(1R,4S,5R)-3-fluoro-1,4,5-trihydroxy-2-cyclohexene-1-carboxylic acid: The fluoro analogue of the enolate intermediate in the reaction catalyzed by type II dehydroquinases

Article abstract of DOI:10.1039/b404535a

The fluoro analogue of the enolate intermediate in the reaction catalyzed by type II dehydroquinases has been prepared from naturally occuring (-)-quinic acid over seven steps and has been shown to be the most potent inhibitor reported to date of the type

Full text of DOI:10.1039/b404535a

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(1R,4S,5R)-3-Fluoro-1,4,5-trihydroxy-2-cyclohexene-1-carboxylic
acid: the fluoro analogue of the enolate intermediate in the
reaction catalyzed by type II dehydroquinases
Martyn Frederickson,a Aleksander W. Roszak,b John R. Coggins,c Adrian J. Lapthornb
and Chris Abell*a
a University Chemical Laboratory, Lensfield Road, Cambridge, UK CB2 1EW.
E-mail: ca26@cam.ac.uk; Fax: ϩ44 (0)1223 336362; Tel: ϩ44 (0)1223 336405
b Department of Chemistry, University of Glasgow, Glasgow, UK G12 8QQ
c Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow, UK G12 8QQ
Received 29th March 2004, Accepted 19th April 2004
First published as an Advance Article on the web 6th May 2004
The fluoro analogue of the enolate intermediate in the reaction catalyzed by type II dehydroquinases has been
prepared from naturally occuring (Ϫ)-quinic acid over seven steps and has been shown to be the most potent
inhibitor reported to date of the type II enzyme from Mycobacterium tuberculosis.
Introduction
Dehydroquinase [3-dehydroquinate dehydratase; E.C. 4.2.1.10]
is common to both the shikimate1,2 and quinate3 pathways. It
catalyses the reversible interconversion of 3-dehydroquinate
(DHQ) 1 and 3-dehydroshikimate (DHS) 2. The enzyme occurs
in two chemically and biochemically distinct forms4 (type I and
type II) that catalyse the same overall chemical transformation
via different mechanisms. Type I enzymes are heat labile protein
dimers5 that catalyse a syn elimination6 of water through
Schiff’s base intermediates7 involving a conserved lysine. Type
II dehydroquinases are more thermally robust dodecameric
species8 that effect an anti dehydration9 via an enzyme-stabil-
ized enolate anion 3 (Scheme 1).
Fig. 1 Selective inhibitors of type II dehydroquinases.
quinases from Streptomyces coelicolor and Mycobacterium
tuberculosis (PDB accession codes for 4: 1GU1 and 1H0R; for
5: 1H0S) allowed us to fully determine their modes of action.
Our search for even more effective inhibitors focused on
derivatives in which the true geometric and electronic features
of enolate 3 were more accurately mimicked, particularly the
electron distribution and high electron density generated at C-3
during the course of the reaction. We thus chose to replace the
enolate oxyanion with its closest chemical equivalent, the iso-
steric and isoelectronic fluorine atom and sought to prepare
vinyl fluoride 6. In a recent communication12 we briefly out-
lined the synthesis of 6 (together with the 3,3-difluoro acid 7).
Herein we report more fully on our studies towards the
synthesis of 6 and describe in detail the inhibitory profiles of
both 6 and 7 against dehydroquinases (both types I and II) from
a variety of bacterial sources. In addition we comment briefly
on the X-ray crystallographic structure of the co-complex of 6
with the type II dehydroquinase from Streptomyces coelicolor.
Results and discussion
Scheme 1
Acyclic vinyl fluorides have gained popularity in recent years
as isosteric and isoelectronic replacements for scissile amide
bonds in non-hydrolysable protease inhibitors.13,14 In contrast,
functionalized endocyclic vinyl fluorides remain far less well
exemplified.15,16 We sought a reliable and relatively mild method
for fluorine incorporation as we were wary of the fact that
the desired polyfunctionalized acid 6 (and closely related
derivatives) might prove to be prone to aromatization if
subjected to more forcing reaction conditions; we thus looked
to the very well developed chemistry of the closely related
semi-aromatic shikimate ring system for inspiration.
The mechanistic differences of the two enzyme types led us to
design inhibitors that were selective for the type II proteins
which are found in a number of clinically important bacterial
strains including Mycobacterium tuberculosis and Helicobacter
pylori. Based upon the idea that compounds which mimicked
the enolate intermediate 3 were likely to show such selectivity
we sought derivatives which, when compared to DHQ 1, pos-
sessed either increased sp2 character at C-2 and C-3 or had
superior hydrogen bonding potential at C-3; accordingly we
prepared alkene 4 and oxime 5 (Fig. 1). We were thus able to
demonstrate that 4 and 5 were potent and highly selective (up to
1000 fold) competitive reversible inhibitors of type II dehydro-
quinases.10 X-ray crystallographic studies11 on the complexes
formed between alkene 4 and oxime 5 and the type II dehydro-
Methods for the introduction of fluorine into the shikimate
nucleus have been investigated by several research groups
with 2-fluoro,15,17 3-fluoro,18,19 3,5-difluoro,20 6-fluoro,21–24 6,6-
difluoro25 and 6-trifluoromethyl26 derivatives having been
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 9 2 – 1 5 9 6
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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described. Amongst these endeavours is work by the group of
Bartlett15 describing the synthesis of 2-fluoroshikimate 11
(Scheme 2). In the key synthetic step incorporation of fluorine
was effected upon treatment of cyclohexanone 8 with diethyl-
aminosulfur trifluoride (DAST) in hot 1,2-dimethoxyethane
(DME). The reaction afforded a mixture of gem-difluoride 9
(28%) and the desired vinyl fluoride 10 (40%) which was
transformed into the fluoro acid 11 over four steps; conversion
of the undesired product 9 to 11 was achieved via aldehyde 12
which readily eliminated hydrogen fluoride under mildly basic
conditions. We thus envisaged a synthesis of fluoro acid
6 involving reaction between DAST and an appropriately
protected cyclohexanone derivative of naturally occuring
(Ϫ)-quinic acid 13.
Scheme 2
(Ϫ)-Quinic acid 13 was smoothly protected as the cyclic
bis-ketal 14 with concomitant protection of the C-1 carboxylate
functionality as the methyl ester upon treatment with 2,3-
butanedione ketal27 in an acidified mixture of trimethyl
orthoformate and methanol at reflux (Scheme 3). The reaction
afforded essentially a single product in high yield (79%), the
ketal protecting group being selective for the vicinal trans-
diequatorial diol with a double anomeric effect controlling both
ketal stereogenic centres. Oxidation of the C-3 secondary
alcohol of 14 was readily achieved upon treatment with RuCl3
and KIO4 under biphasic conditions in a mixture of chloro-
form and aqueous potassium carbonate. Cyclohexanone 1528
(obtained as a colourless solid in 80% yield after column
chromatography) is an exceptionally crystalline compound with
a propensity to crystallize directly from concentrated solutions
in diethyl ether obtained upon purification on silica. Protection
of the remaining C-1 tertiary alcohol as the methoxymethyl
(MOM) ether was effected with dimethoxymethane and excess
phosphorus pentoxide in anhydrous chloroform. The resulting
fully protected product 16 was obtained in essentially quanti-
tative yield.
Scheme 3
starting material. DAST has been observed29,30 to decompose
rapidly and with great exotherm at elevated temperatures
resulting in violent decomposition of the contents of reaction,
however, it is far more stable at moderate reaction temperatures
(below 90 ЊC) in dilute solution and we thus suspected other
factors to be the cause of such poor yields and reproducibility.
Factors that we considered included purity of starting
material, purity and moisture content of the solvent and the age
of DAST samples; to ensure purity of starting material ketone
16 was recrystallized from a mixture of hexane and diethyl
ether as fine colourless needles (yields of 16 from 15 after crys-
tallization ∼80%). Reactions were performed using fresh sam-
ples of DAST dissolved in fresh samples of anhydrous DME
under an argon atmosphere to maintain anhydrous conditions.
A solution of ketone 16 in boiling DME was treated with 3
portions of DAST (1.2 equivalents each) at 2 hourly intervals.
After 6 h the reaction was cooled to room temperature and
quenched by the addition of a saturated aqueous solution of
sodium bicarbonate. Under these conditions we were success-
fully able to control the yield of fluorinated products; moreover
the reaction was shown to be very reproducible.
Initial attempts to introduce fluorine into the cyclohexanone
ring of 16 (DAST, DME, 80 ЊC, nitrogen atmosphere) proved
to be frustratingly poor in terms of both the yields and ratios
of products 17 and 18 so obtained; in addition reaction repro-
ducibility was found to be extremely capricious with some
reactions failing altogether with rapid decomposition of
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Extensive analysis (1H, 13C and 19F NMR spectra recorded in
CDCl3 and mass spectra) of the two reaction products obtained
(3 : 1 ratio) after column chromatography on silica (with
particular reference to H–F, C–F and F–F coupling constants)
allowed for elucidation of their structures. The more chromato-
graphically mobile isomer (colourless oil) contained a vicinal
fluorine coupled olefinic hydrogen (δH 5.62, JH–F 15 Hz) and one
fluorine atom (δF Ϫ115.9) coupled to an olefinic carbon
(δC 159.6, JC–F 277 Hz). Mass spectral data (m/z 333.1349,
M Ϫ OMeϩ) confirmed the product to be the desired vinyl
fluoride 17. The less mobile isomer on silica (colourless solid)
lacked olefinic hydrogens, contained a pair of coupled fluorine
atoms (δF Ϫ103.3, JF–F 245 Hz; Ϫ113.3, JF–F 245 Hz) that were
both coupled to the same sp3 carbon (δC 119.7, JC–F 251 and
245 Hz) and gave mass spectral data (m/z 385.1692, MϩHϩ)
indicative of a difluoride; this product was subsequently
unequivocally shown to be the C-3 gem-difluoride 18 by single
crystal X-ray analysis.12,31
Table 1 Ki values (µM) for 6 and 7 against bacterial dehydroquinases
in comparison to those reported previously10 for 4 and 5
Type I
S. typhi
Type II
M. tuberculosis
Type II
S. coelicolor
Km
4
1832
3000 1000
> 25000
6433
200 20
20
10
65034
30 10
500 200
5
2
2
6
7
1500 500
8000 1000
15
2
700 100
700 100
Somewhat disappointingly, under these conditions the
desired vinyl fluoride 17 proved to be the minor product (13%)
with the difluoride 18 (45%) being the major component of the
mixture, an isomeric reversal compared to the two products
formed in the key fluorination step of the 2-fluoroshikimate
synthesis (9 and 10, 28% and 40% respectively). Despite
numerous attempts we were unable to discover conditions to
increase the yield and proportion of 17 relative to 18. Attempts
to effect conversion of 18 to 17 were similarly unsuccessful. A
plethora of basic conditions were tested in an attempt to induce
elimination of hydrogen fluoride from 18; in all cases 18 was
recovered unchanged. Addition of soluble silver salts to these
reactions (e.g. AgPF6, AgBF4 and AgOTf ) to encourage
precipitation of fluoride anion (as insoluble AgF) also proved
unsuccessful. In spite of these results the conditions developed
for incorporation of fluorine at C-3 of the quinate ring were
robust and reproducible enough to consistently afford accept-
able quantities of material (up to 3 mmol scale) and we were
thus routinely able to prepare hundreds of milligrams of pure
samples of both 17 and 18.
Both fluorides 17 and 18 were readily deprotected over three
steps to afford the desired vinyl fluoride 6 and the difluoro acid
7 in good yields. Treatment of 17 and 18 with 50% aqueous
trifluoroacetic acid at 60 ЊC resulted in the removal of both
the MOM group protecting the C-1 tertiary alcohol and the
2,3-butanedione derived bis-ketal protecting the 4,5-diol
functionality. The colour of the solutions changed dramatically
during the course of the reactions from colourless to an intense
yellow due to the liberation of 2,3-butanedione. Upon cooling
of the reaction mixtures and removal of solvent under reduced
pressure, purification was effected simply by partitioning
between ethyl acetate and water followed by lyophilization of
the resulting colourless aqueous phase. The intermediate
methyl esters were readily saponified upon treatment with
aqueous sodium hydroxide and the free acids 6 and 7 obtained
after cation exchange [Amberlite IR-120(H)] and lyophilization
of the resulting aqueous solutions (63% and 96% yields respect-
ively over the three steps).
Fig. 2 Dixon plot of reciprocal of rate versus inhibitor concentration
[I] for four differing concentrations of 1 showing inhibition of the type
II dehydroquinase from Mycobacterium tuberculosis by 6.
profiles. Both compounds were selective for the type II enzymes
over the type I but in each case showed essentially no selectivity
between the two type II proteins. As expected the vinyl fluoride
6 was significantly more potent than the difluoride 7 (partic-
ularly so against the type II enzymes) consistent with our
previous findings10 that inhibitors with sp2 character at C-2 and
C-3 had significantly greater potency.
The inhibitory behaviour of vinyl fluoride 6 towards the type
II proteins contrasts sharply with those of the low micromolar
inhibitors 4 and 5,10 both of which show a marked selectivity
(up to 25 fold) amongst type II dehydroquinases from a variety
of sources; the ‘ring flattened’ alkene inhibitor 4 is more potent
against the enzyme from S. coelicolor whereas the ‘enhanced
hydrogen bonding’ mimicking oxime 5 shows selectivity for the
M. tuberculosis protein. Vinyl fluoride 6 is the first effective
pan-type II dehydroquinase inhibitor reported to date with, in
all cases, a Ki significantly lower than the corresponding Km but
with a molecular mass and degree of molecular complexity
essentially identical to the natural substrate DHQ 1. As such,
fluoride 6 would appear to effectively mimic structural features
that are critical to turnover for all type II dehydroquinases.
Similar subtleties in the kinetic profiles of type II dehydro-
quinases determined in the presence of simple polydentate
anions (phosphate and sulfate) have also been observed35 and
have been rationalized in terms of their binding characteristics
with particular reference to data obtained from X-ray crystallo-
graphic studies. The results of these structural studies,
when taken in conjunction with the findings outlined herein,
strongly suggest that the binding characteristics of type II
dehydroquinases are acutely sensitive to both the precise
geometric features as well as the chemical functionalities
present within the ligand.
Acids 6 and 7 were assayed against purified samples of both
type I and type II dehydroquinases from a variety of bacterial
sources (Type I: from Salmonella typhi; Type II: from Myco-
bacterium tuberculosis and Streptomyces coelicolor). As
expected from a compound specifically designed to closely
mimic the reaction intermediate, the kinetics observed for 6
were indicative of it being a competitive reversible inhibitor
of each of the enzymes. Difluoride 7, the 3,3-difluoro analogue
of 3-deoxyquinic acid 1910 (a weak competitive reversible
inhibitor), also showed the expected competitive reversible
inhibitory behaviour. Ki values for 6 and 7 against each of
the three dehydroquinases (Table 1) were determined directly
from Dixon plots (Fig. 2).
Vinyl fluoride 6 is the most potent inhibitor of the type II
dehydroquinase from Mycobacterium tuberculosis reported
to date and the second most potent36 for the enzyme from
Streptomyces coelicolor. The increase in potency shown by
6 over both 4 and 5 is only relatively modest (approximately
twofold) for the enzymes against which 4 and 5 are most potent,
The two fluoro acids 6 and 7 showed similar inhibitory
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 9 2 – 1 5 9 6
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however fluoride 6 offers a much more significant increase in
potency (20–30 fold) against the enzymes for which 4 and 5 are
less effective.
specified at ambient temperature on a Perkin Elmer 241 instru-
ment and are quoted in 10Ϫ1 deg cm2 gϪ1. Microanalyses were
determined by the Departmental Microanalytical Service, Uni-
versity Chemical Laboratory, Cambridge using an Exeter Ana-
lytical 440 elemental analyzer. Infrared spectra were recorded
on an ATI Mattson Genesis Series FTIR spectrometer as a thin
film or Nujol mull as stated. NMR spectra were recorded on a
Bruker AC-250 spectrometer operating at 250.1 MHz (for 1H),
62.9 MHz (for 13C) or 234.5 MHz (for 19F) in the deuterated
solvents stated. High resolution mass spectra were recorded on
a Kratos MS890 or Kratos Concept 1H instrument under posi-
tive electron impact (ϩEI) conditions or under positive fast
atom bombardment (ϩFAB) conditions (using xenon as the
ionizing source) as stated. Compounds were assayed for inhibi-
tory activity against dehydroquinases in the presence of 1 by
monitoring changes in the rate of increase in absorbance at 234
nm in UV spectra due to formation of 2 (ε 12000 MϪ1cmϪ1).
Kinetic assays were performed on a Varian Carey spectro-
photometer in 50 mM pH 7 buffer (potassium phosphate and
Tris-HCl for type I and type II dehydroquinases respectively) at
25 ЊC using black walled quartz cuvettes. The assay mixture was
prepared in situ and the reaction initiated by addition of the
enzyme solution to the mixture. Concentrations of solutions of
1 were accurately determined by equilibration with type I dehy-
droquinase and measurement of the change in UV absorbance
at 234 nm. To measure inhibition the concentration of inhibitor
(I) was varied at a constant concentration of substrate and
enzyme and assays were repeated for a number of different
concentrations of substrate. Ki values were obtained through
Dixon plots (1/v versus [I]).
Vinyl fluoride 6 was co-crystallized with the type II dehydro-
quinase from Streptomyces coelicolor (Fig. 3). The structure was
collected at room temperature as the ligand could not be
detected in data collected from frozen crystals (at 100 K). In
accord with both the previously reported apo structure11 (PDB:
1GU0) and co-complexes with phosphate anion36 (PDB:
1D0I), 2 (PDB: 1GTZ) and 4 (PDB: 1GU1) the co-complex
(2.2 Å resolution) is a homo-dodecamer and contains an
equivalent copy of 6 bound at each of the twelve separate active
sites within the structure. The orientation of the vinyl fluoride
6 is essentially identical to that of the alkene 4 with the
additional fluorine atom involved in a long hydrogen bond
(3.2 Å) to a conserved water in the active site. The absence of
any tartrate or glycerol in the active site (both of which are
present in the alkene 4 inhibitor structure 1GU1) results in a
shift in the lid domain and brings the Arg23 residue into the
active site. Although the positively charged guanidinium
sidechain of R23 is still relatively distant from the vinyl fluoride
6 (over 4.5 Å away from the fluorine atom) this observation
may have implications in the binding of the natural substrate 1
and its subsequent turnover to 2.
Methyl (1R,3S,4S,6S,9R)-7-fluoro-3,4-dimethoxy-9-(meth-
oxymethyl)oxy-3,4-dimethyl-2,5-dioxabicyclo[4.4.0]dec-7-ene-9-
carboxylate 17 and methyl (1R,3S,4S,6S,9R)-7,7-difluoro-
3,4-dimethoxy-9-(methoxymethyl)oxy-3,4-dimethyl-2,5-dioxa-
bicyclo[4.4.0]decane-9-carboxylate 18. A stirred solution of
methyl (1R,3S,4S,6S,9R)-3,4-dimethoxy-9-(methoxymethyl)-
oxy-3,4-dimethyl-2,5-dioxabicyclo[4.4.0]decan-7-one-9-carb-
oxylate 1628 (1.12 g, 3.09 mmol) in anhydrous 1,2-
dimethoxyethane (20 cm3) under an argon atmosphere was
treated with (diethylamino)sulfur trifluoride (490 µl, 600 mg,
3.71 mmol) and the resulting mixture was stirred and held at
reflux for 2 h whereupon the mixture was treated with two
further portions of (diethylamino)sulfur trifluoride (490 µl, 600
mg, 3.71 mmol) at 2 h intervals. After cooling saturated
aqueous sodium hydrogen carbonate (100 cm3) was added and
the mixture extracted with ethyl acetate (2 × 200 cm3). The
combined organic extracts were dried over anhydrous sodium
sulfate, filtered, the solvent removed under reduced pressure
and the residue subjected to column chromatography on silica.
Elution with 2 : 1 v/v hexane–diethyl ether afforded the vinyl
fluoride 17 (146 mg, 13%), Rf 0.34, as a colourless oil. [α]D
ϩ175.8 (c 0.48 in CH2Cl2); (Found: C, 51.52; H, 6.83.
C16H25FO8ؒ½H2O requires C, 51.47; H, 7.02%); νmax(film)/cmϪ1
1745 and 1690; δH (250 MHz, CDCl3) 5.62 (1H, dt, J 15, 1.5, 8-
H), 4.82 (1H, d, J 7.5, OCHaHbOMe), 4.58 (1H, d, J 7.5,
OCHaHbOMe), 4.38 (1H, ddd, J 9, 3, 1.5, 6-H), 4.15 (1H, ddd,
J 12.5, 9, 3.5, 1-H), 3.75 (3H, s, CO2Me), 3.36 (3H, s, OMe),
3.28 (3H, s, OMe), 3.26 (3H, s, OMe), 2.26 (1H, ddd, J 12.5, 3.5,
1.5, 10α-H), 1.92 (1H, t, J 12.5, 10β-H), 1.37 (3H, s, Me) and
1.30 (3H, s, Me); δC (100.6 MHz, CDCl3) 172.3, 159.6 (d, J 277),
103.8 (d, J 19), 100.6, 100.3, 93.0, 75.9 (d, J 11.5), 67.3 (d,
J 18.5), 65.2 (d, J 5.5), 56.5, 52.8, 48.1, 48.0, 35.9, 17.7 and 17.6;
δF (235.4 MHz, CDCl3) Ϫ115.9 (dd, J 15, 3); m/z (ϩEI)
333.1349 (M Ϫ OMeϩ), C15H22FO7 requires 333.1334. Further
elution afforded the difluoride 18 (539 mg, 45%), Rf 0.22, as a
colourless solid. mp 93 ЊC (colourless prisms from hexane–
diethyl ether); [α]D ϩ166.0 (c 0.05 in CH2Cl2); (Found: C, 49.96;
H, 6.74. C16H26F2O8 requires C, 50.00; H, 6.82%); νmax(Nujol)/
cmϪ1 1755; δH (250 MHz, CDCl3) 4.83 (1H, d, J 7.5, OCHaHb-
Fig. 3 X-ray crystal structure (PDB: 1V1J) of the co-complex between
vinyl fluoride 6 and the type II dehydroquinase from Streptomyces
coelicolor. The conserved water molecule described in the text is shown
as a red sphere. The figure was prepared using PyMol.37
Dehydroquinases are utilized by bacteria and fungi for the
synthesis of essential aromatics and, in common with the other
enzymes of the shikimate and quinate pathways, are foreign to
all mammalian systems. Type II dehydroquinases are present in
a number of bacterial strains that are responsible for a variety
of disease states; they have therefore been seen as potential
targets for novel therapeutics, particularly given the recently
reported structural data for type II dehydroquinases complexed
to small molecule inhibitors. The availability of this wealth
of data opens up the possibility of rational drug design38 using
a
structure-aided approach against clinically important
pathogens responsible for a number of increasingly prevalent
diseases that include tuberculosis, ulceration and stomach
cancer.
Experimental
All organic solvents were freshly distilled prior to use and Milli-
Q deionized water was used for all biochemical work. Amberlite
IR-120(H) was washed alternately with water, 5 M sodium
hydroxide, water, 6 M HCl and water prior to use. Flash column
chromatography was carried out using silica gel 60 (Merck
9385). Rf values are quoted with respect to the solvent used for
elution. Melting points were recorded on a Kofler hot-stage and
are uncorrected. Specific rotations were recorded in the solvent
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OMe), 4.62 (1H, d, J 7.5, OCHaHbOMe), 4.16 (1H, dddd,
J 12, 10, 4, 1.5, 1-H), 3.78 (1H, ddd, J 20.5, 10, 5, 6-H), 3.74
(3H, s, CO2Me), 3.39 (3H, s, OMe), 3.29 (3H, s, OMe), 3.26
(3H, s, OMe), 2.70 (1H, dddd, J 15.5, 11, 7, 4, 8α-H), 2.39 (1H,
dm, J 14, 10α-H), 2.18 (1H, ddd, J 32, 15.5, 5.5, 8β-H), 1.86
(1H, dd, J 14, 12, 10β-H), 1.38 (3H, s, Me) and 1.29 (3H, s, Me);
δC (100.6 MHz, CDCl3) 171.2, 119.7 (dd, J 251, 245), 100.2,
99.6, 93.1, 72.3 (t, J 39), 63.6 (d, J 8), 56.8, 52.8, 48.0, 48.0, 38.0
(dd, J 27, 24), 35.8, 17.7 and 17.5; δF (235.4 MHz, CDCl3)
Ϫ103.3 (1F, dq, J 245, 5) and Ϫ113.3 (1F, dddd, J 245, 32, 20.5,
11); m/z (ϩFAB) 385.1692 (MϩHϩ), C16H27F2O8 requires
385.1674.
References
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Structure, 2002, 10, 493.
(1R,4S,5R)-3-Fluoro-1,4,5-trihydroxy-2-cyclohexene-1-carb-
oxylic acid 6. A solution of methyl (1R,3S,4S,6S,9R)-7-fluoro-
3,4-dimethoxy-9-(methoxymethyl)oxy-3,4-dimethyl-2,5-dioxa-
bicyclo[4.4.0]dec-7-ene-9-carboxylate 17 (211 mg, 0.58 mmol)
in 50% aqueous trifluoroacetic acid (6 cm3) was stirred and held
at 60 ЊC for 1 h. After cooling the solvent was removed under
reduced pressure, the residue redissolved in water (10 cm3),
filtered and the solution extracted with ethyl acetate (3 ×
10 cm3). The aqueous layer was lyophilized to afford a colour-
less foam that was redissolved in water (5 cm3) and stirred with
aqueous sodium hydroxide solution (2.5 M, 260 µl, 0.65 mmol)
for 10 min. Amberlite IR-120(H) (1.0 g) was added and the
mixture stirred for a further 5 min. The resin was removed by
filtration, washed with water (5 cm3) and the combined filtrates
lyophilized to afford the product 6 (68 mg, 61%), as a pale
yellow oil. [α]D Ϫ83.3 (c 0.12 in H2O); νmax(film)/cmϪ1 3395,
2930, 2600, 1725 and 1640; δH (250 MHz, D2O) 5.53 (1H, d,
J 15.5, 2-H), 4.24 (1H, d, J 7.5, 4-H), 4.03 (1H, ddd, J 10, 7.5,
5.5, 5-H), 2.20 (2H, m, 6α-H and 6β-H); δC (62.9 MHz, D2O)
176.5, 160.3 (d, J 267), 105.3 (d, J 17.5), 71.3 (d, J 13), 69.8 (d,
J 20.5), 68.5 (d, J 7), 38.2; δF (235.4 MHz, D2O) Ϫ115.9
(d, J 15.5); m/z (ϩEI) 147.0457 (M Ϫ CO2Hϩ), C6H8FO3
requires 147.0450.
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(1R,4S,5R)-3,3-Difluoro-1,4,5-trihydroxycyclohexane-1-carb-
oxylic acid 7. A solution of methyl (1R,3S,4S,6S,9R)-7,7-
difluoro-3,4-dimethoxy-9-(methoxymethyl)oxy-3,4-dimethyl-
2,5-dioxabicyclo[4.4.0]decane-9-carboxylate 18 (399 mg, 1.04
mmol) in 50% aqueous trifluoroacetic acid (5 cm3) was stirred
and held at 60 ЊC for 1 h. After cooling the solvent was removed
under reduced pressure and the residue partitioned between
water (10 cm3) and ethyl acetate (10 cm3). The aqueous layer
was lyophilized to afford a colourless foam that was redissolved
in water (5 cm3) and stirred with aqueous sodium hydroxide
solution (1 M, 1.2 cm3, 1.2 mmol) for 30 min. Amberlite
IR-120(H) (1.0 g) was added and the mixture stirred for a
further 15 min. The resin was removed by filtration, washed
with water (5 cm3) and the combined filtrates lyophilized to
afford the product 7 (212 mg, 96%) as a colourless solid. mp
179–181 ЊC (decomp.); [α]D Ϫ20.6 (c 0.34 in H2O); (Found: C,
38.18; H, 4.86. C16H26F2O8ؒ½H2O requires C, 38.02; H, 5.01%);
νmax(Nujol)/cmϪ1 3600, 3360, 2600 and 1705; δH (250 MHz,
D2O) 3.94 (1H, dddd, J 12, 10, 4, 1.5, 5-H), 3.77 (1H, ddd,
J 20.5, 10, 5.5, 4-H), 2.51 (1H, ddd, J 33, 15, 5.5, 2β-H), 2.40
(1H, m, 2α-H), 2.20 (1H, dm, J 14, 6α-H) and 1.98 (1H, dd,
J 14, 12, 6β-H); δC (62.9 MHz, D2O) 176.3, 121.3 (dd, J 246,
243.5), 74.6 (d, J 20), 72.2 (d, J 8.5), 66.8 (d, J 8.5), 39.2 and
38.5 (dd, J 23.5, 22); δF (235.4 MHz, D2O) Ϫ98.6 (1F, dq, J 247,
5.5) and Ϫ110.7 (1F, dddd, J 247, 33, 20.5, 15); m/z (ϩFAB)
213.0562 (MϩHϩ), C7H11O5F2 requires 213.0575.
Acknowledgements
We thank the BBSRC and the Wellcome Trust for postdoctoral
support (to M.F. and A.W.R. respectively).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 9 2 – 1 5 9 6
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