3. Materials and Methods
3.1. General Methods
Reactants and reagents were purchased from standard suppliers (Sigma-Aldrich (St. Louis, MO, USA), Alfa-Aesar (Haverhill, MA, USA), Fisher Scientific (Waltham, MA, USA)) and were used without further purification. Methanol, dichloromethane, tetrahydrofuran, and diethyl ether were dried on a Pure Solv MD system (Innovative Technology, Inc., Oldham, UK). Reactions were monitored on TLC plates using Silica gel F254 (0.2 mm) (Macherey-Nagel SAS, Hoerdt, France), detection was carried out by spraying with an aqueous solution of KMnO4 (2%)/Na2CO3 (4%) or alcoholic solutions of phosphomolybdic acid or p-anisaldehyde, followed by heating. Column chromatography purifications were performed over silica gel M 9385 (40–63 µm) Kieselgel 60 (Macherey-Nagel SAS, Hoerdt, France). NMR spectra were recorded on Bruker AC 500 (Billerica, MA, USA, 500 MHz for 1H, 126 MHz for 13C) or 600 (600 MHz for 1H and 150 MHz for 13C) spectrometers. Chemical shifts are expressed in parts per million (ppm) and were calibrated to deuterated or residual non-deuterated solvent peaks for 1H and 13C spectra. Coupling constants are in Hz and splitting pattern abbreviations are: br, broad; s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet. DEPT and JMOD 1D NMR experiments, COSY, HSQC, HMBC, and NOESY 2D NMR experiments were used to confirm the NMR peak assignments for all compounds. Optical rotations were determined at 25 °C with an Anton Paar Model MCP 5100 polarimeter (Graz, Austria). Mass Spectra (MS) and High Resolution Mass Spectra (HRMS) were performed on a Waters Corp. Q-TOF Micro micromass positive ESI (CV = 30 V) (Milford, MA, USA).
3.2. General Procedure for the Synthesis of Bis-Glycosylamines 10a–e
To a solution of 2,3,5-tri-O-benzyl-l-xylofuranose 2 (1 equiv) in anhydrous dichloromethane was added molecular sieves (4 Å) then diamine 9a–e (0.5 equiv), and the mixture was stirred at room temperature until total consumption of 2. The solution was filtrated and concentrated under reduced pressure to afford an anomeric mixture of the bis-glycosylamine which was not purified further.
(10a) According to general procedure, the reaction of 2 (200 mg, 0.475 mmol) and 9a (0.237 mmol, 32.3 mg) in dichloromethane (3.7 mL) in the presence of molecular sieves (450 mg) afforded after 48 h 10a as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.40–7.24 (m, 34 H, Ar-H), 5.03 (d, J = 3.5 Hz, 0.7 H, 2 × 1-Hmin), 4.76 (d, J = 2.2 Hz, 1.3 H, 2 × 1-Hmaj), 4.64–4.45 (m, 12 H, 6 × CH2-Phmaj + 6 × CH2-Phmin), 4.41 (q, J = 6.0 Hz, 0.7 H, 2 × 4-Hmin), 4.34 (q, J = 5.3 Hz, 1.3 H, 2 × 4-Hmaj), 4.15 (d, J = 13.5 Hz, 0.7 H, 2 × CHaHb-NHmin), 4.07 (dd, J = 13.4, 3.6 Hz, 1.3 H, 2 × CHaHb-NHmaj), 4.01–3.98 (m, 2 H, 2 × 3-Hmaj + 2 × 3-Hmin), 3.95–3.93 (m, 1.3 H, 2 × 2-Hmaj), 3.91–3.89 (m, 0.7 H, 2 × 2-Hmin), 3.82–3.68 (m, 6 H, 2 × CHaHb-NHmaj + 2 × CHaHb-NHmin + 2 × 5-Hmaj + 2 × 5-Hmin) ppm; 13C NMR (125 MHz, CDCl3): δ 140.7 (CIV), 140.4 (CIV), 138.5 (CIV), 138.4 (CIV), 138.1 (CIV), 138.0 (CIV), 137.9 (CIV), 128.7, 128.6, 128.5, 128.5, 128.2, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7, 127.7, 127.1, 126.8, 126.7 (C-Ar), 94.6 (C-1maj), 90.5 (C-1min), 86.1 (C-2maj), 81.6 (C-3maj), 81.4 (C-3min), 81.4 (C-2min), 78.9 (C-4maj), 77.2 (C-4min), 73.6 (CH2-Ph), 72.9 (CH2-Phmin), 72.4 (CH2-Phmin), 72.2 (CH2-Phmaj), 71.7 (CH2-Phmaj), 69.3 (C-5maj), 69.0 (C-5min), 50.2 (CH2-NHmin), 49.9 (CH2-NHmaj) ppm; HRMS (ESI) m/z calcd for [C60H64N2O8 + H]+: 941.4741, found 941.4745.
(10b) According to general procedure, the reaction of 2 (436 mg, 1.04 mmol) and 9b (71 mg, 0.52 mmol) in dichloromethane (7.8 mL) in the presence of molecular sieves (950 mg) afforded after 27 h 10b as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.40–7.27 (m, 34 H, Ar-H), 5.00–4.98 (m, 0.7 H, 2 × 1-Hmin), 4.73–4.72 (m, 1.3 H, 2 × 1-Hmaj), 4.68–4.47 (m, 12 H, 6 × CH2-Phmaj + 6 × CH2-Phmin), 4.39 (q, J = 5.7 Hz, 0.7 H, 2 × 4-Hmin), 4.36 (q, J = 5.2 Hz, 1.3 H, 2 × 4-Hmaj), 4.16 (d, J = 13.5 Hz, 0.7 H, 2 × CHaHb-NHmin), 4.08 (d, J = 13.2 Hz, 1.3 H, 2 × CHaHb-NHmaj), 4.03–4.00 (m, 2 H, 2 × 3-Hmaj + 2 × 3-Hmin), 3.96–3.95 (m, 1.3 H, 2 × 2-Hmaj), 3.92–3.71 (m, 6.7 H, 2 × 2-Hmin + 2 × CHaHb-NHmaj + 2 × CHaHb-NHmin + 2 × 5-Hmaj + 2 × 5-Hmin) ppm; 13C NMR (125 MHz, CDCl3): δ 139.1 (CIV), 138.9 (CIV), 138.5 (CIV), 138.3 (CIV), 138.0 (CIV), 138.0 (CIV), 137.9 (CIV), 128.7, 128.7, 128.6, 128.5, 128.5, 128.5, 128.4, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7, 127.7 (C-Ar), 94.4 (C-1maj), 90.4 (C-1min), 86.0 (C-2maj), 81.6 (C-3maj), 81.4 (C-3min), 81.3 (C-2min), 78.9 (C-4maj), 77.2 (C-4min), 73.6 (CH2-Ph), 72.8 (CH2-Phmin), 72.3 (CH2-Phmin), 72.2 (CH2-Phmaj), 71.7 (CH2-Phmaj), 69.2 (C-5maj), 69.0 (C-5min), 49.9 (CH2-NHmin), 49.6 (CH2-NHmaj) ppm; HRMS (ESI) m/z calcd for [C60H64N2O8 + H]+: 941.4741, found 941.4742.
(10c) According to general procedure, the reaction of 2 (600 mg, 1.43 mmol) and 9c (103 mg, 0.71 mmol) in dichloromethane (11.1 mL) in the presence of molecular sieves (1.35 g) afforded after 23 h 10c as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.40–7.26 (m, 30 H, Ar-H), 5.04 (d, J = 3.4 Hz, 0.7 H, 2 × 1-Hmin), 4.77 (d, J = 2.2 Hz, 1.3 H, 2 × 1-Hmaj), 4.66–4.47 (m, 12 H, 6 × CH2-Phmaj + 6 × CH2-Phmin), 4.39 (q, J = 5.5 Hz, 0.7 H, 2 × 4-Hmin), 4.34 (q, J = 5.4 Hz, 1.3 H, 2 × 4-Hmaj), 4.01–3.99 (m, 2 H, 2 × 3-Hmaj + 2 × 3-Hmin), 3.92–3.89 (m, 2 H, 2 × 2-Hmaj + 2 × 2-Hmin), 3.80–3.67 (m, 4 H, 2 × 5-Hmaj + 2 × 5-Hmin), 3.01–2.87 (m, 2 H, 2 × CHaHb-NHmaj + 2 × CHaHb-NHmin), 2.69–2.57 (m, 2 H, 2 × CHaHb-NHmaj + 2 × CHaHb-NHmin), 1.53–1.28 (m, 12 H, 6 × CH2maj + 6 × CH2min) ppm; 13C NMR (125 MHz, CDCl3): δ 138.5 (CIV), 138.4 (CIV), 138.1 (CIV), 138.0 (CIV), 137.9 (CIV), 137.9 (CIV), 128.7, 128.6, 128.5, 128.5, 128.5, 128.5, 128.4, 128.4, 128.1, 128.1, 128.1, 128.0, 128.0, 128.0, 127.9, 127.9, 127.8, 127.8, 127.7, 127.7 (C-Ar), 95.1 (C-1maj), 91.0 (C-1min), 86.1 (C-2maj), 81.6 (C-3maj), 81.5 (C-3min), 81.2 (C-2min), 78.8 (C-4maj), 77.2 (C-4min), 73.5 (CH2-Ph), 72.8 (CH2-Phmin), 72.4 (CH2-Phmin), 72.2 (CH2-Phmaj), 71.7 (CH2-Phmaj), 69.2 (C-5maj), 68.9 (C-5min), 46.6 (CH2-NHmin), 46.1 (CH2-NHmaj), 30.8 (CH2), 30.5 (CH2), 29.6 (CH2), 29.6 (CH2), 27.4 (CH2), 27.4 (CH2), 27.4 (CH2), 27.3 (CH2) ppm; HRMS (ESI) m/z calcd for [C60H72N2O8 + H]+: 949.5372, found 949.5367.
(10d) According to general procedure, the reaction of 2 (100 mg, 0.24 mmol) and 9d (18 mg, 0.12 mmol) in dichloromethane (2 mL) in the presence of molecular sieves (227 mg) afforded after 24 h 10d as a brown oil. 1H NMR (500 MHz, CDCl3): δ 7.39–7.25 (m, 30 H, Ar-H), 4.97 (d, J = 3.6 Hz, 0.6 H, 2 × 1-Hmin), 4.72–4.71 (m, 1.4 H, 2 × 1-Hmaj), 4.65–4.45 (m, 12 H, 6 × CH2-Phmaj + 6 × CH2-Phmin), 4.37 (q, J = 5.9 Hz, 0.6 H, 2 × 4-Hmin), 4.34 (q, J = 5.4 Hz, 1.4 H, 2 × 4-Hmaj), 3.99–3.98 (m, 2 H, 2 × 3-Hmaj + 2 × 3-Hmin), 3.93 (m, 1.4 H, 2 × 2-Hmaj), 3.90–3.89 (m, 0.6 H, 2 × 2-Hmin), 3.80–3.68 (m, 4 H, 2 × 5-Hmaj + 2 × 5-Hmin), 3.62–3.52 (m, 8 H, 4 × CH2maj + 4 × CH2min), 3.19–3.07 (m, 2 H, 2 × CHaHb-NHmaj + 2 × CHaHb-NHmin), 2.88–2.75 (m, 2 H, 2 × CHaHb-NHmaj + 2 × CHaHb-NHmin) ppm; 13C NMR (125 MHz, CDCl3): δ 138.5 (CIV), 138.4 (CIV), 138.1 (CIV), 138.0 (CIV), 137.9 (CIV), 137.9 (CIV), 128.7, 128.7, 128.5, 128.5, 128.4, 128.4, 128.0, 127.9, 127.9, 127.8, 127.7 (C-Ar), 95.3 (C-1maj), 90.9 (C-1min), 86.1 (C-2maj), 81.6 (C-3min), 81.5 (C-3maj), 81.3 (C-2min), 78.9 (C-4maj), 77.2 (C-4min), 73.5 (CH2-Phmaj), 73.5 (CH2-Phmin), 72.8 (CH2-Phmin), 72.4 (CH2-Phmin), 72.2 (CH2-Phmaj), 71.7 (CH2-Phmaj), 71.5 (CH2min), 71.3 (CH2maj), 70.4 (CH2min), 70.3 (CH2min), 70.2 (CH2maj), 70.2 (CH2maj), 69.2 (C-5maj), 68.9 (C-5min), 46.1 (CH2-NHmin), 45.7 (CH2-NHmaj) ppm; HRMS (ESI) m/z calcd for [C58H68N2O10 + H]+: 953.4952, found 953.4957.
(10e) According to general procedure, the reaction of 2 (157 mg, 0.37 mmol) and 9e (41 mg, 0.18 mmol) in dichloromethane (3.1 mL) in the presence of molecular sieves (356 mg) afforded after 27 h 10e as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.39–7.26 (m, 30 H, Ar-H), 4.95 (d, J = 3.6 Hz, 0.7 H, 2 × 1-Hmin), 4.68 (d, J = 2.2 Hz, 1.3 H, 2 × 1-Hmaj), 4.65–4.45 (m, 12 H, 6 × CH2-Phmaj + 6 × CH2-Phmin), 4.36 (q, J = 6.0 Hz, 0.7 H, 2 × 4-Hmin), 4.30 (q, J = 5.4 Hz, 1.3 H, 2 × 4-Hmaj), 3.99–3.97 (m, 2 H, 2 × 3-Hmaj + 2 × 3-Hmin), 3.87 (m, 2 H, 2 × 2-Hmaj + 2 × 2-Hmin), 3.76–3.65 (m, 4 H, 2 × 5-Hmaj + 2 × 5-Hmin Hmin), 3.62–3.48 (m, 12 H, 6 × CH2maj + 6 × CH2min), 3.06–2.93 (m, 2 H, 2 × CHaHb-NHmaj + 2 × CHaHb-NHmin), 2.78–2.66 (m, 2 H, 2 × CHaHb-NHmaj + 2 × CHaHb-NHmin), 1.81–1.69 (m, 4 H, 2 × CH2-CH2-NHmaj + 2 × CH2-CH2-NHmin) ppm; 13C NMR (125 MHz, CDCl3): δ 138.5 (CIV), 138.4 (CIV), 138.1 (CIV), 138.0 (CIV), 137.9 (CIV), 128.7, 128.6, 128.5, 128.5, 128.5, 128.0, 127.9, 127.9, 127.9, 127.8, 127.8, 127.7, 127.7 (C-Ar), 95.2 (C-1maj), 91.0 (C-1min), 86.2 (C-2maj), 81.6 (C-3maj), 81.4 (C-2min), 81.2 (C-3min), 78.7 (C-4maj), 77.2 (C-4min), 73.6 (CH2-Phj), 72.8 (CH2-Phmin), 72.4 (CH2-Phmin), 72.2 (CH2-Phmaj), 71.7 (CH2-Phmaj), 70.7 (CH2), 70.3 (CH2), 69.8 (CH2), 69.7 (CH2), 69.6 (CH2), 69.2 (C-5maj), 68.9 (C-5min), 43.7 (CH2-NHmin), 43.3 (CH2-NHmaj), 30.7 (CH2-CH2-NHmin), 30.6 (CH2-CH2-NHmaj) ppm; HRMS (ESI) m/z calcd for [C62H76N2O11 + H]+: 1025.5527, found 1025.5531.
3.3. Representative Procedure for the Reduction of Bis-Glycosylamines 10a–e
To a solution of bis-glycosylamine 10a–e (1 equiv) in anhydrous diethyl ether at 0 °C under argon atmosphere was added dropwise lithium aluminium hydride 1 M in diethyl ether (4 equiv). The mixture was stirred for 1 h at the same temperature and then sequentially supplemented dropwise with water (38 µL per mL of LiAlH4 1 M used), an aqueous solution of sodium hydroxide 3 M (38 µL per mL of LiAlH4 1 M used) and water again (114 µL per mL of LiAlH4 1 M used). The resulting mixture was filtered, the inorganic residue was washed with ether, and the resulting ether solution was then combined with the organic layer of the filtrate. The solution was dried with magnesium sulfate, filtered, and concentrated under reduced pressure, and finally, the crude was purified by column chromatography on silica gel.
(11a) According to general procedure, the reaction of 10a (232 mg, 0.246 mmol) and LiAlH4 1 M in diethyl ether (984 µL, 0.984 mmol) in diethyl ether (2.5 mL) afforded after purification (eluent: EtOAc/PE gradient, 80/20 to 95/5) 11a (149 mg, 0.158 mmol, 64%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.36–7.15 (m, 34 H, Ar-H), 4.56–4.42 (m, 12 H, 6 × CH2-Ph), 4.09–4.06 (m, 2 H, 2 × 4-H), 3.83–3.76 (m, 4 H, 2 × 3-H + 2 × NH-CHaHb-Ph), 3.72–3.52 (m, 8 H, 2 × 2-H + 2 × NH-CHaHb-Ph + 2 × 5-H), 2.97 (dd, J = 12.2, 1.4 Hz, 2 H, 2 × 1-HaHb), 2.83 (dd, J = 12.2, 5.1 Hz, 2 H, 2 × 1-HaHb) ppm; 13C NMR (125 MHz, CDCl3): δ 138.5 (CIV), 138.4 (CIV), 138.1 (CIV), 129.2, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.0, 128.0, 127.9, 127.9, 127.9, 127.8, 127.7, 127.7, 127.7, 127.7 (C-Ar), 76.8 (C-3), 76.2 (C-2), 73.8 (CH2-Ph), 73.3 (CH2-Ph), 72.1 (CH2-Ph), 70.9 (C-5), 66.7 (C-4), 53.6 (NH-CH2-Ph), 46.5 (C-1) ppm; = +6.3 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C60H68N2O8 + H]+: 945.5054, found 945.5063.
(11b) According to general procedure, the reaction of 10b (583 mg, 0.619 mmol) and LiAlH4 1 M in diethyl ether (2.48 mL, 2.476 mmol) in diethyl ether (6 mL) afforded after purification (eluent: EtOAc/PE gradient, 80/20 to 95/5) 11b (357 mg, 0.377 mmol, 61%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.36–7.22 (m, 34 H, Ar-H), 4.56–4.41 (m, 12 H, 6 × CH2-Ph), 4.09–4.06 (m, 2 H, 2 × 4-H), 3.81–3.77 (m, 4 H, 2 × 3-H + 2 × NH-CHaHb-Ph), 3.68–3.61 (m, 6 H, 2 × 2-H + 2 × NH-CHaHb-Ph + 2 × 5-HaHb), 3.58–3.54 (m, 2 H, 2 × 5-HaHb), 2.95 (dd, J = 12.2, 1.4 Hz, 2 H, 2 × 1-HaHb), 2.83 (dd, J = 12.2, 5.0 Hz, 2 H, 2 × 1-HaHb) ppm; 13C NMR (125 MHz, CDCl3): δ 138.6 (CIV), 138.4 (CIV), 138.1 (CIV), 137.9 (CIV), 128.8, 128.7, 128.7, 128.6, 128.6, 128.5, 128.4, 128.4, 128.0, 127.9, 127.9, 127.6 (C-Ar), 76.7 (C-3), 76.2 (C-2), 73.8 (CH2-Ph), 73.3 (CH2-Ph), 72.1 (CH2-Ph), 70.9 (C-5), 66.5 (C-4), 53.5 (NH-CH2-Ph), 46.3 (C-1) ppm; = +14.8 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C60H68N2O8 + H]+: 945.5054, found 945.5051.
(11c) According to general procedure, the reaction of 10c (597 mg, 0.629 mmol) and LiAlH4 1 M in diethyl ether (2.52 mL, 2.516 mmol) in diethyl ether (6 mL) afforded after purification (eluent: EtOAc/PE 70/30 then EtOAc/MeOH 95/5) 11c (388 mg, 0.407 mmol, 65%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.37–7.25 (m, 30 H, Ar-H), 4.57–4.47 (m, 12 H, 6 × CH2-Ph), 4.09–4.06 (m, 2 H, 2 × 4-H), 3.76 (d, J = 5.8 Hz, 2 H, 2 × 3-H), 3.67–3.61 (m, 4 H, 2 × 2-H + 2 × 5-HaHb), 3.56 (t, J = 8.5 Hz, 2 H, 2 × 5-HaHb), 2.90 (dd, J = 12.2, 1.3 Hz, 2 H, 2 × 1-HaHb), 2.79 (dd, J = 12.2, 5.0 Hz, 2 H, 2 × 1-HaHb), 2.55 (t, J = 7.4 Hz, 4 H, 2 × NH-CH2-CH2), 1.53–1.38 (m, 4 H, 2 × NH-CH2-CH2), 1.28–1.24 (m, 8 H, 4 × CH2) ppm; 13C NMR (125 MHz, CDCl3): δ 138.6 (CIV), 138.5 (CIV), 138.2 (CIV), 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.0, 127.9, 127.9, 127.9, 127.6 (C-Ar), 76.7 (C-3), 76.2 (C-2), 73.7 (CH2-Ph), 73.3 (CH2-Ph), 72.0 (CH2-Ph), 71.0 (C-5), 66.3 (C-4), 49.6 (NH-CH2-CH2), 46.5 (C-1), 29.5 (NH-CH2-CH2), 29.4 (CH2), 27.3 (CH2) ppm; = +20.3 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C60H76N2O8 + H]+: 953.5680, found 953.5685.
(11d) According to general procedure, the reaction of 10d (1.07 g, 1.12 mmol) and LiAlH4 1 M in diethyl ether (4.48 mL, 4.48 mmol) in diethyl ether (11 mL) afforded after purification (eluent: EtOAc/MeOH gradient, 80/20 to 50/50) 11d (773 mg, 0.806 mmol, 72%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.37–7.25 (m, 30 H, Ar-H), 4.57–4.45 (m, 12 H, 6 × CH2-Ph), 4.10 (t, J = 6.7 Hz, 2 H, 2 × 4-H), 3.76 (dm, J = 5.9 Hz, 2 H, 2 × 3-H), 3.68–3.66 (m, 2 H, 2 × 2-H), 3.64–3.49 (m, 12 H, 2 × 5-H + 4 × CH2), 2.90 (dd, J = 12.5, 1.6 Hz, 2 H, 2 × 1-HaHb), 2.83 (dd, J = 12.5, 4.8 Hz, 2 H, 2 × 1-HaHb), 2.80–2.70 (m, 4 H, 2 × NH-CH2-CH2) ppm; 13C NMR (125 MHz, CDCl3): δ 138.5 (CIV), 138.4 (CIV), 138.1 (CIV), 128.5, 128.4, 128.3, 128.3, 127.8, 127.8, 127.8, 127.5 (C-Ar), 76.9 (C-3), 76.2 (C-2), 73.6 (CH2-Ph), 73.2 (CH2-Ph), 71.8 (CH2-Ph), 71.0 (C-5), 70.3 (CH2), 69.7 (CH2), 66.4 (C-4), 48.7 (NH-CH2-CH2), 46.2 (C-1) ppm; = +14.9 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C58H72N2O10 + H]+: 957.5265, found 957.5270.
(11e) According to general procedure, the reaction of 10e (0.578 g, 0.564 mmol) and LiAlH4 1 M in diethyl ether (2.26 mL, 2.26 mmol) in diethyl ether (5 mL) afforded after purification (eluent: EtOAc/MeOH gradient, 80/20 to 50/50) 11e (336 mg, 0.327 mmol, 58%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.37–7.25 (m, 30 H, Ar-H), 4.56–4.45 (m, 12 H, 6 × CH2-Ph), 4.10–4.07 (m, 2 H, 2 × 4-H), 3.77 (dm, J = 5.8 Hz, 2 H, 2 × 3-H), 3.69–3.67 (m, 2 H, 2 × 2-H), 3.65–3.45 (m, 16 H, 2 × 5-H + 6 × CH2), 2.91 (dd, J = 12.2, 1.1 Hz, 2 H, 2 × 1-HaHb), 2.82 (dd, J = 12.2, 4.9 Hz, 2 H, 2 × 1-HaHb), 2.75–2.64 (m, 4 H, 2 × NH-CH2-CH2), 1.79–1.74 (m, 4 H, 2 × NH-CH2-CH2) ppm; 13C NMR (125 MHz, CDCl3): δ 138.5 (CIV), 138.3 (CIV), 138.1 (CIV), 128.4, 128.4, 128.3, 128.3, 128.3, 128.0, 127.8, 127.8, 127.7, 127.7, 127.5 (C-Ar), 76.6 (C-3), 76.0 (C-2), 73.5 (CH2-Ph), 73.1 (CH2-Ph), 71.8 (CH2-Ph), 70.9 (C-5), 70.5 (CH2), 70.1 (CH2), 69.5 (CH2), 66.3 (C-4), 46.9 (NH-CH2-CH2), 46.4 (C-1), 29.2 (NH-CH2-CH2) ppm; = +8.0 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C62H80N2O11 + H]+: 1029.5840, found 1029.5833.
3.4. Representative Procedure for the Synthesis of Dimeric Iminosugars 12a,b,d
To a solution of 11a,b,d (1 equiv) in pyridine at 0 °C was added dropwise methanesulfonyl chloride (2.4 equiv). The mixture was stirred for 2.5 h at the same temperature then the reaction was quenched with water (10 mL) and extracted with ethyl acetate (3 × 5 mL). The organic phase was dried with magnesium sulfate, filtered, and concentrated under reduced pressure, and finally, the crude was purified by column chromatography on silica gel.
(12a) According to general procedure, the reaction of 11a (187 mg, 0.198 mmol) and methanesulfonyl chloride (38 µL, 0.475 mmol) in pyridine (2.5 mL) afforded after purification (eluent: EtOAc/PE gradient, 20/80 to 30/70) 12a (33 mg, 0.036 mmol, 19%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.33–7.27 (m, 34 H, Ar-H), 4.54–4.53 (m, 8 H, 4 × CH2-Ph), 4.46 (d, J = 12.2 Hz, 2 H, 2 × CHaHbPh), 4.38 (d, J = 12.2 Hz, 2 H, 2 × CHaHbPh), 4.14 (d, J = 12.2 Hz, 2 H, 2 × N-CHaHbAr), 3.93–3.91 (m, 4 H, 2 × 3-H + 2 × 4-H), 3.64–3.62 (m, 4 H, 2 × 6-H), 3.49 (d, J = 12.2 Hz, 2 H, 2 × N-CHaHbAr), 3.05 (d, J = 10.7 Hz, 2 H, 2 × 2-HaHb), 2.89 (q, J = 5.2 Hz, 2 H, 2 × 5-H), 2.58 (dd, J = 10.7, 5.1 Hz, 2 H, 2 × 2-HaHb) ppm; 13C NMR (125 MHz, CDCl3): δ 138.7 (CIV), 138.6 (CIV), 138.4 (CIV), 129.7, 128.5, 128.4, 128.2, 127.9, 127.9, 127.8, 127.7, 127.7, 127.6 (C-Ar), 86.1 (C-4), 81.7 (C-3), 73.3 (CH2-Ph), 71.5 (CH2-Ph), 71.4 (C-6), 71.0 (CH2-Ph), 68.6 (C-5), 59.2 (N-CH2-Ar), 57.1 (C-2) ppm; = +22.2 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C60H64N2O6 + H]+: 909.4843, found 909.4849.
(12b) According to general procedure, the reaction of 11b (166 mg, 0.176 mmol) and methanesulfonyl chloride (33 µL, 0.422 mmol) in pyridine (2 mL) afforded after purification (eluent: EtOAc/PE gradient, 20/80 to 30/70) 12b (65 mg, 0.071 mmol, 41%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.34–7.27 (m, 34 H, Ar-H), 4.54–4.53 (m, 8 H, 4 × CH2-Ph), 4.47 (d, J = 12.2 Hz, 2 H, 2 × CHaHbPh), 4.39 (d, J = 12.2 Hz, 2 H, 2 × CHaHbPh), 4.13 (d, J = 13.2 Hz, 2 H, 2 × N-CHaHbAr), 3.94–3.92 (m, 4 H, 2 × 3-H + 2 × 4-H), 3.65–3.61 (m, 4 H, 2 × 6-H), 3.49 (d, J = 13.2 Hz, 2 H, 2 × N-CHaHbAr), 3.06 (d, J = 10.7 Hz, 2 H, 2 × 2-HaHb), 2.88 (q, J = 5.4 Hz, 2 H, 2 × 5-H), 2.58 (dd, J = 10.7, 5.2 Hz, 2 H, 2 × 2-HaHb) ppm; 13C NMR (125 MHz, CDCl3): δ 138.6 (CIV), 138.4 (CIV), 137.5 (CIV), 129.0, 128.5, 127.9, 127.8, 127.7, 127.7, 127.6 (C-Ar), 86.0 (C-4), 81.7 (C-3), 73.4 (CH2-Ph), 71.6 (CH2-Ph), 71.4 (C-6), 71.0 (CH2-Ph), 68.6 (C-5), 59.0 (N-CH2-Ar), 57.1 (C-2) ppm; = +18.0 (c 1, CHCl3).
(12d) According to general procedure, the reaction of 11d (197 mg, 0.206 mmol) and methanesulfonyl chloride (39 µL, 0.494 mmol) in pyridine (3 mL) afforded after purification (eluent: EtOAc/PE 70/30) 12d (66 mg, 0.072 mmol, 35%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.37–7.28 (m, 30 H, Ar-H), 4.57–4.50 (m, 10 H, 4 × CH2-Ph + 2 × CHaHb-Ph), 4.46 (d, J = 12.3 Hz, 2 H, 2 × CHaHbPh), 3.94 (d, J = 5 Hz, 2 H, 2 × 3-H), 3.89 (d, J = 3.9 Hz, 2 H, 2 × 4-H), 3.64–3.53 (m, 12 H, 2 × 6-H + 4 × CH2), 3.27 (d, J = 10.6 Hz, 2 H, 2 × 2-HaHb), 3.11 (dt, J = 12.7, 6.3 Hz, 2 H, 2 × N-CHaHb-CH2), 2.82 (q, J = 5.0 Hz, 2 H, 2 × 5-H), 2.71 (dd, J = 10.6, 5.1 Hz, 2 H, 2 × 2-HaHb), 2.65 (dt, J = 12.7, 6.3 Hz, 2 H, 2 × N-CHaHb-CH2) ppm; 13C NMR (125 MHz, CDCl3): δ 138.5 (CIV), 138.4 (CIV), 138.3 (CIV), 128.4, 127.9, 127.9, 127.9, 127.8, 127.7, 127.7, 127.7, 127.6 (C-Ar), 85.3 (C-4), 81.8 (C-3), 73.3 (CH2-Ph), 71.4 (CH2-Ph), 71.1 (CH2-Ph), 71.0 (C-6), 70.4 (CH2), 70.3 (CH2), 69.5 (C-5), 58.2 (C-2), 54.5 (N-CH2-CH2) ppm; = −14.1 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C58H69N2O8 + H]+: 921.5054, found 921.5046.
3.5. Representative Procedure for the Synthesis of Dimeric Iminosugars 12c,e
Step 1, protection of amine functions: To a solution of 11c,e (1 equiv) in anhydrous methanol (10 mL) at room temperature were successively added triethylamine (4 equiv), 4-(dimethylamino)pyridine (0.2 equiv) and di-tert-butyl dicarbonate (6 equiv). The resulting mixture was stirred until the total consumption of the starting material (typically 12 h for 11c and 1h for 11e) then the reaction was quenched with water (10 mL) and extracted with dichloromethane (3 × 5 mL). The organic phase was dried with magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting product was used in the next step without further purification.
Step 2, O-mesylation: To the crude obtained in step 1 was added dropwise at 0 °C triethylamine (20 equiv) then methanesulfonyl chloride (16 equiv). The mixture was warmed to room temperature and was stirred for 40 min. The reaction was quenched with water (10 mL) and extracted with dichloromethane (3 × 5 mL). The organic phase was dried with MgSO4, filtered, and concentrated under reduced pressure. The resulting product was used in the last step without further purification.
Step 3, cyclization: To the crude obtained in step 2 and dissolved in ethanol (20 mL) was added hydrochloric acid 37% in water (18 mL). Then, the solution was stirred for 12 h at 60 °C (11c) or 29 h at room temperature (11e). Ethanol was removed under reduced pressure, the crude was diluted with dichloromethane (40 mL) and washed with a saturated aqueous solution of sodium hydrogen carbonate until the acid was neutralized. The organic phase was dried with MgSO4, filtered, and concentrated under reduced pressure, and finally, the crude was purified by column chromatography on silica gel.
(12c) According to general procedure, the reaction of 11c (553 mg, 0.580 mmol) afforded after the three steps and purification (eluent: EtOAc/PE 20/80) 12c (302 mg, 0.329 mmol, 56%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.37–7.28 (m, 30 H, Ar-H), 4.59–4.51 (m, 10 H, 4 × CH2-Ph + 2 × CHaHb-Ph), 4.47 (d, J = 12.3 Hz, 2 H, 2 × CHaHbPh), 3.94 (d, J = 5 Hz, 2 H, 2 × 3-H), 3.92 (d, J = 3.6 Hz, 2 H, 2 × 4-H), 3.62 (dd, J = 9.5, 4.8 Hz, 2 H, 2 × 6-HaHb), 3.55 (dd, J = 9.5, 6.9 Hz, 2 H, 2 × 6-HaHb), 3.25 (d, J = 10.3 Hz, 2 H, 2 × 2-HaHb), 2.88–2.82 (m, 2 H, 2 × N-CHaHb-CH2), 2.75–2.72 (m, 2 H, 2 × 5-H), 2.58 (dd, J = 10.4, 5.0 Hz, 2 H, 2 × 2-HaHb), 2.37–2.31 (m, 2 H, 2 × N-CHaHb-CH2), 1.54–1.48 (m, 4H, 2 × N-CH2-CH2), 1.31–1.28 (m, 8H, 4 × CH2) ppm; 13C NMR (125 MHz, CDCl3): δ 138.6 (CIV), 138.4 (CIV), 138.4 (CIV), 128.5, 128.5, 128.4, 128.4, 128.4, 127.9, 127.9, 127.9, 127.7, 127.6 (C-Ar), 85.6 (C-4), 81.7 (C-3), 73.3 (CH2-Ph), 71.4 (CH2-Ph), 71.1 (C-6), 71.0 (CH2-Ph), 69.5 (C-5), 57.4 (C-2), 55.8 (N-CH2-CH2), 29.7 (CH2), 28.3 (N-CH2-CH2), 27.7 (CH2) ppm; = −26.6 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C60H72N2O6 + H]+: 917.5469, found 917.5472.
(12e) According to general procedure, the reaction of 11e (1.7 g, 1.672 mmol) afforded after the three steps and purification (eluent: EtOAc/PE gradient, 50/50 to 100/00) 12e (349 mg, 0.352 mmol, 21%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.37–7.27 (m, 30 H, Ar-H), 4.57–4.49 (m, 10 H, 4 × CH2-Ph + 2 × CHaHb-Ph), 4.45 (d, J = 12.3 Hz, 2 H, 2 × CHaHbPh), 3.94 (d, J = 5.1 Hz, 2 H, 2 × 3-H), 3.91 (d, J = 3.9 Hz, 2 H, 2 × 4-H), 3.65–3.47 (m, 16 H, 6 × CH2 + 2 × 6-H), 3.23 (d, J = 10.3 Hz, 2 H, 2 × 2-HaHb), 2.95 (dt, J = 11.9, 8.0 Hz, 2 H, 2 × N-CHaHb-CH2), 2.76–2.73 (m, 2 H, 2 × 5-H), 2.59 (dd, J = 10.4, 5.1 Hz, 2 H, 2 × 2-HaHb), 2.43 (dt, J = 12.4, 6.7 Hz, 2 H, 2 × N-CHaHb-CH2), 1.80 (dq, J = 14.4, 7.0 Hz, 4 H, 2 × N-CH2-CH2) ppm; 13C NMR (125 MHz, CDCl3): δ 138.6 (CIV), 138.4 (CIV), 138.4 (CIV), 128.5, 128.5, 128.4, 128.0, 127.9, 127.9, 127.8, 127.8, 127.8, 127.7, 127.7, 127.6 (C-Ar), 85.6 (C-4), 81.7 (C-3), 73.3 (CH2-Ph), 71.4 (CH2-Ph), 71.1 (C-6 + CH2-Ph), 70.7 (CH2), 70.3 (CH2), 69.8 (CH2), 69.5 (C-5), 57.3 (C-2), 52.4 (N-CH2-CH2), 28.4 (N-CH2-CH2) ppm; = −29.3 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C62H76N2O9 + H]+: 993.5629, found 993.5627.
3.6. Representative Procedure for the Synthesis of Deprotected Bis-Iminosugars 13a–e
To a solution of dimeric iminosugars 12a–e (1 equiv) in anhydrous dichloromethane at 0 °C and under argon atmosphere was added dropwise boron trichloride solution 1 M in dichloromethane (10 equiv). The mixture was stirred for 2 h 30 at the same temperature and then was quenched with methanol (3 mL). The solvent was removed under reduced pressure and the residue was washed with diethyl ether (3 × 1 mL) and then chloroform (1.5 mL). Finally, the crude was purified by column chromatography on silica gel (13a–c,e) or passed through an ion exchange resin (DOWEX 50WX8 (NH4+) column (Sigma-Aldrich: St. Louis, MO, USA), elution with a solution of 0.2% ammonium bicarbonate) and lyophilized (13d).
(13a) According to general procedure, the reaction of 12a (57 mg, 0.063 mmol) and boron trichloride solution 1 M in dichloromethane (630 µL, 0.630 mmol) in dichloromethane (0.5 mL) afforded after purification (eluent: CHCl3/MeOH/NH4OH 0.8 M 60/40/10) 13a (8.3 mg, 0.023 mmol, 36%) as a yellow oil. 1H NMR (500 MHz, D2O): δ 7.55–7.54 (m, 4 H, Ar-H), 4.38 (d, J = 12.7 Hz, 2 H, 2 × N-CHaHbAr), 4.21–4.20 (m, 2 H, 3-H), 4.10–4.05 (m, 4 H, N-CHaHbAr + 4-H), 3.83 (dd, J = 12.1, 5.4 Hz, 2 H, 2 × 6-HaHb), 3.77 (dd, J = 12.1, 6.1 Hz, 2 H, 2 × 6-HaHb), 3.30–3.15 (m, 6 H, 2 × 2-H + 2 × 5-H) ppm; 13C NMR (125 MHz, D2O): δ 133.3 (CIV), 132.6, 131.3, 129.5 (C-Ar), 77.6 (C-4), 74.3 (C-3), 72.8 (C-5), 59.6 (C-6), 58.9 (N-CH2-Ar), 58.3 (C-2) ppm; = +3.5 (c 1, MeOH). HRMS (ESI) m/z calcd for [C18H28N2O6 + H]+: 369.2026, found 369.2025.
(13b) According to general procedure, the reaction of 12b (115 mg, 0.127 mmol) and boron trichloride solution 1 M in dichloromethane (1.27 mL, 1.27 mmol) in dichloromethane (2 mL) afforded after purification (eluent: CHCl3/MeOH gradient, 75/25 to 60/40) 13b (18 mg, 0.049 mmol, 39%) as a yellow oil. 1H NMR (500 MHz, D2O): δ 7.61 (s, 4 H, Ar-H), 4.57 (d, J = 12.8 Hz, 2 H, 2 × N-CHaHbAr), 4.34 (d, J = 12.8 Hz, 2 H, 2 × N-CHaHbAr), 4.29–4.28 (m, 2 H, 3-H), 4.11–4.10 (m, 2 H, 4-H), 3.89 (dd, J = 12.3, 5.2 Hz, 2 H, 2 × 6-HaHb), 3.83 (dd, J = 12.3, 6.9 Hz, 2 H, 2 × 6-HaHb), 3.54–3.49 (m, 4 H, 2 × 2-HaHb + 2 × 5-H), 3.37–3.34 (m, 2 H, 2 × 2-HaHb) ppm; 13C NMR (125 MHz, D2O): δ 132.2 (CIV), 131.6 (C-Ar), 76.7 (C-4), 73.8 (C-3), 73.7 (C-5), 59.0 (C-6), 58.9 (N-CH2-Ar), 58.5 (C-2) ppm; = –1.4 (c 1, MeOH). HRMS (ESI) m/z calcd for [C18H28N2O6 + H]+: 369.2026, found 369.2023.
(13c) According to general procedure, the reaction of 12c (117 mg, 0.127 mmol) and boron trichloride solution 1 M in dichloromethane (1.27 mL, 1.27 mmol) in dichloromethane (2 mL) afforded after purification (eluent: CHCl3/MeOH gradient, 70/30 to 50/50) 13c (42 mg, 0.110 mmol, 87%) as a yellow oil. 1H NMR (500 MHz, D2O): δ 4.36–4.35 (m, 2 H, 3-H), 4.12–4.11 (m, 2 H, 4-H), 4.01 (dd, J = 12.5, 4.9 Hz, 2 H, 2 × 6-HaHb), 3.94 (dd, J = 12.5, 7.6 Hz, 2 H, 2 × 6-HaHb), 3.68 (d, J = 12.4 Hz, 2 H, 2 × 2-HaHb), 3.55–3.44 (m, 6 H, 2 × 2-HaHb + 2 × 5-H + 2 × N-CHaHb-CH2), 3.24–3.17 (m, 2 H, 2 × N-CHaHb-CH2), 1.78–1.72 (m, 4 H, 2 × N-CH2-CH2), 1.40–1.37 (m, 8 H, 4 × CH2) ppm; 13C NMR (125 MHz, D2O): δ 78.4 (C-4), 77.7 (C-5), 76.3 (C-3), 61.1 (C-2), 61.0 (C-6), 59.4 (N-CH2-CH2), 30.4 (CH2), 28.1 (CH2), 27.1 (N-CH2-CH2) ppm; = −18.9 (c 0.45, H2O). HRMS (ESI) m/z calcd for [C18H36N2O6 + H]+: 377.2652, found 377.2650.
(13d) According to general procedure, the reaction of 12d (90 mg, 0.098 mmol) and boron trichloride solution 1 M in dichloromethane (980 µL, 0.98 mmol) in dichloromethane (1 mL) afforded after purification 13d (13 mg, 0.034 mmol, 35%) as a yellow oil. 1H NMR (500 MHz, D2O): δ 4.11 (dt, J = 5.4, 2.5 Hz, 2 H, 2 × 3-H), 3.92 (dd, J = 5.0, 2.8 Hz, 2 H, 2 × 4-H), 3.72–3.63 (m, 12 H, 2 × 6-H + 4 × CH2), 3.11–3.04 (m, 4 H, 2 × 2-HaHb + 2 × N-CHaHb-CH2), 2.83–2.79 (m, 2 H, 2 × 2-HaHb), 2.64–2.61 (m, 2 H, 2 × N-CHaHb-CH2), 2.58 (q, J = 5.0 Hz, 2 H, 2 × 5-H) ppm; 13C NMR (125 MHz, D2O): δ 78.8 (C-4), 75.4 (C-3), 72.0 (C-5), 69.4 (CH2), 68.6 (CH2), 60.9 (C-6), 58.8 (C-2), 53.8 (N-CH2-CH2) ppm; = −14.6 (c 1, MeOH). HRMS (ESI) m/z calcd for [C16H32N2O8 + H]+: 381.2237, found 381.2236.
(13e) According to general procedure, the reaction of 12e (277 mg, 0.279 mmol) and boron trichloride solution 1 M in dichloromethane (2.79 mL, 2.79 mmol) in dichloromethane (3 mL) afforded after purification (eluent: EtOAc/MeOH/NH4OH 0.8 M 50/50/5) 13e (78 mg, 0.173 mmol, 62%) as a yellow oil. 1H NMR (500 MHz, D2O): δ 4.33–4.32 (m, 2 H, 2 × 3-H), 4.10–4.09 (m, 2 H, 2 × 4-H), 3.98 (dd, J = 12.4, 4.9 Hz, 2 H, 2 × 6-HaHb), 3.91 (dd, J = 12.4, 6.8 Hz, 2 H, 2 × 6-HaHb), 3.70–3.61 (m, 14 H, 2 × 2-HaHb + 6 × CH2), 3.52–3.56 (m, 2 H, 2 × N-CHaHb-CH2), 3.41 (dd, J = 12.1, 4.4 Hz, 2 H, 2 × 2-HaHb), 3.36–3.35 (m, 2 H, 2 × 5-H), 3.21–3.17 (m, 2 H, 2 × N-CHaHb-CH2), 2.10–1.95 (m, 4 H, 2 × N-CH2-CH2) ppm; 13C NMR (125 MHz, D2O): δ 76.5 (C-4), 74.7 (C-5), 74.0 (C-3), 69.5 (CH2), 69.5 (CH2), 68.4 (CH2), 58.7 (C-6), 58.6 (C-2), 54.2 (N-CH2-CH2), 25.1 (N-CH2-CH2) ppm; = –5.7 (c 0.37, H2O). HRMS (ESI) m/z calcd for [C20H40N2O9 + H]+: 453.2812, found 453.2814.
3.7. Procedure for Allylation of Bis-Glycosylamine 10d
To a solution of dry lithium chloride (650 mg, 15.24 mmol, 6 equiv) in anhydrous tetrahydrofuran (10 mL) at −78 °C and under argon atmosphere was added dropwise a solution of allyl magnesium chloride 2 M in tetrahydrofuran (7.62 mL, 15.24 mmol, 6 equiv). The mixture was stirred for 20 min then a solution of the bis-glycosylamine 10d (2.42 g, 2.54 mmol, 1 equiv) in anhydrous tetrahydrofuran (20 mL) was added dropwise. The mixture was stirred at −78 °C for 30 min then was warmed at room temperature and stirred until total consumption of starting material (typically 2 h). The reaction was quenched with a saturated aqueous solution of ammonium chloride and extracted with ethyl acetate (3 × 30 mL). The organic phase was dried with magnesium sulfate, filtered, and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel (eluent: EtOAC/MeOH 97:3) to afford a mixture of 14d(R,R) and 14d(R,S) (85/15) (2.24 g, 2.16 mmol, 85%) as a yellow oil.
(14d) 1H NMR (500 MHz, CDCl3): δ 7.37–7.22 (m, 30 H, Ar-H), 5.80–5.72 (m, 1.7 H, 2 × 2-Hmaj), 5.60–5.51 (m, 0.3 H, 2 × 2-Hmin), 5.14–5.09 (m, 3.4 H, 2 × 1-Hmaj), 5.02 (d, J = 10.2 Hz, 0.3 H, 2 × 1-HaHbmin), 4.90 (d, J = 17 Hz, 0.3 H, 2 × 1-HaHbmin), 4.68–4.32 (m, 12 H, 6 × CH2-Phmaj + 6 × CH2-Phmin), 4.16–4.13 (m, 0.3 H, 2 × 6-Hmin), 4.08 (d, J = 6.7 Hz, 1.7 H, 2 × 6-Hmaj), 3.83–3.81 (m, 2 H, 2 × 7-Hmaj +2 × 7-Hmin), 3.65–3.39 (m, 14 H, 2 × 5-Hmaj + 2 × 5-Hmin + 2 × 8-Hmaj + 2 × 8-Hmin + 4 × CH2maj + 4 × CH2min), 3.06–2.93 (m, 4 H, 2 × 4-Hmaj + 2 × 4-Hmin + 2 × NH-CHaHbmaj + 2 × NH-CHaHbmin), 2.81–2.77 (m, 1.7 H, 2 × NH-CHaHbmaj), 2.56–2.45 (m, 4 H, 2 × 3-Hmaj + 2 × 3-HaHbmin + 2 × NH-CHaHbmin), 2.15–2.07 (m, 0.3 H, 2 × 3-HaHbmin) ppm; 13C NMR (125 MHz, D2O): δ 138.7 (CIV), 138.6 (CIV), 138.6 (CIV), 138.5 (CIV), 138.4 (CIV), 138.3 (CIV), 138.3 (CIV), 138.3 (CIV), 138.1 (CIV), 138.0 (CIV), 137.9 (CIV), 135.7 (C-2min), 135.3 (C-2maj), 128.8, 128.7, 128.5, 128.4, 128.4, 128.3, 128.0, 127.9, 127.9, 127.8, 127.6, 127.6 (C-Ar), 118.2 (C-1maj), 117.7 (C-1min), 78.4 (C-7maj), 77.1 (C-5min), 75.1 (C-5maj + C-7min), 73.9 (CH2-Phmaj), 73.6 (CH2-Phmin), 73.2 (CH2-Phmin), 73.2 (CH2-Phmaj), 73.0 (CH2-Phmaj), 72.8 (CH2-Phmin), 71.1 (C-8maj), 70.8 (C-8min), 70.3 (CH2), 70.2 (CH2), 67.2 (C-6maj), 66.3 (C-6min), 58.9 (C-4min), 54.5 (C-4maj), 46.8 (NH-CH2maj), 45.6 (NH-CH2min), 34.6 (C-3min), 34.2 (C-3maj) ppm; HRMS (ESI) m/z calcd for [C64H80N2O10 + H]+: 1037.5891, found 1037.5898.
3.8. Procedure for the Synthesis of Bis-Allylpyrrolidines 15d(R,R) and 15d(R,S)
To a solution of the bis-glycosylamines 14d(R,R) and 14d(R,S) (2.24 g, 2.16 mmol, 1 equiv) in anhydrous tetrahydrofuran (10 mL) under argon atmosphere was added pyridine (10 mL). The mixture was cooled to −78 °C then methanesulfonyl chloride (0.84 mL, 10.81 mmol, 5 equiv) was added dropwise. The mixture was stirred for 1 h at the same temperature, and then was warmed to 0 °C and stirred for another 2 h. The reaction was quenched with water (10 mL) and extracted with ethyl acetate (4 × 40 mL). The organic phase was dried with magnesium sulfate, filtered, and concentrated under reduced pressure, and finally, the crude was purified by column chromatography on silica gel (eluent: PE/EtOAc 70/30) to afford 15d(R,R) (731 mg, 0.731 mmol), 15d(R,S) (100 mg, 0.1 mmol) and a mixture of the two diastereomers (869 mg, 0.869 mmol, 15d(R,R)/15d(R,S): 83/17)). Global yield 79% yellow oils.
(15d(R,R)) 1H NMR (500 MHz, CDCl3): δ 7.37–7.28 (m, 30 H, Ar-H), 5.83–5.74 (m, 2 H, 2 × 2a-H), 5.08–5.05 (m, 4 H, 2 × 3a-H), 4.57–4.41 (m, 12 H, 6 × CH2-Ph), 3.90 (s, 2 H, 2 × 4-H), 3.80 (s, 2 H, 2 × 3-H), 3.70–3.52 (m, 12 H, 2 × 6-H + 4 × CH2), 3.29–3.26 (m, 2 H, 2 × 5-H), 3.19–3.17 (m, 2 H, 2 × 2-H), 3.01–2.91 (m, 4 H, 2 × N-CH2), 2.54–2.49 (m, 2 H, 2 × 1a-HaHb), 2.23–2.17 (m, 2 H, 2 × 1a-HaHb) ppm; 13C NMR (125 MHz, D2O): δ 138.5 (CIV), 135.6 (C-2a), 128.4, 128.4, 128.4, 128.0, 127.9, 127.9, 127.8, 127.6, (C-Ar), 117.2 (C-3a), 85.7 (C-3), 85.6 (C-4), 73.4 (CH2-Ph), 71.4 (CH2-Ph), 71.3 (CH2-Ph), 70.6 (CH2), 70.5 (CH2), 69.5 (C-6), 66.1 (C-5), 65.7 (C-2), 46.7 (N-CH2), 32.1 (C-1a) ppm; = −8.4 (c 1, MeOH). HRMS (ESI) m/z calcd for [C64H76N2O8 + H]+: 1001.5680, found 1001.5682.
(15d(R,S)) 1H NMR (500 MHz, CDCl3): δ 7.38–7.25 (m, 30 H, Ar-H), 5.82–5.68 (m, 2 H, 2a-H + 2a′-H), 5.10–5.04 (m, 3 H, 3a-H + 3a′-HaHb), 4.99 (dd, J = 10.2, 1.5 Hz, 1 H, 3a′-HaHb), 4.62–4.41 (m, 11 H, 5 × CH2-Ph + CHaHb-Ph), 4.32 (d, J = 11.7 Hz, 1 H, CHaHb-Ph), 3.89–3.88 (m, 2 H, 4-H + 4′-H), 3.79–3.77 (m, 2 H, 3-H + 3′-H), 3.69–3.51 (m, 11 H, 6-H + 6′-HaHb + 4 × CH2), 3.43 (t, J = 9.3 Hz, 1 H, 6′-HaHb), 3.28–3.25 (m, 1 H, 5-H), 3.18–3.16 (m, 1 H, 2-H), 3.14–3.10 (m, 1 H, 5′-H), 3.09–3.05 (m, 1 H, 2′-H), 3.04–2.87 (m, 4 H, 2 × N-CH2), 2.53–2.43 (m, 2 H, 1a-HaHb + 1a′-HaHb), 2.34–2.29 (m, 1 H, 1a′-HaHb), 2.23–2.16 (m, 1 H, 1a-HaHb) ppm; 13C NMR (125 MHz, D2O): δ 138.6 (CIV), 138.5 (CIV), 138.65 (CIV), 138.2 (CIV), 136.1 (C-2a′), 135.6 (C-2a), 128.5, 128.5, 128.4, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2, 128.2, 128.2, 128.0, 128.0, 127.9, 127.9, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6, (C-Ar), 117.2 (C-3a), 116.5 (C-3a′), 85.7 (C-3), 85.6 (C-4), 82.3 (C-4′), 82.3 (C-3′), 73.4 (CH2-Ph), 73.2 (CH2′-Ph), 72.4 (C-6′), 71.7 (CH2′-Ph), 71.4 (CH2-Ph), 71.3 (CH2-Ph), 70.7 (CH2′-Ph), 70.5 (CH2), 70.2 (CH2), 69.7 (C-5′), 69.5 (C-6), 66.9 (C-2′), 66.1 (C-5), 65.7 (C-2), 53.1 (N-CH2′), 46.7 (N-CH2), 32.8 (C-1a′), 32.1 (C-1a) ppm; = +1.9 (c 1, MeOH). HRMS (ESI) m/z calcd for [C64H76N2O8 + H]+: 1001.5680, found 1001.5682.
3.9. Procedure for the Synthesis of Deprotected Bis-Allylpyrrolidine 16d(R,R)
According to general procedure described in 3.6, the reaction of 15d(R,R) (27 mg, 0.027 mmol, 1 equiv) and boron trichloride solution (270 µL, 0.27 mmol) in anhydrous dichloromethane (3 mL) afforded after purification (eluent: AcOEt/MeOH/NH4OH 0.8 M 40/60/5) 16d(R,R) (7 mg, 0.015 mmol, 56%) as a yellow oil.
(16d(R,R)) 1H NMR (600 MHz, D2O): δ 5.91–5.84 (m, 2 H, 2 × 2a-H), 5.19 (dd, J = 19.9, 13.8 Hz, 4 H, 2 × 3a-H), 3.96–3.94 (s, 2 H, 2 × 4-H), 3.91–3.89 (s, 2 H, 2 × 3-H), 3.80–3.74 (m, 4 H, 2 × 6-H), 3.70–3.63 (m, 8 H, 4 × CH2), 3.05 (dt, J = 8.7, 4.0 Hz, 2 H, 2 × 2-H), 2.99–2.91 (m, 6 H, 2 × 5-H + 2 × N-CH2), 2.43 (dt, J = 14.0, 4.9 Hz, 2 H, 2 × 1a-HaHb), 2.12 (dt, J = 14.0, 8.8 Hz, 2 × 1a-HaHb) ppm; 13C NMR (151 MHz, D2O): δ 135.1 (C-2a), 117.6 (C-3a), 79.7 (C-3), 79.1 (C-4), 69.6 (CH2), 68.7 (CH2), 68.2 (C-5), 66.2 (C-2), 59.7 (C-6), 45.7 (N-CH2), 31.4 (C-1a) ppm; = −17.6 (c 0.10, H2O). HRMS (ESI) m/z calcd for [C22H40N2O8 + Na]+: 483.2682, found 483.2691.
3.10. Procedure for the Synthesis of 19d(R,R) and 19d(R,S)
To a solution of 14d(R,R) and 14d(R,S) (85/15) (150 mg, 0,145 mmol, 1 equiv) in anhydrous dichloromethane (10 mL) at 0 °C and under argon atmosphere was added dropwise boron trichloride solution 1 M in dichloromethane (1.45 mL, 1.45 mmol, 10 equiv). The mixture was stirred for 2 h 30 at the same temperature and then was quenched with methanol (2 mL). The solvent was removed under reduced pressure and the residue was washed with chloroform (3 × 1 mL) then dichloromethane (3 × 1 mL) to afford a mixture of 19d(R,R) and 19(R,S) (85/15) (55 mg, 0.112 mmol, 77%) as a yellow oil.
(19d(R,R)) 1H NMR (500 MHz, MeOD): δ 5.92–5.77 (m, 2 H, 2 × 2-H), 5.29 (d, J = 17.1 Hz, 2 H, 2 × 1-HaHb), 5.19 (d, J = 10.2 Hz, 2 H, 2 × 1-HaHb), 3.98 (dd, J = 5.3, 3.1 Hz, 2 H, 2 × 5-H), 3.87 (t, J = 3.3 Hz, 2 H, 2 × 6-H), 3.82–3.55 (m, 14 H, 2 × 7-H + 2 × 8-H + 4 × CH2), 3.52 (q, J = 6.5 Hz, 2 H, 2 × 4-H), 3.42–3.34 (m, 2 H, 2 × NH-CHaHb), 3.30–3.25 (m, 2 H, 2 × NH-CHaHb), 2.68–2.54 (m, 4 H, 2 × 3-H) ppm; 13C NMR (125 MHz, MeOD): δ 133.8 (C-2), 120.3 (C-1), 72.9 (C-7), 71.8 (C-6), 71.4 (CH2), 70.1 (C-5), 66.8 (CH2), 64.0 (C-8), 62.8 (C-4), 47.7 (NH-CH2), 33.3 (C-3) ppm; HRMS (ESI) m/z calcd for [C22H44N2O10 + H]+: 497.3074, found 497.3070.
3.11. Procedure for Ethynylation of Bis-Glycosylamine 10d
To a solution of trimethylsilylacetylene (1.65 mL, 11.6 mmol, 8 equiv) in anhydrous tetrahydrofuran (10 mL) at 0 °C and under argon atmosphere was added dropwise ethyl magnesium bromide 3 M in diethylether (3.9 mL, 11.6 mmol, 8 equiv). The mixture was stirred for 30 min at the same temperature then a solution of the bis-glycosylamine 10d (1.38 g, 1.45 mmol, 1 equiv) in anhydrous tetrahydrofuran (20 mL) was added dropwise. The mixture was stirred at 0 °C for 30 min then was finally warmed at room temperature and stirred for 16 h. The reaction was quenched with a saturated aqueous solution of ammonium chloride and extracted with ethyl acetate (2 × 40 mL). The organic phase was dried with magnesium sulfate, filtered, and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel (eluent: PE/EtOAc 70/30) to afford 20d (605 mg, 0.53 mmol, 37%) as a mixture of two diastereoisomers (95:5) and a yellow oil.
(20d) 1H NMR (500 MHz, CDCl3) major diastereomer: δ 7.36–7.27 (m, 28 H, Ar-H), 7.18–7.16 (m, 2 H, Ar-H), 4.73 (d, J = 11.8 Hz, 2 H, 2 × CHaHb-Ph), 4.64 (d, J = 11.8 Hz, 2 H, 2 × CHaHb-Ph), 4.51 (d, J = 12.0 Hz, 2 H, 2 × CHaHb-Ph), 4.46 (d, J = 12.0 Hz, 2 H, 2 × CHaHb-Ph), 4.42 (d, J = 12.1 Hz, 2 H, 2 × CHaHb-Ph), 4.38 (d, J = 12.1 Hz, 2 H, 2 × CHaHb-Ph), 4.09 (dd, J = 8.1, 5.8 Hz, 2 H, 2 × 6-H), 3.81–3.79 (m, 4 H, 2 × 3-H + 2 × 4-H), 3.63–3.44 (m, 14 H, 2 × 5-H + 2 × 7-H + 4 × CH2), 3.22–3.17 (m, 2 H, 2 × NH-CHaHb), 2.62 (dt, J = 12.1, 4.2 Hz, 2 H, 2 × NH-CHaHb), 0.2 (s, 18 H, 2 × Si(CH3)3) ppm; 13C NMR (125 MHz, CDCl3) major diastereomer: δ 138.7 (CIV), 138.3 (CIV), 138.0 (CIV), 128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 128.2, 128.0, 127.9, 127.9, 127.8, 127.8, 127.7, 127.5 (C-Ar), 106.5 (CIV, C≡C-Si), 89.0 (CIV, C≡C-Si), 79.7 (C-4), 76.4 (C-5), 74.2 (CH2-Ph), 73.7 (CH2-Ph), 73.1 (CH2-Ph), 70.6 (C-7), 70.2 (CH2), 70.0 (CH2), 65.9 (C-6), 48.3 (C-3), 46.4 (NH-CH2), 0.1 (Si(CH3)3) ppm; HRMS (ESI) m/z calcd for [C68H88N2O10Si2 + H]+: 1149.6056, found 1149.6057.
3.12. Procedure for the Synthesis of Bis-Ethynylpyrrolidine 21d
Step 1, O-mesylation and cyclization: according to the procedure described in 3.8, the reaction of 20d (550 mg, 0.48 mmol) and methanesulfonyl chloride (0.19 mL, 2.4 mmol) in pyridine (3 mL) and THF (3 mL) afforded the cyclized intermediate (235 mg, 0.21 mmol, 44%) as a yellow oil after purification (eluent: PE/EtOAc 90/10).
Step 2, TMS removing: To a solution of the pyrrolidine obtained in step 1 (200 mg, 0.18 mmol, 1 equiv) in anhydrous tetrahydrofuran (10 mL) was added a solution of tetrabutylammonium fluoride 1 M in tetrahydrofuran (0.45 mL, 0.447 mmol, 2.5 equiv). The mixture was stirred for 3 h at room temperature and then was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (eluent: EP/EtOAc 70:30) to afford 21d (140 mg, 0.144 mmol, 69%) as a colorless oil.
(21d) 1H NMR (500 MHz, CDCl3): δ 7.39–7.26 (m, 30 H, Ar-H), 4.64–4.58 (m, 6 H, 2 × CH2Ph + 2 × CHaHb-Ph), 4.54–4.45 (m, 6 H, 2 × CH2Ph + 2 × CHaHb-Ph), 3.90–3.89 (m, 4 H, 2 × 2-H + 2 × 3-H), 3.87–3.86 (m, 2 H, 2 × 4-H), 3.74–3.67 (m, 4 H, 2 × CH2), 3.63–3.53 (m, 8 H, 2 × CH2 + 6-H), 3.17–3.03 (m, 6 H, 2 × N-CH2 + 2 × 5-H), 2.39 (s, 2 H, 2 × C≡CH) ppm; 13C NMR (125 MHz, CDCl3): δ 138.5 (CIV), 138.1 (CIV), 138.0 (CIV), 128.4, 128.3, 128.3, 128.3, 128.0, 127.8, 127.8, 127.7, 127.6, 127.6, 127.6, 127.5 (C-Ar), 83.6 (C-4), 82.3 (C-3), 81.2 (CIV, C≡CH), 73.7 (C≡CH), 73.1 (CH2-Ph), 72.1 (CH2-Ph), 71.5 (C-6), 70.9 (CH2-Ph), 70.3 (CH2), 69.5 (CH2), 67.6 (C-5), 58.5 (C-2), 52.1 (N-CH2) ppm; = −13.6 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C62H68N2O8 + H]+: 969.5054, found 969.5056.
3.13. Procedure for the Synthesis of Glycophane 22d
To a solution of the bis-ethynylpyrrolidine 21d (80 mg, 0.083 mmol, 1 equiv) in a mixture of ethanol (20 mL) and chloroform (5 mL) were successively added pyridine (340 μL, 0.42 mmol, 5 equiv), triethylamine (350 μL, 0.25 mmol, 3 equiv), copper(II) acetate monohydrate (17 mg, 0.083 mmol, 1 equiv) and nickel chloride (11 mg, 0.083 mmol, 1 equiv). The mixture was heated to 60 °C and stirred for 16 h. The solvents were removed under reduced pressure and the crude was purified by column chromatography on silica gel (eluent: EP/EtOAc 70/30) to afford 22d (67 mg, 0.068 mmol, 82%) as a colorless oil.
(22d) 1H NMR (500 MHz, CDCl3): δ 7.37–7.27 (m, 26 H, Ar-H), 7.21–7.19 (m, 4 H, Ar-H), 4.65 (d, J = 12.5 Hz, 2 H, 2 × CHaHb-Ph), 4.57 (d, J = 12.5 Hz, 2 H, 2 × CHaHb-Ph), 4.54 (d, J = 12.0 Hz, 2 H, 2 × CHaHb-Ph), 4.47 (d, J = 12.0 Hz, 2 H, 2 × CHaHb-Ph), 4.41 (d, J = 12.1 Hz, 2 H, 2 × CHaHb-Ph), 4.38 (d, J = 12.1 Hz, 2 H, 2 × CHaHb-Ph), 3.85–3.82 (m, 4 H, 2 × 2-H + 2 × 3-H), 3.74 (dd, J = 3.3, 1.2 Hz, 2 H, 2 × 4-H), 3.60–3.48 (m, 6 H, 2 × CH2 + 2 × CHaHb), 3.60–3.50 (m, 6 H, 2 × 6-H + 2 × CHaHb), 3.09–3.04 (m, 2 H, 2 × N-CHaHb), 2.91–2.83 (m, 4 H, 2 × 5-H + 2 × N-CHaHb) ppm; 13C NMR (125 MHz, CDCl3): δ 138.4 (CIV), 138.0 (CIV), 138.0 (CIV), 128.5, 128.5, 128.5, 128.3, 127.9, 127.8, 127.8 (C-Ar), 83.9 (C-4), 82.5 (C-3), 78.0 (CIV, C≡C-C≡C), 73.3 (CH2-Ph), 72.3 (CH2-Ph), 71.7 (C-6), 71.4 (CH2-Ph), 71.0 (CIV, C≡C-C≡C), 70.9 (CH2), 70.0 (C-5), 69.7 (CH2), 60.6 (C-2), 54.5 (N-CH2) ppm; = −84.2 (c 1, CHCl3). HRMS (ESI) m/z calcd for [C62H67N2O8 + H]+: 967.4897, found 967.4896.
3.14. Biological Assays Towards Glycosidases
All the enzymes were purchased from Sigma Chemical Co. In a typical experiment, the glycosidase (0.013 U/mL) was pre-incubated at 33 °C for 5 min in the presence of the inhibitor in 50 mM acetate buffer (pH 5.6, except for rice α-glucosidase pH 5.1 and yeast α-glucosidase pH 6.2). The reaction was started by the addition of the appropriate substrate (p-nitrophenyl glycoside, 1 mM concentration) to a final volume of 250 µL. The reaction was stopped after 10–15 min (depending on the enzyme) by the addition of 350 µL of 0.4 M Na2CO3. The released p-nitrophenolate was quantified spectrophotometrically at 415 nm with a microplate reader (300 µL of the reaction mixture per well). In cases where inhibition was greater than 95%, IC50 values were determined further after assaying decreasing concentrations of inhibitor. All the assays were performed in duplicate (less than 10% variability in each case).