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

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 Nicola D’Antona a,⇑, Raffaele Morrone a, Paolo Bovicelli b, Giovanni Gambera a, David Kubácˇ c, Ludmila Martínková c a

Istituto di Chimica Biomolecolare del CNR—UOS Catania, Via P. Gaifami 18, I-95126 Catania, Italy Istituto di Chimica Biomolecolare del CNR—UOS Sassari, Traversa La Crucca 3-Baldanica, I-07040 Sassari, Italy c Laboratory of Biotransformation, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Vídenˇská 1083, CZ-142 20 Prague 4, Czech Republic b

a r t i c l e

i n f o

Article history: Received 13 July 2010 Accepted 15 October 2010

a b s t r a c t 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,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 The development of molecules with enhanced biological activity 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, minimizing collateral effects due to different isomeric forms, and reducing the amount of drug administered. Over the last few decades, 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 processing3 and their therapeutic potential.4,5 (+)-Cyclophellitol 1, (1S,2R,3S,4R,5R,6R)-5-(hydroxymethyl)-7oxabicyclo[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 bglucosidases7,8 and also to inhibit human glucocerebrosidase.9 Moreover since glucosidase inhibitors have the interesting property of inhibiting HIV infection and metastasis formation, cyclophellitol represents a promising anti-HIV agent.8 Interestingly, the unnatural (+)-1,6-epi-isomer 2, which has an a-epoxide configuration, displays potent a-glucosidase inhibitory activity (Fig. 1).10 Since (+)-cyclophellitol and its stereoisomers appear as promising candidates for biological applications, they have been the focus of extensive synthetic efforts.11 In general a cyclitol preparation is ⇑ Corresponding author. Tel.: +39 095 7338342; fax: +39 095 7338310. E-mail address: [email protected] (N. D’Antona). 0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetasy.2010.10.010

CH2OH O

6 5 4 1 2 3

CH2OH

OH

OH O

OH

OH

OH

OH

(+)-1

(+)-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 functionality largely by the use of protecting groups. Only two preparations of (+)-cyclophellitol starting from achiral precursors have been reported in the literature,23,24 and these are based on typical asymmetric 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 enzymes for the preparation of enantiomerically pure bioactive molecules 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. 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-

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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 reaction,27 it requires strong acid concentrations and high temperatures. Under these harsh operative conditions, a sample of (+)-4 underwent fast and complete degradation, thus being unsuitable for the classic chemical approach. 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. erythropolis A4 whole cells, to ‘integrate’ the synthetic pathway for conversion 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 cosolvent, were incubated in the presence of bacterial whole-cell preparations 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 obtained with 78% ee.25 Esterification followed by reduction of compound (+)-3 gave access to the key alcohol synthon ()-13 (Scheme 5). Direct epoxidation of substrate ()-13 with mCPBA was largely stereocontrolled by the free allylic hydroxyl group to furnish enantiopure epicyclophellitol (+)-2 as the main product. Conversely in order to reverse the facial selectivity of the epoxidation, a protection–deprotection sequence of secondary hydroxyl groups, as proposed by Trost,24 was developed (Scheme 6): protection of triol (+)-12 as the benzyl ether, as in (+)-14, followed by reduction of the ester moiety afforded alcohol (+)-15. Finally, epoxidation with mCPBA, stereochemically directed by the free homoallylic 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 altogether, 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.

2. Results and discussion 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 easily available p-benzoquinone 6 in five synthetic steps (see Scheme 2).25 The resolution of acid 3 could be achieved, in principle, 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 unsuitable. To overcome this problem, we decided to obtain enantiomerically pure 3 via a different enzyme-catalyzed biotransformation, by exploiting the enantioselective action of a lipase in the esterification 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. Conversely, when lipase from Candida antarctica B or Rhizomucor 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 unreacted compound ()-4 with an enantiomeric excess >98%. The 1H NMR spectroscopic analysis of compound 10 gave evidence 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 rotation and by gas chromatographic analysis, the stereochemical configuration 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 accordance with the data in the literature26 which reported a similar behavior of these enzymes toward different derivatives of conduritol B and C (note that compound 4 can be considered as a direct derivative of conduritol B). With the enantiopure nitriles ()- and (+)-4 in hand, we attempted to convert them into the corresponding desired enantiopure chiral acids ()- and (+)-3. Unfortunately, although CH2OH

CH2OH

OH O

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 biocatalytic synthetic strategy for the preparation of cyclophellitols, and the first general example of the synergic use of lipase, nitrile COOH OH

OH O

OH

CH2OH

OH

OH

(+)-2

(+)-3

CH2OH

OH O

OH

OH

OH (+)-1

OH OH

COOH

OH

OH

OH (±)-4

O OH OH (-)-1

CN

OH OH (-)-2

OH OH (-)-3

Only one isomer is drawn for racemic compounds. Scheme 1. Retrosynthetic strategy for the preparation of enantiomeric series of 1 and 2.

O

O (±)-5

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O

O

OH Br

Br2, CHCl3 0°C, 3 h

0°C, 3 h

Br

O

O

6

(±)-7 (yield 87%)

Br

NaBH4, Et2O/H2O

Br OH (±)-8 (yield 86%)

KOH, THF 0°C, 4 h

CN

CN OH

CF3COOH THF/H2O

OH

rt, 2 h

OSiMe3

O

CaO, Me3SiCN CH2Cl2/Hexane rt, 24 h

O

O

OH

(±)-9

(±)-4 (yield 25%)

(±)-5 (yield 96%)

Only one isomer is drawn for each pair of enantiomers. Scheme 2. Synthetic pathway for the preparation of (±)-4.

CN OH OH

CN

Rhizomucor miehei or Candida antarctica B Lipase

CN OH

OH +

THF, Vinyl acetate

OH

OH

OAc

(±)-4

(+)-10 (ee 98%, yield 45-48%)

OH OH (-)-4 (ee 80-90 %)

Only one isomer is drawn for racemic compounds Scheme 3. Biocatalyzed resolution of compound (±)-4.

CN

CONH2

COOH

OH

OH OH

OH

OH

OH

OH (+)-4

OH

(+)-11

(+)-3

R.erythropolis A4

R.erythropolis A4

(Nitrile hydratase) CN

(Amidase) COOH

CONH2 OH

OH

OH

OH

OH OH

OH

OH (-)-11

(-)-4

OH OH (-)-3

Scheme 4. Biotransformation pathway for the preparation of (+)- and ()-3.

hydratase, and amidase. This coupling of different enzymatic species shows the great potential of biocatalytical tools in organic synthesis, in terms of efficiency, selectivity, and ‘green’ features. The application of this strategy and the extension of biotransformations to the synthesis of various chiral cyclitols that could find use for the preparation of other enantiopure bioactive compounds, are currently being investigated in our laboratories.

4. Experimental 4.1. General 1

H and 13C NMR spectra were recorded in the indicated deuterated solvents on a Bruker Avance™ 400 spectrometer at 400.13 and 100.62 MHz, respectively. Chemical shifts (d) are given as parts

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COOH OH OH

COOCH3

CH2OH

OH

MeOH/H+ Reflux, 8 h

NaBH4 THF/MeOH, 30 m

OH

CH2OH

OH

OH

mCPBA

O

CH2Cl2, 0°C

OH

OH

OH

OH

OH

OH

(+)-3

(+)-12

(-)-13

(+)-2

Scheme 5. Synthetic pathway for the preparation of (+)-2.

OH OH

CH2OH

COOCH3

COOCH3

OBn

BnBr, NaH DMF, 0°C

OBn

OBn

NaBH4 THF/MeOH, 30 m

OBn

OH

OBn

OBn

(+)-12

(+)-14

(+)-15

mCPBA CH2Cl2, 0°C

CH2OH OH O OH

CH2OH H2, 5% Pd/C MeOH, rt

OBn O OBn

OH

OBn

(+)-1

(+)-16

Scheme 6. Synthetic pathway for the preparation of (+)-1.

per million relative to the residual solvent peak and coupling constants (J) are in Hertz. Column chromatography was performed on Silica Gel 60 (70–230 mesh) using the specified eluants. GC analyses 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 Perkin–ElmerÒ 8500 equipped with a FID detector and a MEGAÒ dimethyl-tert-butylsilyl-b-cyclodextrin (coated on OV 1701) column. Optical densities (OD) were measured on a DAD-UV–Visible 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 ratio (E) was calculated from the experimental values of conversion and enantiomeric excess using the equation reported in the literature.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 Sigma–AldrichÒ. P. cepacia was obtained from Amano.

4.2. General protocol for lipase catalyzed esterification of compound (±)-4 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 tetrahydrofuran (3 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 1:1) and gas

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-dihydroxycyclohex2-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 reaction was quenched, the catalyst filtered off, and the filtrate evaporated 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). ½a25 D = +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. 4.4. Hydrolysis of (1S,4R,5R,6S)-4-cyano-5,6-dihydroxycyclohex2-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-

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ness in vacuum, affording (+)-4 {27.12 mg, 0.175 mmol, 95% yield, [a]D = +135.6 (c 0.26, MeOH, ee = 98%)}.25 4.5. Microorganisms and cultures 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 glycerol and 3 g/L of yeast extract. 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 assays.29,31 The nitrile hydratase and amidase activities in whole cells of R. erythropolis A4 were approx. 1.4 and 0.3 U mg1 of dry cell weight, respectively. 4.7. Biotransformation of (+)-4 or ()-4 catalyzed by whole cells from R. erythropolis A4 Compound (+)-4 or ()-4 (112 mg, 0.72 mmol) and undecane (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 ml1) 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 aliquots at regular time intervals that, after centrifugation, lyophilization, and derivatization (see below), were analyzed by gas chromatography. Derivatization protocol: a 200 ll sample was added to 100 ll of a silylating mixture (hexamethyldisilazane–trimethylchlorosilane– pyridine, 3:1:9) and stirred at 40 °C for 30 min. Fast-GC conditions: injector temperature 250 °C; detector temperature 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. 4.7.1. (+)-(1S,4S,5R,6R)-4,5,6-Trihydroxycyclohex-2-enecarboxylic acid, (+)-3 MS (ESI-): m/z 173 [MH]; 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 (1C, C-4), 71.7 (1C, C-5), 52.9 (1C, C-1). ½a25 D = +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-enecarboxylic acid, ()-3 MS (ESI-): m/z 173 [MH]; 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

(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 (1C, C-4), 71.7 (1C, C-5), 52.9 (1C, C-1). ½a25 D = 168.4 (c 0.230, MeOH). Anal. Calcd for C7H10O5: C, 48.29; H, 5.79. Found: C, 48.48; H, 5.74. 4.8. Synthesis of (+)-(1S,4S,5R,6R)-methyl 4,5,6-trihydroxycyclohex-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 repeatedly partitioned with AcOEt; the final organic phase was dried over anhydrous Na2SO4 and taken to dryness to yield (+)-(1S,4S,5R,6R)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). ½a25 D = +110.3 (c 0.3, MeOH). Anal. Calcd for C8H12O5: C, 51.06; H, 6.43. Found: C, 50.87; H, 6.50. 4.9. Synthesis of ()-(1R,2R,3S,6R)-6-(hydroxymethyl)cyclohex4-ene-1,2,3-triol ()-13 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-enecarboxylate (+)-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 addition of water, the reaction mixture was extracted with AcOEt; the final organic phase was dried over anhydrous Na2SO4 and taken 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 21 ½a25 Anal. D = 12.9 (c 0.3, MeOH), lit. [a]D = 13.4 (c 1.1, MeOH). Calcd for C7H12O4: C, 52.49; H, 7.55. Found: C, 52.62; H, 7.61. 4.10. Synthesis of epi-cyclophellitol (+)-2 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 literature data.10 MS (ESI+): m/z 199 [M+Na]+; ½a25 D = +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. 4.11. Synthesis of (1S,4S,5R,6R)-methyl 4,5,6-tris(benzyloxy)cyclohex-2-enecarboxylate, (+)-14 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 diluted with EtOAc and successively washed with 1 M HCl, aq NaHCO3, 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-

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3), 5.10–4.8 (4H, m), 4.65 (1H, dd, J = 10.0, 7.8 Hz, H-6), 4.66 (1H, d, 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. ½a25 D = +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)cyclohex2-enyl)methanol, (+)-15 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 organic 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+): m/z 453 [M+Na]+; ½a25 D = +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). 13 C 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. 4.13. Synthesis of ((1R,2R,3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-7oxabicyclo[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  10 ml) and brine (10 ml). The aqueous 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 13 C NMR data (not reported) are in accordance with the literature data.24 ½a25 D = +71.5 (c 1.3, CHCl3), lit. [a]D = +71.0 (c 0.9, CHCl3).24 Anal. Calcd for C28H30O5: C, 75.31; H, 6.77. Found: C, 75.02; H, 6.55. 4.14. Synthesis of cyclophellitol, (+)-1 Epoxide (+)-16 (97.70 mg, 0.219 mmol) was dissolved in methanol (5 ml) and palladium hydroxide on carbon (Pearlman’s catalyst, 40 mg) was added. The flask was evacuated, purged with H2 three times and then the mixture was stirred under an atmosphere of H2. After 12 h, the hydrogen was removed, and the solution was filtered through a plug of Celite and rinsed with methanol (12 ml). The filtrate was concentrated in vacuum to give a colorless oil. Chromatography on silica gel (10:1–3:1 CH2Cl2/MeOH gradient) provided (+)-cyclophellitol, 1 (34.8 mg, 0.198 mmol, yield 91%) as a white crystalline solid. MS (ESI+): m/z 199 [M+Na]+; 1H NMR and 13C NMR data (not reported) are in accordance with the literature data.24 Mp 146–147 °C; ½a25 D = +102 (c 0.7, H2O), lit. [a]D = +103 (c 0.5, H2O);24 Anal. Calcd for C7H12O5: C, 47.72; H, 6.87. Found: C, 47.58; H, 6.80.

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4.15. Synthesis of epi-cyclophellitol ()-2 Compound ()-3 was subjected to the procedures described in Sections 4.13–4.15, affording epi-cyclophellitol ()-2 with comparable 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 synthesized and described. MS (ESI+): m/z 199 [M+Na]+; 1H NMR and 13C NMR data (not reported) are in accordance with the literature data.10 ½a25 D = 77.4 (c 0.3, H2O). Anal. Calcd for C7H12O5: C, 47.73; H, 6.87. Found: C, 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. MS (ESI+): m/z 199 [M+Na]+; 1H NMR and 13C NMR data (not reported) are in accordance with the literature data.24 ½a25 D = 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 (Institute of Microbiology), and COST/ESF action CM0701 is gratefully acknowledged. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

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