A novel chemo-multienzymatic synthesis of bioactive cyclophellitol and epi-cyclophellitol in both enantiopure forms

Article abstract of DOI:10.1016/j.tetasy.2010.10.010

A new route to synthesize cyclophellitol and epi-cyclophellitol from racemic starting materials in enantiopure forms has been developed. The synthesis involves a multi-enzymatic biotransformation pathway of the novel cyano-cyclitol (1R,4S,5R,6R)/(1S,4R,5S

Full text of DOI:10.1016/j.tetasy.2010.10.010

Tetrahedron: Asymmetry 21 (2010) 2448–2454
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
A novel chemo-multienzymatic synthesis of bioactive cyclophellitol
and epi-cyclophellitol in both enantiopure forms
ˇ c
Nicola D’Antona a, , Raffaele Morrone , Paolo Bovicelli , Giovanni Gambera , David Kubác ,
⇑
a
b
a
Ludmila Martínková c
a Istituto di Chimica Biomolecolare del CNR—UOS Catania, Via P. Gaifami 18, I-95126 Catania, Italy
b Istituto di Chimica Biomolecolare del CNR—UOS Sassari, Traversa La Crucca 3-Baldanica, I-07040 Sassari, Italy
c
ˇ
Laboratory of Biotransformation, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-142 20 Prague 4, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2010
Accepted 15 October 2010
A new route to synthesize cyclophellitol and epi-cyclophellitol from racemic starting materials in enan-
tiopure forms has been developed. The synthesis involves a multi-enzymatic biotransformation pathway
of the novel cyano-cyclitol (1R,4S,5R,6R)/(1S,4R,5S,6S)-4,5,6-trihydroxycyclohex-2-enecarbonitrile by a
cooperative use of lipase, nitrile hydratase, and amidase.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
CH2OH
CH2OH
OH
OH
5
2
The development of molecules with enhanced biological activ-
ity is a continuing primary goal in biomedical research. The need
for compounds interacting with biological receptors characterized
by a high grade of asymmetry has led researchers to synthesize
molecules possessing well defined stereochemical patterns, with
the aim of obtaining better substrate–receptor interactions, mini-
mizing collateral effects due to different isomeric forms, and
reducing the amount of drug administered. Over the last few dec-
ades, there has been considerable interest in the synthesis and
testing of glycosidases inhibitors; this interest is not only based
upon the inherent usefulness of these compounds as probes of
mechanism and as active site structures of glycosidases,1,2 but also
upon their utility in probing mechanisms of glycoprotein process-
ing3 and their therapeutic potential.4,5
(+)-Cyclophellitol 1, (1S,2R,3S,4R,5R,6R)-5-(hydroxymethyl)-7-
oxabicyclo[4.1.0]heptane-2,3,4-triol, is a cyclitol6 isolated from
the culture filtrate of the mushroom Phellinus sp.7 It has been found
to be a highly specific and an effective irreversible inactivator of b-
glucosidases7,8 and also to inhibit human glucocerebrosidase.9
Moreover since glucosidase inhibitors have the interesting prop-
erty of inhibiting HIV infection and metastasis formation, cyclop-
hellitol represents a promising anti-HIV agent.8 Interestingly, the
6
1
4
3
O
O
OH
OH
OH
(+)-1
OH
(+)-2
Figure 1. Structure of (+)-cyclophellitol 1 and (+)-epi-cyclophellitol 2.
challenging due to the dense stereochemistry of the hydroxylated
carbon cycle; this is even more so for cyclophellitols that possess a
carbon substituent in place of a hydroxyl group, since they require
the creation of a carbon–carbon bond with stereocontrol. To date
most syntheses of cyclophellitol start with enantiomerically pure
natural products, most notably carbohydrates,12–22 and require
long synthetic sequences due to the need to manipulate function-
ality largely by the use of protecting groups. Only two preparations
of (+)-cyclophellitol starting from achiral precursors have been re-
ported in the literature,23,24 and these are based on typical asym-
metric synthesis methods such as ‘asymmetric allylic alkylations’.
A possible alternative route for the synthesis of enantiopure
cyclophellitols, not yet explored, is biocatalysis. The use of en-
zymes for the preparation of enantiomerically pure bioactive mol-
ecules is nowadays a well established method among organic
chemists due to the number of advantages it offers (‘soft’ and green
working conditions, high chemo- regio- and stereoselectivity, and
unique chemical reactivities) when compared to classic organic
synthesis methods.
unnatural (+)-1,6-epi-isomer 2, which has an
tion, displays potent
Since (+)-cyclophellitol and its stereoisomers appear as promis-
ing candidates for biological applications, they have been the focus
of extensive synthetic efforts.11 In general a cyclitol preparation is
a-epoxide configura-
a
-glucosidase inhibitory activity (Fig. 1).10
Recently we have reported the preparation of a number of new
nitrile conduritols and we have found that the nitrile hydratase/
amidase bienzymatic system from Rhodococcus erythropolis A4
recognizes these cyclitols and is able to catalyze their biotransfor-
⇑
Corresponding author. Tel.: +39 095 7338342; fax: +39 095 7338310.
E-mail address: nicola.dantona@icb.cnr.it (N. D’Antona).
0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.10.010

N. D’Antona et al. / Tetrahedron: Asymmetry 21 (2010) 2448–2454
2449
mations.25 Starting from these considerations, we herein report a
novel chemoenzymatic synthesis of both enantiomeric series of
cyclophellitol and epi-cyclophellitol in optically active form.
conventional chemical hydrolysis of nitriles is a well known reac-
tion,27 it requires strong acid concentrations and high tempera-
tures. Under these harsh operative conditions, a sample of (+)-4
underwent fast and complete degradation, thus being unsuitable
for the classic chemical approach.
2. Results and discussion
Considering the enzymatic trend observed in our previous
work,25 we decided to exploit the hydrolytic catalysis of the nitrile
hydratase/amidase bienzymatic system expressed by R. erythropo-
lis A4 whole cells, to ‘integrate’ the synthetic pathway for conver-
sion of the enantiopure (ꢀ)- and (+)-4 into the corresponding (ꢀ)-
and (+)-3. With this in mind, single enantiomers of 4 dissolved in
Tris–HCl buffer (pH 8) containing up to 5% of methanol as cosol-
vent, were incubated in the presence of bacterial whole-cell prep-
arations of R. erythropolis A4 (Scheme 4). As expected, the
hydrolysis of the two nitriles enantiomers occurred with different
reaction rates; (+)-4 was completely converted in 10 h while (ꢀ)-4
required 24 h. These results are in agreement with that previously
observed during the resolution of ( )-4, in which acid (+)-3 was ob-
tained with 78% ee.25
The great chemical versatility of the cyano group makes the
conduritol derivative ( )-4 a suitable precursor in the preparation
of cyclophellitol framework, as shown by the retrosynthetic
Scheme 1.
We prepared nitrile conduritol ( )-4 from inexpensive and eas-
ily available p-benzoquinone
6 in five synthetic steps (see
Scheme 2).25 The resolution of acid 3 could be achieved, in princi-
ple, by enzymatic nitrile hydrolysis of ( )-4 but, as evidenced in a
previous investigation,25 the reaction occurs with unsatisfactory
enantioselectivity, thus indicating that this approach was unsuit-
able. To overcome this problem, we decided to obtain enantiome-
rically pure 3 via a different enzyme-catalyzed biotransformation,
by exploiting the enantioselective action of a lipase in the esterifi-
cation reaction in an organic solvent. With this in mind, lipases
from Candida rugosa or Pseudomonas cepacia were assayed in the
direct esterification of ( )-4 but no product was obtained after
12 h.
Esterification followed by reduction of compound (+)-3 gave ac-
cess to the key alcohol synthon (ꢀ)-13 (Scheme 5). Direct epoxida-
tion of substrate (ꢀ)-13 with mCPBA was largely stereocontrolled
by the free allylic hydroxyl group to furnish enantiopure epi-
cyclophellitol (+)-2 as the main product.
Conversely, when lipase from Candida antarctica B or Rhizomu-
cor miehei were used as biocatalysts (Scheme 3), a main monoester
product 10 with yields in the range of 45–48% and ee >98% (E >100)
was recovered. Moreover, by prolonging the reaction time and
achieving a conversion value >50%, it was possible to recover unre-
acted compound (ꢀ)-4 with an enantiomeric excess >98%.
The 1H NMR spectroscopic analysis of compound 10 gave evi-
dence that only the less hindered secondary hydroxy group at
the 4-position was subjected to esterification, while both groups
at the 5- and 6-positions remained unaltered. Chemical hydrolysis
of compound 10 afforded the optically active compound (+)-4,
which allowed us to establish, both by the sign of the specific rota-
tion and by gas chromatographic analysis, the stereochemical con-
figuration of this enantiopure product as (1R,4S,5R,6R). The
observed regio- and stereopreference shown by C. antarctica B
and R. miehei lipases exclusively toward the secondary hydroxyl
groups with an (S)-configuration of the stereocenter, is in accor-
dance with the data in the literature26 which reported a similar
behavior of these enzymes toward different derivatives of condur-
itol B and C (note that compound 4 can be considered as a direct
derivative of conduritol B).
Conversely in order to reverse the facial selectivity of the epox-
idation, a protection–deprotection sequence of secondary hydroxyl
groups, as proposed by Trost,24 was developed (Scheme 6): protec-
tion of triol (+)-12 as the benzyl ether, as in (+)-14, followed by
reduction of the ester moiety afforded alcohol (+)-15. Finally, epox-
idation with mCPBA, stereochemically directed by the free homoal-
lylic alcohol, and hydrogenolysis of the benzyl groups gave access
to the enantiopure cyclophellitol (+)-1.
Considering that the enantiomers of biologically active products
very often exhibit improved potencies or even novel activities alto-
gether, we applied the same synthetic strategies illustrated in
Scheme 5 and Scheme 6 to compound (ꢀ)-3, to gain access to
the corresponding pure enantioforms (ꢀ)-cyclophellitol (ꢀ)-1
and (ꢀ)-epi-cyclophellitol (ꢀ)-2.
3. Conclusion
A novel chemo-multienzymatic route toward the synthesis of
(+)-cyclophellitol (+)-1 and (+)-epi-cyclophellitol (+)-2, and their
enantiomers (ꢀ)-cyclophellitol (ꢀ)-1 and (ꢀ)-epi-cyclophellitol
(ꢀ)-2 has been achieved. This represents the first example of a bio-
catalytic synthetic strategy for the preparation of cyclophellitols,
and the first general example of the synergic use of lipase, nitrile
With the enantiopure nitriles (ꢀ)- and (+)-4 in hand, we at-
tempted to convert them into the corresponding desired enantio-
pure chiral acids (ꢀ)- and (+)-3. Unfortunately, although
CH2OH
OH
CH2OH
OH
COOH
OH
O
O
O
O
OH
OH
OH
CN
O
O
OH
(+)-1
OH
(+)-2
OH
(+)-3
OH
OH
CH2OH
OH
CH2OH
OH
COOH
OH
OH
( )-4
( )-5
OH
OH
OH
OH
OH
OH
(-)-1
(-)-2
(-)-3
Only one isomer is drawn for racemic compounds.
Scheme 1. Retrosynthetic strategy for the preparation of enantiomeric series of 1 and 2.

2450
N. D’Antona et al. / Tetrahedron: Asymmetry 21 (2010) 2448–2454
O
O
OH
Br
Br
Br
Br
Br2, CHCl3
0°C, 3 h
NaBH4, Et2O/H2O
0°C, 3 h
O
O
OH
6
( )-7
(yield 87%)
( )-8
(yield 86%)
KOH, THF
0°C, 4 h
CN
CN
O
O
CaO, Me3SiCN
CH2Cl2/Hexane
CF3COOH
THF/H2O
OH
OH
OSiMe3
rt, 2 h
rt, 24 h
O
OH
( )-4
(yield 25%)
( )-9
( )-5
(yield 96%)
Only one isomer is drawn for each pair of enantiomers.
Scheme 2. Synthetic pathway for the preparation of ( )-4.
CN
CN
CN
Rhizomucor miehei or
OH
OH
OH
OH
OH
OH
Candida antarctica B Lipase
+
THF, Vinyl acetate
OH
OAc
OH
(+)-10
( )-4
(-)-4
(ee 98%, yield 45-48%)
(ee 80-90 %)
Only one isomer is drawn for racemic compounds
Scheme 3. Biocatalyzed resolution of compound ( )-4.
CN
CONH2
OH
COOH
OH
OH
OH
OH
OH
OH
OH
OH
(+)-3
(+)-4
(+)-11
R.erythropolis A4
R.erythropolis A4
(Nitrile hydratase)
(Amidase)
CN
OH
CONH2
OH
COOH
OH
OH
OH
OH
OH
OH
OH
(-)-3
(-)-11
(-)-4
Scheme 4. Biotransformation pathway for the preparation of (+)- and (ꢀ)-3.
hydratase, and amidase. This coupling of different enzymatic spe-
cies shows the great potential of biocatalytical tools in organic syn-
thesis, in terms of efficiency, selectivity, and ‘green’ features.
The application of this strategy and the extension of biotrans-
formations to the synthesis of various chiral cyclitols that could
find use for the preparation of other enantiopure bioactive com-
pounds, are currently being investigated in our laboratories.
4. Experimental
4.1. General
1H and 13C NMR spectra were recorded in the indicated deuter-
ated solvents on a Bruker Avance™ 400 spectrometer at 400.13
and 100.62 MHz, respectively. Chemical shifts (d) are given as parts

N. D’Antona et al. / Tetrahedron: Asymmetry 21 (2010) 2448–2454
2451
COOH
OH
COOCH3
OH
CH2OH
OH
CH2OH
OH
MeOH/H+
Reflux, 8 h
mCPBA
NaBH4
O
CH2Cl2, 0°C
THF/MeOH, 30 m
OH
OH
OH
OH
OH
OH
OH
OH
(+)-12
(+)-3
(-)-13
(+)-2
Scheme 5. Synthetic pathway for the preparation of (+)-2.
COOCH3
OH
COOCH3
CH2OH
OBn
OBn
NaBH4
BnBr, NaH
DMF, 0°C
THF/MeOH, 30 m
OH
OH
OBn
OBn
OBn
OBn
(+)-15
(+)-12
(+)-14
mCPBA
CH2Cl2, 0°C
CH2OH
OH
CH2OH
OBn
H2, 5% Pd/C
MeOH, rt
O
O
OH
OH
OBn
OBn
(+)-1
(+)-16
Scheme 6. Synthetic pathway for the preparation of (+)-1.
per million relative to the residual solvent peak and coupling con-
stants (J) are in Hertz. Column chromatography was performed on
Silica Gel 60 (70–230 mesh) using the specified eluants. GC analy-
ses were carried out in a ShimadzuÒ Fast-GC-17A equipped with a
FID detector and a SUPELCOÒ Fast-SPB™-5, using n-undecane as
the internal standard. Chiral-GC analyses were carried out in a Per-
kin–ElmerÒ 8500 equipped with a FID detector and a MEGAÒ di-
methyl-tert-butylsilyl-b-cyclodextrin (coated on OV 1701)
column. Optical densities (OD) were measured on a DAD-UV–Vis-
ible AgilentÒ spectrophotometer at 610 nm. Optical rotations were
measured on a DIP 135 JASCOÒ instrument at 25 °C. ESI-MS spectra
were acquired in positive mode in a WatersÒ Micromass ZQ2000,
using a 10 V cone voltage, 3 kV capillary voltage and 150 °C source
temperature. Melting points are uncorrected. The enantiomeric ra-
tio (E) was calculated from the experimental values of conversion
and enantiomeric excess using the equation reported in the litera-
ture.28 The chemicals, solvents, and materials for microbiological
cultures were obtained from Sigma–AldrichÒ, FlukaÒ, Carlo ErbaÒ,
Riedel-de HaenÒ, DifcoÒ and used without further purification. C.
Antarctica B, R. miehei and C. rugosa lipases were obtained from Sig-
ma–AldrichÒ. P. cepacia was obtained from Amano.
chromatography. After 24 h the reaction was quenched, filtering
off the catalyst, and the filtrate evaporated to dryness in vacuum.
4.3. Synthesis of (1S,4R,5R,6S)-4-cyano-5,6-dihydroxycyclohex-
2-enyl acetate, (+)-10
Lipase from R. miehei (50 mg) was added to a solution of ( )-4
(62 mg, 0.400 mmol) in tetrahydrofuran (5 ml) containing vinyl
acetate (3 equiv). The suspension was shaken (300 rpm) at 45 °C,
aliquots were drawn at regular time intervals and monitored by
TLC (MeOH/CH2Cl2) and gas chromatography. After 24 h the reac-
tion was quenched, the catalyst filtered off, and the filtrate evapo-
rated to dryness in vacuum. The residue was purified by flash
chromatography on silica gel (MeOH/CH2Cl2 gradient) affording
compound (+)-10 (36.32 mg, 0.184 mmol, 46% yield) as an oil.
MS (ESI+): m/z 220 [M+Na]+; 1H NMR (CD3OD): 5.85 (1H, ddd,
J = 10.0, 2.2, 2.7 Hz, H-3), 5.57 (1H, ddd, J = 10.0, 2.0, 2.1 Hz, H-2),
4.72 (1H, ddd, J = 9.7, 2.9, 2.2 Hz, H-4), 3.92 (1H, dd, J = 2.0,
7.9 Hz, H-1), 3.58 (1H, t, J = 9.7 Hz, H-5), 3.30 (1H, dd, J = 9.7,
8.0 Hz, H-6) 2.08 (3H, s, Ac). 13C NMR (CD3OD): 171.2 (1C, CO),
125.8 (1C, C-3), 122.5 (1C, C-2), 120.9 (1C, CN), 76.4 (1C, C-6),
71.4 (1C, C-5), 70.1 (1C, C-4), 36.7 (1C, C-1), 20.9 (1C, –CH3).
½
a 2D5
= +102 (c 0.3, H2O). Anal. Calcd for C9H11NO4: C, 54.82; H,
5.62; N, 7.10. Found: C, 54.61; H, 5.91; N, 7.00.
ꢁ
4.2. General protocol for lipase catalyzed esterification of
compound ( )-4
4.4. Hydrolysis of (1S,4R,5R,6S)-4-cyano-5,6-dihydroxycyclohex-
The lipase of choice (from C. rugosa, P. cepacia, R. miehei, or C.
Antarctica B, 20 mg) was added to a solution of ( )-4 (20 mg) in tet-
rahydrofuran (3 ml) containing vinyl acetate (3 equiv). The suspen-
sion was shaken (300 rpm) at 45 °C, aliquots were drawn at regular
time intervals and monitored by TLC (MeOH/CH2Cl2 1:1) and gas
2-enyl acetate, (+)-10
Compound (+)-10 (36.32 mg, 0.184 mmol) was dissolved in
MeOH (5 ml) and added with 30% v/v NH4OH (1 ml). The solution
was stirred at room temperature for 12 h, then evaporated to dry-

2452
N. D’Antona et al. / Tetrahedron: Asymmetry 21 (2010) 2448–2454
ness in vacuum, affording (+)-4 {27.12 mg, 0.175 mmol, 95% yield,
D = +135.6 (c 0.26, MeOH, ee = 98%)}.25
(1H, m, H-1), 3.84 (1H, t, J = 9.6 Hz, H-5), 3.47 (1H, dd, J = 10.2,
7.6 Hz, H-6), 3.05–2.90 (1H, m, H-4). 13C NMR (CD3OD): 178.6
(1C, –COOH), 128.3 (1C, C-3), 126.3 (1C, C-2), 76.9 (1C, C-6), 72.2
[a]
4.5. Microorganisms and cultures
(1C, C-4), 71.7 (1C, C-5), 52.9 (1C, C-1). ½a D25
= ꢀ168.4 (c 0.230,
ꢁ
MeOH). Anal. Calcd for C7H10O5: C, 48.29; H, 5.79. Found: C,
48.48; H, 5.74.
The bacterial strains were maintained at 4 °C on meat peptone
agar (in g/L, Bacto beef extract 3, peptone 10, NaCl 5, agar 15). R.
erythropolis A429 was grown for 2 days at 28 °C in shaken 500-ml
Erlenmeyer flasks containing 100 ml of basal salts broth according
to Di Geronimo and Antoine,30 supplemented with 10 g/L of glyc-
erol and 3 g/L of yeast extract.
4.8. Synthesis of (+)-(1S,4S,5R,6R)-methyl 4,5,6-trihydroxycyclo-
hex-2-enecarboxylate, (+)-12
At first, HCl (150 ll) was added to a solution of (+)-3 (13.5 mg,
0.075 mmol) in 3 ml of MeOH, and the mixture stirred at reflux for
8 h. After the addition of water, the reaction mixture was repeat-
edly partitioned with AcOEt; the final organic phase was dried over
anhydrous Na2SO4 and taken to dryness to yield (+)-(1S,4S,5R,6R)-
4.6. Preparation of whole-cell catalysts
Whole cells of bacteria were harvested by centrifugation and
washed with Tris–HCl buffer (50 mM, pH 8). The activities of nitrile
hydratase and amidase were determined with benzonitrile and
benzamide, respectively, using the previously described as-
says.29,31 The nitrile hydratase and amidase activities in whole cells
of R. erythropolis A4 were approx. 1.4 and 0.3 U mgꢀ1 of dry cell
weight, respectively.
methyl
4,5,6-trihydroxycyclohex-2-enecarboxylate,
(+)-12
(11.1 mg, 0.057 mmol, 76% yield) as an oil. MS (ESI+): m/z 211
[M+Na]+; 1H NMR (CD3OD): 5.75–5.70 (1H, m, H-2), 5.60–5.50
(1H, m, H-3), 4.03–3.97 (1H, m, H-1), 3.75–3.70 (1H, m, H-5),
3.48–3.42 (1H, m, H-6), 2.99–2.94 (1H, m, H-4). 13C NMR (CD3OD):
173.0 (1C, –CO), 127.3 (1C, C-2), 125.3 (1C, C-3), 76.9 (1C, C-6), 72.7
(1C, C-5), 70.2 (1C, C-4), 53.9 (1C, C-1), 52.2 (1C, –OCH3).
4.7. Biotransformation of (+)-4 or (ꢀ)-4 catalyzed by whole cells
from R. erythropolis A4
½
a 2D5
= +110.3 (c 0.3, MeOH). Anal. Calcd for C8H12O5: C, 51.06; H,
6.43. Found: C, 50.87; H, 6.50.
ꢁ
Compound (+)-4 or (ꢀ)-4 (112 mg, 0.72 mmol) and undecane
4.9. Synthesis of (ꢀ)-(1R,2R,3S,6R)-6-(hydroxymethyl)cyclohex-
4-ene-1,2,3-triol (ꢀ)-13
(112 ll, internal standard for gas chromatography analysis) were
dissolved in 2 ml of MeOH, and added to a suspension containing
whole cells from R. erythropolis A4 (OD = 16; approx. 3.7 mg dry
cell weight mlꢀ1) in Tris–HCl buffer (50 mM). The mixture was
shaken (200 rpm) at 35 °C for a period ranging between 12 and
24 h; the reaction was stopped by centrifugation of the solution,
and evaporation under vacuum of the supernatant at 40 °C. The
mixture was purified by silica gel column chromatography using
MeOH/CH2Cl2 1:1 as the eluants, and yielded compound (+)-3
(101.8 mg, 0.576 mmol, 82% yield), or compound (ꢀ)-3 (89.1 mg,
0.504 mmol, 72% yield), respectively.
The course of the reaction, including the concentration and
enantiomeric excesses values, was monitored by withdrawing ali-
quots at regular time intervals that, after centrifugation, lyophiliza-
tion, and derivatization (see below), were analyzed by gas
chromatography.
At first, NaBH4 (12.9 mg, 0.342 mmol) was added to a solution
of (+)-(1S,4S,5R,6R)-methyl 4,5,6-trihydroxycyclohex-2-enecarb-
oxylate (+)-12 (11.1 mg, 0.057 mmol) in 4 ml of distilled THF/
MeOH (1:3), and the mixture agitated for 30 min. After the addi-
tion of water, the reaction mixture was extracted with AcOEt;
the final organic phase was dried over anhydrous Na2SO4 and ta-
ken to dryness to yield compound (ꢀ)-13 (9.0 mg, 0.054 mmol,
96% yield) as an oil. MS (ESI+): m/z 183 [M+Na]+; 1H NMR data
(not reported) are in accordance with the literature data.21
½
a 2D5
ꢁ
= ꢀ12.9 (c 0.3, MeOH), lit. [
a
]
D = ꢀ13.4 (c 1.1, MeOH).21 Anal.
Calcd for C7H12O4: C, 52.49; H, 7.55. Found: C, 52.62; H, 7.61.
4.10. Synthesis of epi-cyclophellitol (+)-2
Derivatization protocol: a 200
silylating mixture (hexamethyldisilazane–trimethylchlorosilane–
pyridine, 3:1:9) and stirred at 40 °C for 30 min.
Fast-GC conditions: injector temperature 250 °C; detector tem-
perature 280 °C; oven 100 °C for 1 min, then 10 °C/min up to
280 °C for 1 min. Chiral-GC conditions: injector temperature
250 °C; detector temperature 250 °C; oven 100 °C for 1 min, then
0.4 °C/min up to 200 °C for 1 min.
l
l sample was added to 100
l
l of a
To a solution of triol (ꢀ)-13 (9.0 mg, 0.054 mmol) in CH2Cl2
(2 ml) was added mCPBA (15.3 mg, 0.081 mmol). The mixture
was heated at reflux for 24 h and then concentrated and purified
by silica gel column chromatography using MeOH/CH2Cl2 1:3 as
the eluants, to give compound (+)-2 (7.6 mg, 0.043 mmol, 80%
yield) as a clear oil. 1H NMR spectra are in accordance with the lit-
erature data.10 MS (ESI+): m/z 199 [M+Na]+; ½a 2D5
ꢁ
= +78.2 (c 0.4,
H2O), lit. [a]
D = +80.0 (c 0.36, H2O).10 Anal. Calcd for C7H12O5: C,
47.73; H, 6.87. Found: C, 47.44; H, 6.50.
4.7.1. (+)-(1S,4S,5R,6R)-4,5,6-Trihydroxycyclohex-2-enecarboxylic
acid, (+)-3
4.11. Synthesis of (1S,4S,5R,6R)-methyl 4,5,6-tris(benzyloxy)-
cyclohex-2-enecarboxylate, (+)-14
MS (ESI-): m/z 173 [MꢀH]ꢀ; 1H NMR (CD3OD): 5.76 (1H, d,
J = 10.1, 2.0 Hz, H-2), 5.55 (1H, d, J = 10.4, 2.3 Hz, H-3), 4.10–4.05
(1H, m, H-1), 3.87 (1H, t, J = 9.6 Hz, H-5), 3.47 (1H, dd, J = 10.1,
7.6 Hz, H-6), 3.00–2.90 (1H, m, H-4). 13C NMR (CD3OD): 178.6
(1C, –COOH), 128.3 (1C, C-3), 126.3 (1C, C-2), 76.9 (1C, C-6), 72.2
To a solution of compound (+)-12 (55 mg, 0.292 mmol) and NaH
(42.0 mg, 1.750 mmol) in DMF (5 ml) at 0 °C was added BnBr
(0.170 ml, 1.460 mmol). After 30 min, the mixture was warmed
to room temperature and stirred overnight. The mixture was di-
luted with EtOAc and successively washed with 1 M HCl, aq NaH-
CO3, and brine. The organic phase was dried (MgSO4),
concentrated, and purified by silica gel column chromatography
using petroleum ether/diethyl ether 2:1–1:1 as the eluants, to give
compound (+)-14 (131.06 mg, 0.286 mmol, yield 98%) as an oil. MS
(ESI+): m/z 481 [M+Na]+; 1H NMR (CD3OD): 7.60–7.20 (15H, m),
5.98 (1H, dt, J = 10.2, 2.3 Hz, H-2), 5.75 (1H, dt, J = 10.2, 2.4 Hz, H-
(1C, C-4), 71.7 (1C, C-5), 52.9 (1C, C-1). ½a D25
ꢁ
= +171.2 (c 0.265,
MeOH), lit. [a]
D = +128.2 (c 0.26 MeOH, ee 74.5%).25 Anal. Calcd
for C7H10O5: C, 48.29; H, 5.79. Found: C, 48.48; H, 5.74.
4.7.2. (ꢀ)-(1R,4R,5S,6S)-4,5,6-Trihydroxycyclohex-2-enecarb-
oxylic acid, (ꢀ)-3
MS (ESI-): m/z 173 [MꢀH]ꢀ; 1H NMR (CD3OD): 5.75 (1H, d,
J = 10.0, 2.0 Hz, H-2), 5.55 (1H, d, J = 10.2, 2.2 Hz, H-3), 4.09–4.06

N. D’Antona et al. / Tetrahedron: Asymmetry 21 (2010) 2448–2454
2453
3), 5.10–4.8 (4H, m), 4.65 (1H, dd, J = 10.0, 7.8 Hz, H-6), 4.66 (1H, d,
4.15. Synthesis of epi-cyclophellitol (ꢀ)-2
J = 11.2 Hz), 4.27–4.24 (1H, m), 4.13–3.90 (1H, m, H-1), 3.91 (3H, s),
3.64–3.56 (1H, m), 2.51–2.49 (1H, m). 13C NMR (CD3OD): 172.3,
138.7, 137,7, 136.6, 128.5, 128.3, 128.0, 127,9, 127,8, 127.5,
127.4, 127.3, 126.7, 126.6, 80.8, 76.8, 73.9, 72.7, 72.6, 51.7, 48.2.
Compound (ꢀ)-3 was subjected to the procedures described in
Sections 4.13–4.15, affording epi-cyclophellitol (ꢀ)-2 with compa-
rable yields. Intermediate products (ꢀ)-12 and (+)-13 showed MS
(ESI+), specific rotations, 1H NMR and 13C NMR data (not reported)
in accordance with data of their enantiomers previously synthe-
sized and described.
½
a 2D5
= +98.6 (c 1.4, MeOH). Anal. Calcd for C29H30O5: C, 75.96; H,
6.59. Found: C, 76.15; H, 6.50.
ꢁ
4.12. Synthesis of ((1R,4S,5R,6R)-4,5,6-tris(benzyloxy)cyclohex-
2-enyl)methanol, (+)-15
MS (ESI+): m/z 199 [M+Na]+; 1H NMR and 13C NMR data (not re-
ported) are in accordance with the literature data.10
½
a 2D5
ꢁ
= ꢀ77.4 (c
0.3, H2O). Anal. Calcd for C7H12O5: C, 47.73; H, 6.87. Found: C,
At first, NaBH4 (10.60 mg, 0.280 mmol) was added to a solution
of (+)-14 (131.06 mg, 0.286 mmol) in 4 ml of distilled THF/MeOH
(1:3), and the mixture agitated for 30 min. After the addition of
water, the reaction mixture was extracted with AcOEt; the final or-
ganic phase was dried over anhydrous Na2SO4, taken to dryness,
and purified by silica gel column chromatography using petroleum
ether/diethyl ether 2:1–1:1 as the eluants, to yield compound (+)-
15 (118.06 mg, 0.274 mmol, 96% yield) as a colorless oil. MS (ESI+):
47.52; H, 6.52.
4.16. Synthesis of cyclophellitol (ꢀ)-1
Compound (ꢀ)-3 was subjected to the procedures described in
paragraphs 4.13, 4.16, 4.17, 4.18, and 4.19, affording cyclophellitol
(ꢀ)-1 with comparable yields. Intermediate products (ꢀ)-12, (ꢀ)-
14, (ꢀ)-15, and (ꢀ)-16 showed MS (ESI+), specific rotations, 1H
NMR and 13C NMR data (not reported) in accordance with data of
their enantiomers previously synthesized and described.
m/z 453 [M+Na]+; ½a D25
ꢁ
= +103.9 (c 1.2, CHCl3), lit. [a]D = +104.5 (c
1.92, CHCl3).24 1H NMR (CD3OD): 7.60–7.20 (15H, m), 5.76 (1H,
dt, J = 10.0, 2.4 Hz, H-2), 5.55 (1H, dt, J = 10.4, 2.4 Hz, H-3), 5.01
(1H, d, J = 11.2 Hz), 4.94 (2H, s), 4.70 (2H, s), 4.66 (1H, d,
J = 11.2 Hz), 4.27–4.24 (1H, m), 3.85 (1H, dd, J = 10.0, 7.8 Hz, H-6),
3.64–3.56 (3H, m), 2.51–2.49 (1H, m), 1.50–1.47 (1H, m, H-1).
13C NMR data (not reported) are in accordance with the literature
data.24 Anal. Calcd for C28H30O4: C, 78.11; H, 7.02. Found: C, 78.34;
H, 7.22.
MS (ESI+): m/z 199 [M+Na]+; 1H NMR and 13C NMR data (not re-
ported) are in accordance with the literature data.24
½
a 2D5
ꢁ
= ꢀ98.5 (c
0.7, H2O), lit. [
a
]
D = ꢀ100.7 (c 0.32, H2O);24 Anal. Calcd for C7H12O5:
C, 47.72; H, 6.87. Found: C, 47.54; H, 6.78.
Acknowledgments
Financial support via project OC09046 (Ministry of Education,
Czech Rep.), institutional research concept AV0Z50200510 (Insti-
tute of Microbiology), and COST/ESF action CM0701 is gratefully
acknowledged.
4.13. Synthesis of ((1R,2R,3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-7-
oxabicyclo[4.1.0]heptan-2-yl)methanol, (+)-16
mCPBA (93.27 mg, 0.548 mmol) was added to a cooled (0 °C)
solution of compound (+)-15 (118.06 mg, 0.274 mmol) in CH2Cl2
(3 ml). The mixture was warmed to room temperature. After
16 h, the mixture was diluted with ethyl acetate (20 ml) and
washed with NaOH 1 M (3 ꢂ 10 ml) and brine (10 ml). The aque-
ous layer was back extracted with ethyl acetate (10 ml). The
combined organic phases were dried on MgSO4, concentrated
in vacuum, and purified by silica gel column chromatography
using petroleum ether/diethyl ether 1:2 as the eluants, yielding
compound (+)-16 (97.70 mg, 0.219 mmol, yield 80%) as a white
solid and its diastereomeric epoxide (6.2 mg, 0.014 mmol, yield
5%). MS (ESI+): m/z 469 [M+Na]+; mp 98–99 °C; 1H NMR and
13C NMR data (not reported) are in accordance with the litera-
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