES): 217
(100), 245 (25), 451 (65).
(4S,5S)-4-Hydroxy-3,5-dimethoxycyclohex-2-enone 34. To a
solution of enone cis-diol 3 (174 mg, 1.10 mmol) in CH2Cl2 (10 mL)
was added Ag2O (260 mg, 1.12 mmol) and methyl iodide (234 mg,
1.65 mmol). After stirring the reaction mixture for 12 h at room
temperature, the insoluble salts were filtered off and the filtrate
concentrated in vacuo, to yield a dark brown colored oil. The crude
product on PLC (50% EtOAc in hexane, 3 elutions) separated into
compounds 34, 35 and 36. Enone 34 was isolated as an oil (70 mg,
37%); Rf 0.26 (75% EtOAc in hexane); [α]D −48.0 (c 0.67, MeOH);
lit.75 [α]D +28.4 (c 1.62, MeOH); HRMS (ES): [M + H]+ found
173.0811, C8H13O4+ calcd. 173.0814; 1H NMR (400 MHz, CDCl3) δH
2.52 (1 H, dd, J 16.6, 4.3, H-6), 2.70 (1 H, dd, J 16.6, 9.2, H-6′), 3.42
(3 H, s, OMe), 3.75−3.78 (1 H, m, H-5), 3.78 (3 H, s, OMe), 4.49 (1
H, d, J 3.5, H-4), 5.39 (1 H, s, H-2); 13C NMR (100 MHz, CDCl3) δC
39.2, 57.0, 57.1, 68.2, 78.3, 103.1, 178.3, 199.5; LRMS (ES): 173 (10),
217 (100), 304 (60); IR (film) νmax /cm−1 3434, 2944, 1637, 1607,
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of all compounds and HMBC spectra
of compounds 3 and 26. This material is available free of charge
via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Authors
*E-mail: dr.boyd@qub.ac.uk.
*E-mail: p.stevenson@qub.ac.uk.
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