Synthesis of (2-azidoethyl) 2-O-(α-D-mannopyranosyl)-5a-carba-α-D- mannopyranoside

67

EXPERIMENTAL

General procedures.

All reactions were performed in flame-dried modified Schlenk (Kjeldahl shape) flasks fitted with a glass stopped or rubber septa under a positive pressure of Argon. Air and moisture-sensitive liquids and solutions were transferred via syringe. Organic solutions were dried on MgSO₄ and concentrate by a rotary evaporator below 40°C al ca. 25 Torr.

Flash chromatography was performed employing 230-400 mesh silica gel. Analytical TLC was performed on Alugram SIL G/UV254 silica gel sheets ( Macherey_Nagel) with detection by 0.5% phosphomolybdic acid solution in 95% EtOH.

Materials.

Tri-O-acetyl-D-glucal, lipase from Candida Rugosa type VII CRL, pyridinium p-toluen sulfonate (PPTS), PMBCl, Ph3PMeI, potassium hexamethyldisilazide (KHMDS), 1,3- dichlorobenzene, NaBH4, BnBr, NaH 60%, 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), MCPBA 70% , NaN3, Cu(OTf)2, TMSOTf, 10% Pd-C, MeONa, Ac2O were purchased from Aldrich and used without purification. 2-Iodoxybenzoic acid (IBX) was synthesized according to the literature methods. 3,4-dihydro-2H-pyran (DHP) and AW 300 molecular, anhydrous CH3CN over molecular sieves, anhydrous N,N- dimethylformamide (DMF) over molecular sieves, anhydrous pyridine over molecular sieves, anhydrous CH₂Cl₂ over molecular sieves were purchased from Aldrich and used without purification. Toluene, Et₂O, THF were distilled from sodium/benzophenone.

Instrumentation.

Infrared (IR) spectra were obtained using Cary 600 Series FTIR spectrometer. Data are presente as frequency absorption (cm^-1). Proton and carbon-13 nuclear magnetic resonance (¹H NMR and ¹³C NMR) spectra were recorded at 250 and 62.5 MHz, respectively using a Bruker Advance 250 MHz spectrometer; chemical shifts are expressed in parts per million (δ scale) and refer to residual protium in the NMR solvent [CDCl3: δ 7.26; CD3CN: δ 1.94, CD3OD: δ 3.31]. Data are presented as follows:

68 chemical shift, multiplicity ( s=singlet, d=doublet, t=triplet, m=multiplet and/or multiple resonances), integration, coupling costant in Hertz (Hz).

3,4-Di-O-acetyl-D-glucal (+)-2.2

A phospate buffer (NaH2PO4∙H2O/Na2HPO4, pH 7.0, 60 mL) was added to a solution of tri-O-acetyl-D-glucal (+)-2.1 (10.0 g, 36.73 mmol)^22-23 in (i-Pr)2O (15.0 mL), and anhydrous acetone (15.0 mL). The mixture was treated with lipase CRL (lipase from Candida Rugosa type VII activity >700 unit/mg) (1.7 g) and anhydrous acetone (5 mL) was added. The reaction mixture was stirred for 12 h at room temperature. Then, the reaction mixture was concentrated by rotary evaporator and the remaining solution was filtered on a pad of Celite^® and extracted with AcOEt. The evaporation of the washed (brine) and dried (MgSO4) organic extracts afforded a crude reaction product (7.75 g, 36.73 mmol, 91% yield), which was subjected to a flash chromatography. Elution with an 6:4 hexane/AcOEt mixture afforded a viscous liquid consisting of a pure alcohol (+)-2.2 (4.667 g, 20.29 mmol, 55% yield).

(+)-2.2: R_f = 0.26 (1:1 hexane/AcOEt). ¹H NMR (CDCl₃) δ 6.49 (dd, 1H, J= 6.1, 1.2 Hz), 5.41-5.50 (m, 1H), 5.22 ( dd, 1H, J=9.0, 6.5 Hz), 4.81 (dd, 1H, J=5.9, 2.9 Hz), 3.98-4.09 (m, 1H), 3.66-3.86 ( m, 2H), 2.13 (s,3H), 2.07 (s, 3H). ¹³C NMR (CDCl₃) δ 170.8, 170.6, 145.9, 99.1, 76.7, 68.4, 67.9, 60.0, 21.2, 20.9. Anal. Calcd for C₁₀H₁₄O₆: C, 52.17; H,6.13. Found: C, 52.23; H, 6.41.

6-O-(2-Tetrahydropyranyl)-3,4-di-O-acetyl-D-glucal (2.3)

A solution of PPTS (0.151 g, 0.602 mmol, 0.02 equiv) in anhydrous CH2Cl2 (150.0 mL) was added dropwise at room temperature to a stirred solution of alcohol (+)-2.2 (6.922 g, 30.0 mmol) in DHP (4.10 mL, 45.14 mmol, 1.5 equiv) and the

69 reaction mixture was stirred at the same temperature for 16 h. Dilution with CH2Cl2

and evaporation of the washed (saturated aqueous NaHCO3 and saturated aqueous NaCl) organic solution afforded a crude reaction product (9.27 g, 29.41 mmol, 98%

yield), a liquid consisting of 6-OTHP protected diacetate 2.3, as a mixture of diasteroisomers, which was use in the next step without any purification.

2.3: Rf =0.16 (8:2 hexane/AcOEt); FTIR (neat film) ν 1743, 1471, 1238, 1033 cm^-1. ¹H NMR (CDCl3) δ 6.48 (dt, 1H, J= 6.0, 1.3 Hz), 5.17-5.38 (m, 2H), 4.75-4.85 (m, 1H), 4.53-4.70 (m, 1H), 4.17-4.30 (m, 1H), 3.69-4.00 (m, 2H), 3.40-3.68 (m, 2H), 2.06 and 2.07 (two singlets corresponding to two diasteroisomers, 3H), 2.03 and 2.04 (two singlets corresponding to two diasteroisomers, 3H), 1.43-1.92 (m, 6H). ¹³C NMR (CDCl₃) δ 170.4, 169.5, 145.8, 99.3, 99.2, 98.6, 98.4, 75.3, 75.1, 74.9, 74.7, 67.7, 67.4, 65.3, 64.8, 62.1, 61.7, 30.3, 25.3, 21.0, 20.8, 19.2, 19.0. Anal. Calcd for. C₁₅H₂₂O₇: C, 57.32; H,7.05. Found: C, 57.38; H, 7.23.

6-O-(2-Tetrahydropyranyl)-D-glucal (2.4)

A solution of diacetate 2.3 (9.27 g, 29.51 mmol, 1.0 equiv) in MeOH (244.0 mL) was treated with MeONa (0.398 g, 7.38 mmol, 0.25 equival) and the reaction mixture was stirred for 12 h at room temperature. Dilution with MeOH and evaporation of the filtered organic solution afforded a crude reaction product, which was subjected to a flash chromatography. Elution with an 2:8 hexane/AcOEt mixture afforded a pale yellow liquid consisting of trans diol 2.4 (4.125 g, 17.93 mmol, 61% yield).

2.4: Rf = 0.10 (8:2 CH2Cl2/AcOEt); FTIR (neat film) ν 3398, 1647, 1350, 1232, 1126, 1080, 1026 cm^-1. ¹H NMR (CDCl3) δ 6.27 (dt, 1H, J= 6.0, 1.9 Hz), 4.65 (dt, 1H, J= 6.0, 1.5 Hz), 4.45-4.62 (m, 1H), 3.37-4.31 (m, 9H), 1.38-1.84 (m, 6H). ¹H NMR (CD3CN) δ 6.29 (d, 1H, J= 6.0 Hz), 4.65 (dd, 1H, J= 6.0, 1.6 Hz), 4.54-4.60 (m, 1H), 4.13-4.38 (m, 9H), 1.41-1.80 (m, 6H). ¹³C NMR (CDCl₃) δ 144.3, 144.2, 103.2, 102.8, 100.8, 100.7, 99.4, 77.5, 77.1, 76.6, 69.6, 69.5, 69.4, 66.7, 66.4, 64.4, 62.6, 31.1, 30.8, 30.4, 25.3, 25.1, 21.3, 20.6, 19.5. ¹³C NMR (CD₃CN) δ 144.4, 144.3, 104.7, 104.6, 103.7, 103.5,

70 102.4, 100.1, 78.7, 78.6, 78.5, 78.0, 75.2, 69.5, 68.6, 67.3, 66.8, 65.6, 65.4, 63.4, 62.8, 31.9, 31.8, 31.4, 31.2, 26.1, 26.0, 20.6, 20.3. Anal. Calcd for C11H18O5: C, 57.38; H, 7.88. Found: C, 56.99; H, 7.95.

3,4-Di-O-(p-methoxybenzyl)-6-O-(2-tetrahydropyranyl)-D-glucal (2.5)

A solution of trans diol 2.4 (4.325 g, 18.80 mmol) in anhydrous DMF (40 mL) was added dropwise at 0°C to a suspension of 60% NaH in mineral oil (2.256 g, 94.04 mmol, 5.0 quiv) in anhydrous DMF (40 mL). After stirring at 0°C for 15 minutes and then at room temperature for 30 minutes, the suspension was cooled at 0°C and p-methoxybenzylchloride (PMBCl) (6.38 mL, 47.02 mmol, 2.5 equiv.) was added dropwise. The reaction mixture was allowed to warmed at room temperature and stirred for further 12 h. Dilution with Et₂O and evaporation of the washed (brine) and dried (MgSO₄) organic solution afforded a crude reaction product (9.145 g), which was subjected to a flash chromatography. Elution with an 7:3 hexane/AcOEt mixture afforded the 3,4-di-O-para-methoxybenzyl (PMB)-derivative 2.5 (7.841 g, 89% yield), pure as a yellow oil.

2.5: Rf = 0.20 (8:2 hexane/AcOEt). ¹H NMR (250 MHz, CDCl3) δ 7.17-7.36 (m, 4H), 6.78-6.97 (m, 4H), 6.40 (dd, 1H, J= 6.2, 3.1 and 1.3 Hz), 4.84 (ddd, 1H, J= 6.2, 2.7, 0.9 Hz), 4.55-4.82 (m, 2H), 4.77 (d, 1H, J= 10.8 Hz), 4.68 (d, 1H, J= 10.8 Hz), 4.49 (dd, 1H, J= 11.3 and 1.9 Hz), 4.13-4.22 (m, 1H), 3.95-4.12 (m, 2H), 3.65-3.93 (m, 3H), 3.80 (s, 6H), 3.42-3.55 (m, 1H), 1.41-1.92 (m, 6H). ¹³C NMR (62.5 MHz, CDCl3) (2 diasteroisomers) δ 159.4, 144.8, 130.6, 129.7, 129.6, 113.9, 100.2, 100.0, 99.4, 99.2, 75.8, 75.6, 74.3, 73.6, 73.5, 70.4, 62.5, 62.2, 55.5, 30.7, 25.6, 19.7, 19.5. Anal. Calcd for C27H34O7: C, 68.92; H, 7.29. Found; C, 69.24; H, 7.55.

71 3,4-Di-O-(p-methoxybenzyl)-D-glucal (-)-2.6

3,4-Di-O-(p-methoxybenzyl)-derivative 2.5 (2.604 g, 6.40 mmol) was dissolved in a 1.5:2:1 AcOH/THF/H2O mixture (70 mL)²⁷, the reaction mixture was heated at 45°C and stirred at the same temperature for 12 h. After dilution with Et2O and neutralization with solid NaHCO3 (60.0 g), evaporation of the washed (satured aqueous NaHCO3 and saturated aqueous NaCl) and dried organic solution afforded a crude solid product (2.20 g), which was subjected to a flash chromatography. Elution with an 7:3 hexane/AcOEt mixture, afforded the primary alcohol (-)-2.6 (1.50 g, 3.90 mmol, 61%), pure as a yellow solid, m.p. 67-69°C.

(-)-2.6: Rf= 0.14 (7:3 hexane/AcOEt); [α]²⁰_D -21.7 (c 0.6, CHCl₃). ¹H NMR (250 MHz, CDCl₃), δ 7.21-7.32 (m, 4H), 6.84-6.92 (m, 4H), 6.39 (dd, 1H, J=6.2, 1.2 Hz), 4.87 (dd, 1H, J= 6.2, 2.7 Hz), 4.78 (d, 1H, J=11.1 Hz), 4.64 (d, 1H, J= 11.1 Hz), 4.60 (d, 1H, J=

11.1 Hz), 4.50 (d, 1H, J=11.1 Hz), 4.16-4.22 (m, 1H), 3.87-3.96 (m, 1H), 3.82-3.86 (m, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.76 (dd, 1H, J= 8.4, 6.5 Hz). ¹³C NMR (62.5 MHz, CDCl3) δ 159.5, 159.4, 144.6, 130.3, 130.2, 129.9, 129.6, 114.0, 100.4, 77.4 , 75.3, 74.3, 73.5, 70.5, 62.0, 55.4. Anal. Calcd for C22H26O6: C, 68.38; H, 6.78. Found: C, 68.82; H, 6.53.

2-Formyl-3,4-di-(p-methoxybenzyloxy)-3,4-dihydro-2H-pyrane (2.7)

IBX²⁸ (3.96 g, 11.64 mmol, 3.0 equiv) was added to a solution of primary alcohol (-)-2.6 (1.50 g, 3.90 mmol) in anhydrous CH3CN and the reaction mixture was stirred at 45°C overnight. After cooling, the reaction mixture was filtered on a pad of Celite^® that was further eluted with AcOEt. Evaporation of the filtered solution afforded aldehyde 2.7 ( 1.50 g, > 99% yield), pure as a liquid, which was used in the next step without any purification.

72 2.7 Rf = 0.22 (7:3 hexane/AcOEt); ¹H NMR (250 MHz, CDCl3) δ 9.53 (d, 1H, J=0.6 Hz), 7.20-7.34 (m, 2H), 7.09-7.17 (m, 2H), 6.79-6.93 (m,4H), 6.65 (d, 1H, J=6.2 Hz), 5.00- 5.09 (m, 1H), 4.62 (d, 1H, J=11.7 Hz), 4.55 (d, 1H, J=11.7 Hz), 4.51-4.59 (m, 1H9, 4.29 (s, 2H), 4.01-4.06 (m, 1H), 3.81 (s, 3H), 3.79 (s, 3H), 3.71-3.78 (m, 1H). ¹³C NMR (62.5 MHz, CDCl3) δ 198.9, 159.6, 159.5, 145.0, 129.7, 129.6, 129.4, 114.0, 100.5, 79.4, 72.2, 71.6, 69.4, 66.9, 55.5. Anal. Calcd for C22H24O6: C, 68.74; H, 6.29. Found:

C, 69.02; H, 6.11.

1,5-Anhydro-di-O-(p-methoxybenzyl)-2,6,7-trideoxy-D-arabino-hept-1,6-dienitol (+)-2.8.

A solution 0. 5 M of KHMDS in THF ( 11.18 mL, 5.59 mmol, 1.4 equiv) was added dropwise to a solution of Ph₃PMe⁺I^- ( 2.36 g, 5.83 mmol, 1.5 equiv) in anhydrous THF (20 mL) at - 78°C, and the mixture was stirred at the same temperature for 30 min and at 0°C for 1 h. After cooling at -78°C, a solution of aldehyde 2.7 ( 1.50 g, 3.50 mmol) in anhydrous THF (19 mL) was added dropwise, and the reaction mixture was stirred at r.t. for 4 h. Dilution with Et2Oand filtration on Celite^® pad afforded an organic solution which was washed ( saturated aqueous NH4Cl, saturated aqueous NaHCO3, and saturated aqueous NaCl) and dried. Evaporation of the organic solution afforded a crude product which was filtered again on Fluorisil^®-silica gel pad using hexane/AcOEt mixture as eluent at three different concentrations: 7:3, 1:1 and 3:7. Evaporation of the filtered solution of the first mixture afforded olefin (+)-2.8, practically pure as a solid ( 0.930 g). Evaporation of the filtered solution of the second mixture afforded a crude product ( 0.320 g) which was subjected to flash chromatography. Elution with 8:2 hexane/AcOEt mixture afforded olefin (+)-2.8, pure as a solid ( 0.070 g, 76% global yield)

(+)-2.8. R_f =0.43 (7:3 hexane/ACOEt); [α]²⁰_D+1.1 (c 0.10, CHCl₃). ¹H NMR (250 MHz, CDCl3) δ 7.17-7.37 (m, 4H), 6.79-6.97 (m, 4H), 6.41 (d, 1H, J=6.2 Hz), 6.03 (ddd, 1H, J=17.2, 10.5 and 6.5 Hz), 4.70 (d, 1H, J=10.7 Hz), 4.60 (d, 1H, J=10.7), 4.57 (d, H, J=11.3 Hz), 4.51 (d, 1H, J=11.3 Hz), 4.29 (t, 1H, J=7.7 Hz), 4.12-4.20 (m, 1H), 3.80 (s, 6H9, 3.56 (dd, 1H, J=8.5 and 6.2 Hz). ¹³C NMR (62.5 MHz, CDCl₃) δ 159.5, 159.4,

73 144.6, 134.6, 130.7, 130.4, 129.8, 129.5, 118.4, 113.9, 100.7, 78.2, 78.1, 75.3, 73.6, 70.6, 55.4 Anal. Calcd for C23H26O5: C, 72.23; H, 6.85. Found: C, 72.82; H, 6.73.

3,4-Di-O-(p-methoxybenzyl)-5a-carba-D-glucal (-)-2.10.

Olefin (+)-2.8 (1.40 g, 3.69 mmol) was dissolved in 1,3- dichlorobenzene (6.0 mL) and the reaction mixture was stirred for 2 h at 240°C. . The obtained aldehyde 2.9 was not isolated but directly reduced with NaBH₄ (0.209 g, 5.53 mmol, 1.5 equiv) in a 2:1 THF/EtOH mixture (7.0 mL) to primary carba alcohol (-)-2.10, which was subjected to flash chromatography. Elution with 7:3 hexane/ACOEt mixture afforded primary alcohol (- )-2.10 (0.725 g, 52 %).

(-)-2.10: R_f = 0.28 (1:1 hexane/AcOEt); [α]²⁰_D= – 14.6 (c 0.52, CHCl₃). ¹H NMR (250 MHz, CDCl₃) δ 7.19-7.39 (m, 4H), 6.80-6.95 (m, 4H), 5.62-5.81 (m, 2H), 4.91 (d, 1H, J= 11.0 Hz), 4.67 (d, 1H, J= 11.0 Hz), 4.66 (d, 1H, J= 11.2 Hz), 4.58 (d, 1H, J= 11.2 Hz), 4.15-4.25 (m, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.49-3.67 (m, 3H), 1.57-2.22 (m, 3H). ¹³C NMR (62.5 MHz, CDCl3) δ 159.5, 159.4, 130.6, 130.1, 129.7, 128.9, 128.2, 127.0, 126.2, 114.1, 114.0, 82.1, 81.1, 74.1, 71.2, 66.0, 55.4, 40.7, 28.2. Anal. Calcd for C23H28O5: C, 71.85; H 7.34. Found: C,72.02; H, 7.43.

6-O-Benzyl-3,4-di-O-(p-methoxybenzyl)-5a-carba-D-glucal (+)-2.11.

A solution of primary alcohol (-)-2.10 (0.700 g, 1.82 mmol) in anhydrous DMF (4 mL) was added dropwise at 0°C to a suspension of 60% NaH in mineral oil ( 0.216 g, 5.47 mmol, 3.0 equiv) in anhydrous DMF (4 mL) and the resulting reaction mixture was stirred at the same temperature for 30 min and then at room temperature for 30 min. After cooling at 0°C, BnBr (0.42 mL, 3.64 mmol, 2.0 equiv) was added dropwise, and the mixture was stirred at room temperature overnight. After dilution with Et₂O and ice, evaporation

74 of the washed (satured aqueous NaCl) and dried organic layer, afforded a crude reaction mixture (0.911 g), which was subjected to flash chromatography. Elution with an 8:2 hexane/AcOEt mixture afforded monobenzyl-derivate (+)-2.11, pure as liquid (0.600 g, 70% yield).

(+)-2.11 Rf = 0.36 (8:2 hexane/AcOEt); [α]²⁰D +2.9 (c 0.83, CHCl3). ¹H NMR (250 MHz, CDCl3) δ 7.15-7.40 (m, 9H), 6.80-6.92 (m, 4H), 5.61-5.81 (m, 2H) 4.81 (d, H, J=

10.5 Hz), 4.62 (s, 2H), 4.55 (d, 1H, J= 10.5 Hz), 4.50 (s, 2H), 4.10.4.21 (m, 1H) 3.81 (s, 3H), 3.53-3.71 (m, 3H), 2.20-2.31 (m, 2H), 1.99-2.15 (m, 1H). ¹³C NMR (62.5 MHz, CDCl₃) δ 159.3, 138.8, 138.4, 132.4, 132.2, 129.8, 129.6, 129.2, 128.9, 128.6, 127.9, 127.8, 127.6, 113.9, 81.0, 79.4, 74.2, 73.2, 71.3, 70.7, 55.5, 39.5, 29.0. Anal. Calcd for C₃₀H₃₄O₅: C, 75.92; H, 7.22. Found. C, 76.04; H, 7.53.

6-O-Benzyl-5a-carba-D-glucal (+)-2.12.

2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (0.404 g, 1.78 mmol, 1.5 equiv) was added at room temperature to a solution of benzyl derivate (+)-2.11 (0.560 g, 1.18 mmol, 1.0 equiv) in 18:1 CH2Cl2/H2O mixture (32 mL) and the reaction solution was stirred at the same temperature for 3 h.

After dilution with CH2Cl2, evaporation of the washed (saturated aqueous NaHCO3, and satured aqueous NaCl) and dried organic solution, afforded a crude product (0.530 g), wich was subjected to flash chromatography.

Elution with 1:1 hexane/AcOEt mixture afforded trans diol (+)-2.12, pure as a liquid (0.183 g, 66% yield).

(+)-2.12: Rf = 0.18 (1:1 hexane/AcOEt); [α]²⁰D +40.8 (CHCl3, c 0.13). ¹H NMR (250 MHz, CDCl3) δ 7.19-7.49 (m, 5H), 5.42-5.71 (m, 2H), 4.55 (s, 2H), 4.12-4.24 (m, 1H), 3.49-3.76 (m, 3H), 1.85-2.24 (m, 3H). ¹³C NMR (62.5 MHz, CDCl3) δ 137.8, 128.7, 128.5, 128.1, 127.9, 127.0, 77.4, 74.0, 73.7, 73.5, 38.4, 28.4. Anal Calcd for C14H18O3: C, 71.77; H 7.74. Found: C, 72.02; H 7.43.

75 6-O-Benzyl-1,2-anhydro-5a-carba-β-D-mannopyranose (+)-2.13.

70% MCPBA (0.151 g, 0.735 mmol, 1.2 equiv) was added to a solution of trans diol (+)-2.12 (0.170 g, 0.73 mol) in anhydrous CH2Cl2 (17 mL) at 0°C and the reaction mixture was stirred at room temperature. After 18h, dilution with CH2Cl2 and evaporation of washed (10% aqueous Na2S2O3, satured aqueous NaHCO3 and brine) organic solution afforded a crude reaction product (0.140 g), which was subjected to a flash chromatography. Elution with 3:7 hexane/AcOEt mixture afforded epoxide β-(+)-2.13 ( 0.084 g, 46% yield), pure as a white solid. M.p. 86-88°C

β-(+)-2.13: Rf=0.13 (3:7 hexane/AcOEt). [α]²⁰_D +8.03 (c 0.61, CHCl₃). FTIR (neat) ν 3356.9, 3249.9, 3060.9, 2922.9, 2854.6, 1950.8, 1453.5, 1113.5, 1058.3, 1015.0 cm^-1.

1H NMR (250 MHz, CDCl₃) δ 7.44 – 7.15 (m, 5H), 4.50 (d, J = 1.6 Hz, 2H), 3.83 (dd, J

= 8.1 Hz, 1H), 3.60 (dd, J = 10.2, 8.3 Hz, 1H), 3.53 – 3.41 (m, 2H), 3.35 (dd, J = 4.1, 1.8 Hz, 1H), 3.26 (t, 1H), 2.14 – 1.97 (m, 1H), 1.95 – 1.79 (m, 1H), 1.74 (dd, J = 14.8, 3.6 Hz, 1H). ¹³C NMR (CDCl3) δ 74.5, 73.5, 72.9, 72.1, 56.8, 53.4, 38.9, 26.4.

Synthesis of (2-azidoethyl) 2-O-(α-D-mannopyranosyl)-5a-carba-α-D- mannopyranoside (+)-2.14

3,4,6-tri-O-benzyl-1,2-anhydro-5a-carba-β-D-mannopyranose (+)-2.16

A 60% dispersion of NaH (0.077, 3.20, 4.0 equiv) was added to a solution of epoxide (+)-2.13 (0.270 g,1.08 mmol) in anhydrous DMF (4 mL) at 0°C and the reaction mixture was stirred at the same temperature for 30 minutes and then at room temperature for 30 minutes. After cooling at 0°C, BnBr (0.32 mL, 2.70 mmol, 2.5 equiv) was added dropwise and was stirred 2 h at room temperature. Dilution with Et2O and ice and the evaporation of the washed (brine) and

76 dried organic solution afforded a crude reaction mixture (0.360 g), which was subjected to a flash chromatography. Elution with an 8:2 hexane/AcOEt mixture afforded epoxide β-(+)-2.16 ( 0.286 g, 62% yield), pure as a white solid. M.p. 53-55°C

(+)-2.16: Rf =0.24 (8:2 hexane/AcOEt); [α]²⁰D +32.7 (c 0.81, CHCl3). FTIR (neat) ν 3025.0, 2852.0, 1946.5, 1873.9, 1744.0, 1453.3, 1366.2, 1102.3, 1081.5, 1023.5, 737.6, 695.5 cm^-1. ¹H NMR (250 MHz, CDCl3) δ 7.45 – 7.26 (m, J = 11.6, 8.8 Hz, 14H), 7.22 – 7.18 (m, 1H), 4.80 (s, 2H), 4.53 (s, 2H), 4.43 (s, 2H), 3.82 (dd, J = 3.9 Hz, 1H), 3.65 (dd, 1H), 3.46 (dd, J = 8.8 Hz, 2H), 3.32 (dd, 1H), 3.28 – 3.21 (m, 1H), 2.31 – 1.98 (m, 2H), 1.88 – 1.67 (m, 1H).

Synthesis of a 1.3 N solution of 2-azido-1-ethanol in CH2Cl2

2-chloro-1-ethanol (1.75 mL, 26.07 mmol) was added to a solution of NaN₃ (2.0 g, 30.77 mmol, 1.18 equiv) in distilled H₂O (8.0 mL) and the resulting reaction mixture was stirred at room temperature for 1 h, and then heated at 70°C for 3 days. After cooling the reaction mixture was diluted with brine and the aqueous phase was extracted 3 times with CH2Cl2. The combined organic extracts were dried over Na2SO4 and concentrated under vacuum (rotating evaporator) until about 20 mL. An analytical amount of this solution was completely evaporated at the pump, and the resulting liquid residue examined by

1H NMR analysis. The concentrate solution of 2-azido-ethanol in CH2Cl2 was used as the reagent in the corresponding glycosylation reaction.

77 (2-azidoethyl)-3,4,6-tri-O-benzyl-5a-carba-α-D,L-mannopyranoside (+)-2.17

Epoxide (+)-2.16 (0.270 g, 0.617 mmol) was dissolved in a solution 1.3 M 2-azido-1-ethanol in CH2Cl2 ( 2.60 mL, 3.36 mmol, 4.0 equiv) and Cu(OTf)2 (0.081 g, 0.22 mmol, 0.4 equiv) was added. The reaction mixture was stirred for 18 h at room temperature. Dilution with CH2Cl2 and evaporation of the washed (1:1 NaHCO3/NH4Cl mixture and brine) and dried organic solution afforded a crude reaction mixture (0.260 g) which was subjected to a flash chromatography. Elution with 7:3 hexane/AcOEt mixture afforded the ring opening product (+)-2.17 (0.158 g, 0.30 mmol), pure as a pale yellow liquid, in 50% yield.

(+)-2.17: R_f = 0.50 (7.3 hexane/AcOEt); [α]²⁰_D +22.34 (c. 0.67, CHCl₃). FTIR (neat) ν 3449.6, 2927.2, 2871.8, 2103.9, 1737.6, 1109.5, 1048.3 cm^-1. ¹H NMR (250 MHz, CDCl₃) δ 7.43 – 7.15 (m, 15H), 4.82 (d, J = 10.9 Hz, 1H), 4.71 (d, J = 11.5 Hz, 1H), 4.64 (d, J = 11.5 Hz, 1H), 4.50 (d, J = 10.9 Hz, 1H), 4.45 (s, 2H), 4.11 (t, J = 3.4 Hz, 1H), 3.79 (dd, J = 9.0, 3.0 Hz, 1H), 3.73 – 3.44 (m, 6H), 3.33 – 3.21 (m, 2H), 2.11 – 2.02 (m, J = 13.0, 3.0 Hz, 1H), 1.92 – 1.82 (m, J = 9.2, 2.6 Hz, 2H). ¹³C NMR (63 MHz, CDCl3) δ 138.87, 138.64, 138.28, 128.52, 128.43, 128.26, 127.88, 127.77, 127.47, 127.39, 126.93, 82.15, 77.70, 76.83, 74.92, 72.93, 72.52, 70.38, 69.51, 68.12, 50.84, 37.32, 26.53.

(2-azidoethyl) 2-O-[2’, 3’, 4’, 6’-tetra-O-benzoyl-α-D-mannopyranosyl]-3,4,6-tri-O- benzyl-5a-carba-α-D-mannopyranoside (-)-2.19

The glycosyl-donor trichloroacetimidate (-)-2.18 (0.208 g, 0.281 mmol, 1.3 equiv) and the glycosyl- acceptor, the partially protected carbamannoside (+)- 2.17 (0.112 g, 0.216 mmol, 1.0 equiv) were dissolved in distilled toluene and dried overnight at the pump. Acid washed molecular sieves AW 300

78 were activated under vacuum and added to the reagents. The mixture was dissolved in dry CH2Cl2 (2.24 mL) and a catalytic amount of TMSOTf in CH2Cl2 (7.81 µL, 0.034 mmol, 0.2 equiv) was added under nitrogen at -20°C and the reaction mixture was stirred at the same temperature for 3h, monitoring by TLC (8:2 CH2Cl2/MeOH, 3 runs ). Et3N (20.0 µL) was added and the mixture was diluted with CH2Cl2 and filtered through a Celite® pad. Evaporation of the solvent afforded a crude product (0.290 g), which was subjected to a flash chromatography. Elution with a 7:3 hexane/acetone mixture afforded the O-protected pseudomannobioside (-)-2.19 (0.208 mg, 0.190 mmol, 88% yield), pure as a white solid. M.p. 41-42°C.

(-)-2.19: R_f = 0.22 (hexane/acetone); [α]²⁰D – 23.0 (c 0.63, CH3Cl₃). FTIR (neat) ν 3069.3, 2961.0, 2929.5, 2106.4, 1724.0, 1601.0, 1259.8, 1065.8, 707.9 cm^-1. ¹H NMR (250 MHz, CDCl₃) δ 8.12 – 7.99 (m, J = 12.0, 4.3 Hz, 6H), 7.98 – 7.88 (m, 3H), 7.88 – 7.78 (m, 3H), 7.65 – 7.45 (m, 6H), 7.44 – 7.27 (m, 13H), 7.19 – 7.01 (m, 4H), 6.02 (s, J

= 9.5 Hz, 1H), 5.93 – 5.84 (m, J = 9.1 Hz, 2H), 5.43 (s, 1H), 4.84 (d, J = 10.8 Hz, 1H), 4.76 – 4.38 (m, 8H), 4.23 – 4.15 (m, 1H), 3.86 – 3.77 (m, J = 6.0 Hz, 2H), 3.73 – 3.65 (m, 1H), 3.63 – 3.48 (m, J = 18.1 Hz, 3H), 3.40 – 3.27 (m, 1H), 3.23 – 3.11 (m, J = 4.0 Hz, 2H), 2.15 – 2.03 (m, 1H), 2.00 – 1.85 (m, 2H). ¹³C NMR (CDCl3) δ 166.4, 165.8, 165.6, 165.3, 139.0, 138.9, 138.6, 133.6, 133.3, 130.1, 129.9, 129.7, 129.4, 129.1, 128.7, 128.5, 127.9, 127.7, 127.6, 98.9, 76.3, 75.2, 73.1, 70.8, 70.4, 70.2, 68.3, 67.4, 63.4, 60.6, 51.0, 37.8, 27.8.

(2-aminoethyl)-2-O-[2’, 3’, 4’, 6’-tetra-O-benzoyl- α-D-mannopyranosyl]-5a-carba- α-D-mannopyranoside (-)-2.20

Pseudomannobioside (-)-2.19 (0.078 g, 0.071 mmol) was dissolved in a 4:1 MeOH/anhydrous DMF mixture (4.0 mL) and 10% Pd-c (0.078 g) was added. The reaction mixture was hydrogenated (1 bar) at room temperature under hydrogen saturated atmosphere until reduction of the azide group and deprotection of the benzyl

79 groups were achieved, monitoring by analytical TLC, 8:2 CH2Cl2/MeOH. After 18 h the mixture was diluted with MeOH and filtered through a Celite^® pad. The evaporation of the solvent afforded a crude reaction product (0.035 g) which was further diluted with MeOH and filtered off using syringe filter, to remove the residue coal.

Evaporation of the solvent afforded a white oil, which was subjected to a crystallization in presence of (i-Pr)2O. Removal of the surnatant and drying of the precipitate, afforded the 1,2-pseudomannobioside (-)-2.20 (0.031 g, 0.039 mmol, 55% yield), pure as a yellow solid. M.p. 38-39°C

(-)-2.20: R_f = 0.3 (8:2 CH₂Cl₂/MeOH + 0.1% Et₃N), R= 0.11 (8:2 Tol/MeOH 0.1%

Et₃N); [α]²⁰_D -34.85 (c 0.68, MeOH). FTIR (neat) ν 3387.6, 3072.1, 2951.0, 1708.2, 1698.2, ¹H NMR (250 MHz, MeOD) δ 8.13 – 8.02 (m, 2H), 8.01 – 7.86 (m, 4H), 7.76 – 7.66 (m, J = 7.3 Hz, 2H), 7.66 – 7.55 (m, 2H), 7.55 – 7.30 (m, J = 24.5, 7.8 Hz, 8H), 7.28 – 7.18 (m, J = 7.8 Hz, 2H), 6.08 (t, 1H), 5.95 – 5.86 (m, 1H), 5.86 – 5.76 (m, J = 10.2 Hz, 1H), 5.46 (s, 1H), 4.80 – 4.68 (m, J = 9.5 Hz, 1H), 4.58 – 4.42 (m, J = 9.4 Hz, 2H), 4.22 (s, 1H), 3.96 – 3.51 (m, 8H), 3.14 – 3.00 (m, 1H), 2.03 (s, 1H), 1.91 – 1.68 (m, 2H). ¹³C NMR (63 MHz, CDCl₃) δ 167.36, 167.15, 166.71, 166.45, 134.88, 134.23, 131.10, 130.85, 130.63, 130.19, 129.70, 129.60, 129.33, 101.30, 80.47, 78.05, 74.02, 72.20, 71.75, 71.55, 70.64, 68.18, 66.15, 64.13, 63.47, 41.09, 40.18, 27.88.

(2-aminoethyl)-2-O-(α-D-mannopyranosyl)-5a-carba-α-D-mannopyranoside (+)- 2.14

1M MeONa (42.0 µL, 0.042 mmol, 1.0 equiv) in MeOH was added to a solution of 1,2- pseudomannobioside (-)-2.20 (0.034 g, 0.043 mmol, 1.0 equiv) in MeOH (0.8 mL). The reaction mixture was stirred for 12 h monitoring by TLC (8:2 CH₂Cl₂/MeOH). The reaction mixture was diluted with MeOH and Amberlite IRC 50 was added until pH=7. The beads were filtered off and washed with MeOH, then, the filtrated solution was evaporated under reduced pressure affording a crude reaction product (0.017 g).

The solid product was dissolved in MeOH and filtered through a syringe filter.

80 Evaporation of the solvent afforded an oil, which was subjected to a crystallization in presence of (i-Pr)2O. Removal of the surnatant and drying of the precipitate, afforded fully O-deprotected 1,2-pseudomannobioside (+)-2.14 (0.011 g, 0.030 mmol, 69 % yield), pure as a yellow solid.

(+)-2.14: Rf = 0.15 (1:1 CH2Cl2/MeOH); [α]²⁰D +16.4 (c 1.14, MeOH). FTIR (neat) ν 3396.5, 2924.9, 1537.7, 1486.7, 1180.4, ¹H NMR (250 MHz, MeOD) δ 5.04 (d, J = 1.4 Hz, 1H), 4.16 – 3.41 (m, 15H), 3.05 (t, J = 4.8 Hz, 1H), 2.05 – 1.71 (m, 2H), 1.63 – 1.49 (m, 1H). ¹³C NMR (63 MHz, CDCl₃) δ 104.27, 79.91, 78.06, 75.71, 74.69, 72.69, 72.19, 68.82, 67.31, 64.76, 63.61, 41.64, 40.61, 28.17. HR-MS (ESI): calculated for C₁₅H₂₉NO₁₀ [M+H]^-: 384.2

Synthesis of (2-azidoethyl) 2-O-(α-D-mannopyranosyl)-5a-carba-α-D- mannopyranoside

1,2-anhydro-5a-carba-β-D-mannopyranose 2.21

Epoxide β (+)-2.13 (0.051 g, 0.204 mmol) was dissolved in EtOH (4.0 mL) and 10% Pd-c (0.020 g) was added. The reaction mixture was hydrogenated (1 bar) at room temperature under hydrogen saturated atmosphere until deprotection of the benzyl group was achieved, monitoring by analytical TLC the disappearance of the starting material, with 7:3 hexane/AcOEt mixture. After 24 h the mixture was diluted with EtOH and filtered through a Celite^® pad. The evaporation of the solvent afforded a crude reaction product, the triol 2.21 (0.032 g, 0.200 mmol, 98% yield ) which was used in the next step without any purification.

2.21: Rf = 0.17 (CH2Cl2/MeOH 9:1). ¹H-NMR and ¹³C-NMR see reference 30 cap.2.

81 3,4,6-tri-O-acetyl-1,2-anhydro-5a-carba-β-D-mannopyranose (+)-2.22.

Triol 2.21 (0.040 g, 0.250 mmol) was dissolved in Pyridine (1.0 mL) and the reaction mixture was cooled at 0°C.

Acetic anhydride (0.5 mL) was added and the reaction was stirred at room temperature overnight. Co-evaporation of the reaction mixture with toluene afforded a crude product (0.060 g), which was subjected to flash chromatograpy.

Elution with 1:1 hexane/AcOEt mixture afforded tri-O-acetyl-β-epoxide (+)-2.22 (0.030 g, 0.010 mmol, 40 % yield), pure as a pale yellow oil.

(+)-3.22: R_f = 0.26 (hexane/AcOEt 1:1). [α]²⁰_D +1.85 (c 2.0, CHCl₃). ¹H-NMR (CDCl₃) δ: 5.23 (dd, 1H, J= 8.7, 1.8 Hz), 5.14 (dd, 1H, J= 10.3, 8.7 Hz), 4.05 (dd, 1H, J= 11.2, 4.7 Hz), 3.85 (dd, 1H, J= 11.2, 3.2 Hz), 3.41 (dd, 1H, J= 4.2, 1.8 Hz), 3.33 (t, 1H, J= 4.2 Hz), 2.15-2.24 (m, 1H), 2.10 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.99-2.08 (m, 2H). ¹³C- NMR (CDCl3)δ: 171.0, 170.0, 73.5, 69.5, 63.1, 54.8, 52.9, 37.5, 26.4, 21.1, 21.0.

(2-azidoethyl)-3,4,6-tri-O-acetyl-5a-carba-α-D-mannopyranoside (+)-2.23

Epoxide (+)-2.22 (0.011 g, 0.038 mmol) was dissolved in a 1.3 M solution of 2-azido-1-ethanol in CH2Cl2 (0.11 mL, 0.143 mmol, 4.0 equiv) and Cu(OTf)2 (0.004 g, 0.015 mmol, 0.4 equiv) was added. The reaction mixture was stirred for 18 h at room temperature. Dilution with CH2Cl2 and evaporation of the washed (1:1 NaHCO3/NH4Cl mixture and brine) and dried organic solution afforded a crude reaction product ( 0.016 g) which was subjected to flash chromatography. Elution with a 1:1 hexane/AcOEt solution afforded the glycosyl acceptor (+)-2.23 ( 0.0067 g, 47% yield), pure as a pale yellow oil.

2.23: Rf = 0.14 ( 1:1 hexane/AcOEt). [α]²⁰D= +21.71 ( c. 0.35, CHCl3) . FTIR (neat) ν 3420.2, 2925.7, 2106.9, 1738.1, 1227.5, 1039.6. ¹H NMR (250 MHz, CDCl₃) δ 5.28 (dd, J = 18.7 Hz, 1H), 5.20 (dd, J = 9.3, 2.9 Hz, 1H), 4.22 – 4.16 (m, 1H), 4.11 (dd, J =

82 11.0, 4.5 Hz, 1H), 3.96 (d, J = 3.7 Hz, 1H), 3.77 – 3.73 (m, 1H), 3.70 (t, J = 5.0 Hz, 2H), 3.42 – 3.33 (m, 2H), 2.21 (d, 1H), 2.08 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.94 – 1.88 (m, 2H). ¹³C NMR (CDCl3) δ 171.2, 170.6, 169.9, 80.71, 73.9, 69.7, 68.7, 64.0, 51.0, 35.2, 26.8, 21.1, 21.0.

(2-azidoethyl)-2-O-[2’,3’,4’,6’-tetra-O-benzoyl-α-D-mannopyranosyl]-3,4,6-tri-O- acetyl-5a-carba-α-D-mannopyranoside (-)-2.24.

The glycosyl-donor trichloroacetimidate (-)-2.18.

(0.047g, 0.063 mmol, 1.3 equiv) and the glycosyl-acceptor, the partially protected carbamannoside 2.23 (0.019 g, 0.049 mmol, 1.0 equiv) were dissolved in distilled toluene and dried overnight at the pump. Acid washed molecular sieves AW 300 were activated under vacuum and added to the reagents. The mixture was dissolved in dry CH2Cl2 (1.0 mL) and a catalytic amount of TMSOTf in CH2Cl2

(1.76 µL, 0.010 mmol, 0.2 equiv) was added under nitrogen at -20°C and the reaction mixture was stirred at the same temperature for 20 minutes, monitoring by TLC (1:1 hexane/AcOEt ). Et3N (10.0 µL) was added and the mixture was diluted with CH2Cl2

and filtered through a Celite^® pad. Evaporation of the solvent afforded a crude reaction product, which was subjected to preparative TLC, using a 1:1 hexane/AcOEt mixture as eluant. Extraction of the most intense band afforded pseudodisaccharide (-)-2.24 (0.025 g, 0.026 mmol, 53% yield).

(-)-2.24: Rf = 0.40 (1:1 hexane/AcOEt). [α]²⁰D - 42.8 (c 1,7, CHCl3).; ¹H NMR (CDCl³) δ 7.92-8.13 (m, 6H), 7.817.86 (m, 2H), 7.21-7.66 (m, 12H), 6.05 (t, 1H, J= 10.0 Hz), 5.91 (dd, 1H, J= 10.0, 2.9 Hz), 5.69 (dd, 1H, J= 2.9, 1.6 Hz), 5.28-5.43 (m, 1H), 5.26 (dd, 1H, J= 9.9, 2.8 Hz), 4.68 (dd, 1H, J= 11.9, 2.6 Hz), 4.49 (dd, 1H, J= 11.9, 5.3 Hz), 4.35-4.45 (m, 1H), 4.31 (t, 1H, J= 3.2 Hz), 4.17 (dd, 1H, J= 11.0, 5.3 Hz), 4.02 (dd, 1H, J= 11.0, 3.2 Hz), 3.72-3.79 (m, 1H), 3.44-3.58 (m, 2H), 3.12-3.35 (m, 2H), 2.22-2.37 (m, 1H), 2.16 (s, 3H), 2.13 (s, 3H), 2.04 (s, 3H), 1.952.02 (m, 2H). ¹³C NMR (CDCl₃) δ

83 171.4, 170.7, 170.1, 166.3, 165.9, 165.5, 133.7, 133.4, 130.0, 129.9, 129.5, 129.2, 129.0, 128.8, 128.7, 128.5, 99.1, 76.8, 76.7, 72.5, 70.9, 70.1, 69.8, 69.3, 68.8, 68.6, 67.5, 63.9, 63.5, 50.9, 35.5, 21.0.

(2-azidoethyl)-2-O-(α-D-mannopyranosyl)-5a-carba-α-D-mannopyranoside 2.15a A solution 1M MeONa in MeOH (35.0 µL, 0.035 mmol, 0.7 equiv) was added to a solution of fully- O-protected 1,2-pseudomannobioside (-)-2.24 (0.048 g, 0.050 mmol) in MeOH (1.0 mL). The reaction was stirred for 12 h monitoring by TLC (1:1 hexane/AcOEt for the disappearance of the starting material, and 9.5:0.5 CHCl3/MeOH for the formation of the product). Amberlite IR 120 H⁺ was added until pH 7, the beads were filtered off and washed with MeOH. Evaporation of the solvent under reduced pressure afforded the pseudomannobioside fully-O- deprotected 2.15a (0.018 g, 88% yield), pure as a white solid.

2.15a: Rf = 0.21 (1:1 CH2Cl2/MeOH); ¹H NMR (D2O) δ 4.98 (s, 1H), 4.05 (bs, 2H), 3.48-3.94 (m, 13H), 3.40 (t, 1H, J= 4.5 Hz), 1.84-1.98 (m, 1H), 1.54-1.83 (m, 2H). ¹³C NMR (D₂O, 0.1% CD₃CN) δ 103.6, 80.4, 77.1, 74.7, 73.4, 71.4, 71.2, 71.0, 71.0, 68.7, 67.9, 63.2, 62.1, 51.5, 39.7, 27.3.

3.1 ¹H NMR spectra

84

85

86

87

88