4. Material and Methods
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a Bruker® Ultrashield 300 NMR spectrometer. 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Bruker® Avance 400 MHz NMR spectrometer. 1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on a Bruker® Avance 500 MHz NMR spectrometer. All spectra were recorded at room temperature (~20 °C) in Sigma Aldrich® deuterated solvents. Chemical shifts (δ) were expressed in parts per million (ppm) relative to the reference peak. Coupling constants (J) were expressed in Hertz (Hz). Splitting patterns in 1H NMR spectra were designated as s (singlet), br s (broad singlet), d (doublet), br d (broad doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), ddd (doublet of doublet of doublets), and m (multiplet). Optical rotations were measured on a Jasco P-2000 polarimeter at 22.5 nd using a sodium lamp and wavelength of 589 nm at 22.5 °C. HPLC purifications were designed in a Shimadzu® SCL-10A HPLC system, Diode Array Detector Shimadzu® SPD-M10A, and processed on Class-VP software. Purification of the compounds 31a and 31b was performed in HPLC using a Macherey–Nagel CLC-ODS semiprep column, Methanol 40%, flowrate 4.0 mL/min, and 200 nm. High-resolution mass spectra (HRMS) were obtained on a Bruker Daltonics MicrOTOF-Q II ESI-TOF mass spectrometer, and an Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Germany) was equipped with an electrospray (H-ESI-II) probe and operated in negative ionization mode. The system was controlled by Xcalibur and Tune (Thermo Scientific). For biological assays, absorbance at 405 nm was measured using SpectraMax M2 Molecular Devices®.
1,2:5,6-di-anhydro-D-mannitol (
25) [
43]: D-Mannitol (5.05 g, 27.4 mmol) was solubilized in pyridine (25.0 mL) and heated to 120 °C for 15 min. After that, the solution was refrigerated to 0 °C, treated dropwise with tosyl chloride solution (13.10 g, 68.7 mmol/10 mL pyridine) for 1 h, and stirred at 0 °C for 3 h then at r.t. for 1 h. The mixture was coevaporated with toluene, and the solid was diluted in dichloromethane and washed with HCl 1 mol·L
−1 and saturated NaHCO
3. The organic layer was dried over MgSO
4 and concentrated. The crude mixture of 1,6-di-O-tosyl-D-mannitol was solubilized in a mixture of acetonitrile and water (38.0 mL, 2:1, v/v), and a small portion of phenolphthalein was added to it. The mixture was stirred at 35–40 °C and titrated with NaOH 5 mol·L
−1 until the solution remained pink. After that, a mixture of Na
2CO
3 (87.4 g) in ethyl acetate (324 mL) was added and stirred vigorously. The solid was filtrated and washed with ethyl acetate. The organic solution was dried over MgSO
4, filtered, and concentrated. The resulting mixture was purified by flash chromatography (hexane and ethyl acetate, 7:3, v/v) to yield
1 (1.12 g, 7.70 mmol, 28%).
1H NMR (400 MHz, CD
3OD): δ 2.77 (2H, dd, J 2.7 Hz, 5.3 Hz, H-1a, H-6a); 2.82 (2H, dd, J 4.0 Hz, 5.3 Hz, H-1b, H-6b); 3.11–3.17 (2H, m, H-2, H-5); 3.46–3.52 (2H, m, H-3, H-4).
13C NMR (100 MHz, CDCl
3): δ 46.1 (C-1, C-6); 52.8 (C-2, C-5); 73.3 (C-3, C-4).
1,2:5,6-Di-O-isopropilidene D-mannitol (32) [
48]
1,2:5,6-Dianhydro-3,4-di-O-benzyl-L-iditol (35): prepared as described by Wilkinson et al. [
47]
General procedure for the synthesis ofL-gulo-piperidine and D-manno-azepane derivatives(26a,b-31a,b): 1,2:5,6-dianhydro-D-glucitol was solubilized in methanol and 2.5 eq of primary amine (propargylamine, ethanolamine, butylamine, or phenethylamine) was added to the solution. The mixture was heated to 90 °C in a microwave for 5 min (150 W) in a sealed vessel. The solvents were evaporated in vacuum. Compounds 27a,b and 29a,b were separated in a flash column (dichloromethane and methanol, 8:2 v/v). Compounds 26a,b and 28a,b were acetylated by the addition of pyridine (8.0 mL) and acetic anhydride (22.8 mL) and stirred at r.t. After 3 h, ice was added to the mixture, the product was extracted with ethyl ether, and the organic layer was evaporated and dried with MgSO4. Compounds 30a,b were separated by column chromatography (toluene and ethyl acetate, 3:7, v/v). Compounds 31a,b were purified in a HPLC C-18 semiprep column in methanol/water 40% and flow rate 4.0 mL/min.
See
1H,
13C and bidimensional NMR and ESI HRMS spectra of compounds
26-31a,b and
36-41a,b in
Supplementary Materials.General procedure for removal of Acetyl groups: Compounds 30a,b and 31a,b were dissolved in methanol (1.0 mL), and sodium methoxide 1 M was added dropwise to the solution until pH 9.0 and checked with Tornassol. After half an hour, TLC showed total consumption of starting material, then it was neutralized with ion exchange resin DOWEX® 50WX4-50. After that, the mixture was filtered through a Celite® pad and concentrated in vacuo.
General procedure for removal o Benzyl groups [50]: The corresponding product was solubilized in dichloromethane, and Me
3SI (4 eq) was added slowly. The reaction was allowed to stir at r.t. for 15 min. TLC showed total consumption of starting material, then the reaction was quenched with methanol. The product was purified in a SPE-C18 silica pad with methanol/water 1:1 v/v.
N-Propynyl-1,5-dideoxy-1,5-imino-L-gulitol (26a) [43]: 80% (0.0082 g, 0.041 mmol):
1H NMR (500 MHz, D
2O): δ 2.71–2.77 (2H, m, H-1a, ≡CH); 2.85 (1H, t,
J 10.9 Hz, H-1b); 2.88–2.93 (1H, m, H-5); 3.46 (1H, d,
J 17.6 Hz, H-7a); 3.68 (1H, d,
J 17.6 Hz, H-7b); 3.83 (1H, dd,
J 5.4 Hz, 11.7 Hz, H-6a); 3.89 (1H, dd,
J 3.9 Hz, 11.7 Hz, H-6b); 3.91–3.94 (1H, m, H-3); 4.05–4.14 (2H, m, H-2, H-4). ESI HRMS: [M+H]
+ calculated for C
9H
15NO
4 202.1074; found 202.1076.
N-Propynyl-1,6-dideoxy-1,6-imino-D-mannitol (26b) [
43]: 86% (0.0088 g, 0.044 mmol):
1H NMR (500 MHz, D
2O): δ 2.66 (1H, t,
J 2.2 Hz, ≡CH); 2.85 (4H, m, H-1a; H-1b; H-6a; H-6b); 3.37 (1H, d,
J 17.0 Hz, H-7a); 3.41 (1H, d,
J 17.0 Hz, H-7b); 3.89–3.91 (2H, m, H-3, H-4); 4.12 (2H, t,
J 4.8 Hz, H-2, H-5). ESI HRMS: [M+H]
+ calculated for C
9H
15NO
4 202.1074; found 202.1073.
N-Butyl-1,5-dideoxy-1,5-imino-L-gulitol (27a): 29.5 (c 1.8, MeOH), 1H NMR (500 MHz, D2O): δ 0.83 (3H, t, J 7.4 Hz, H-10); 1.17–1.28 (2H, m, H-9); 1.37–1.50 (2H, m, H-8); 2.50–2.92 (5H, m, 2xH-1, H-5, 2xH-7); 3.75 (1H, dd, J 5.6. 11.8, H-6a); 3.78–3.85 (2H, m, H-3, H-6b); 3.94–4.00 (1H, m, H-2); 4.05–4.11 (1H, m, H-4). 13C NMR (125 MHz, D2O): δ 13.3 (C-10); 20.0 (C-9); 27.4 (C-8); 53.1 (C-1 or C-7); 55.1 (C-1 or C-7); 58.5 (C-6); 68.2 (C-4); 70.1 (C-3); 70.4 (C-3); 72.8 (C-2). ESI HRMS: [M+H]+ calculated for C10H22NO4 220.1544; found 220.1538.
N-Butyl-1,6-dideoxy-1,6-imino-D-mannitol (27b): −62.0 (c 2.2, MeOH), 1H NMR (500 MHz, D2O): δ 0.76 (3H, t, J 7.3 Hz, 3xH-10); 1.07–1.25 (2H, m, 2xH-9); 1.30–1.44 (2H, m, 2xH-8); 2.47–2.57 (2H, m, 2xH-7); 2.65–2.85 (4H, m, 2xH-1, 2xH-6); 3.73–3.79 (2H, m, H-3, H-4); 3.94–4.04 (2H, m, H-2, H-5). 13C NMR (125 MHz, D2O): δ 13.2 (C-10); 20.0 (C-9); 27.5 (C-8); 55.2 (C-1, C-6); 58.4 (C-7); 68.3 (C-2; C-5); 72.8 (C-4; C-3). ESI HRMS: [M+H]+ calculated for C10H22NO4 220.1544; found 220.1543.
N-Hydroxyethyl-1,5-dideoxy-1,5-imino-L-gulitol (28a): 16.1 (c 0.6, MeOH), 1H NMR (400 MHz, CD3OD): δ 2.53–2.71 (2H, m, H-1a, H-7a); 2.78–2.89 (2H, m, H-5, H-7b); 2.97 (1H, ddd, J 4.9; 7.0; 13.5 Hz, H-1b); 3.55–3.98 (7H, m, H-2, H-3, H-4, H-6a, H-6b, H-8a, H-8b). 13C NMR (100 MHz, CD3OD): δ 51.9 (C-7); 55.1 (C-1); 58.6 (C-8); 60.0 (C-6); 61.0 (C-5); 66.5, 70.9, 71.3 (C-2, C-3, C-4). ESI HRMS: [M+H]+ calculated for C8H18NO5 208.1180; found 208.1177
N-Hydroxyethyl-1,6-imino-D-mannitol (28b): −15.3 (c 0.5, MeOH), 1H NMR (400 MHz, CD3OD): δ 2.58–2.80 (4H, m, H-1a; H-6ª; 2xH-7); 2.89 (2H, dd, J 4.3; 13.2 Hz, H-1b, H-6b); 3.59 (2H, dd, J 6.0; 12.5 Hz, 2xH-8); 3.86–3.92 (2H, m, H-3, H-4); 4.00–4.08 (2H, m, H-2, H-5). 13C NMR (100 MHz, CD3OD): δ 58.4 (C-1; C-6); 60.4 (C-8); 61.8 (C-7); 70.9 (C-2; C-5); 74.0 (C-3; C-4). ESI HRMS: [M+H]+ calculated for C8H18NO5 208.1180; found 208.1182.
N-Phenethyl-1,5-dideoxy-1,5-imino-L-gulitol (29a) [
46]
N-Phenethyl-1,6-dideoxy-1,6-imino-D-mannitol (29b) [
46]
N-Propynyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-L-gulitol (30a) [43]:
1H NMR (400 MHz, CDCl
3): δ 2.03, 2.09, 2.13, 2.14 (12H, 4s, 4×CH
3); 2.33 (1H, t,
J 2.3 Hz, ≡CH); 2.80 (1H, dd,
J 4.7 Hz, 11.2 Hz, H-1a); 2.97 (1H, dd,
J 9.6 Hz, 11.2 Hz, H-1b); 3.28 (1H, ddd,
J 3.3 Hz, 5.8 Hz, 10.0 Hz, H-5); 3.44 (1H, dd,
J 2.3 Hz, 17.8 Hz, H-7a); 3.67 (1H, dd,
J 2.3 Hz, 17.8 Hz, H-7b); 4.20 (1H, dd,
J 5.8 Hz, 11.6 Hz, H-6b); 4.24 (1H, dd,
J 3.3 Hz, 11.6 Hz, H-6a); 5.20–5.27 (3H, m, H-2, H-3, H-4).
13C NMR (100 MHz, CDCl
3): δ 20.8; 20.9 (CH
3); 43.9 (C-1); 49.6 (C-7); 55.5 (C-5); 61.4 (C-6); 66.5 (C-3, C-4); 68.7 (C-2); 77.2 (≡CH); 169.3; 169.7; 170.5 (C=O). ESI HRMS: [M+H]
+ calculated for C
17H
24NO
8 370.1496; found 370.1496.
N-Propynyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-D-mannitol (30b) [
43]:
1H NMR (400 MHz, CDCl
3): δ 2.05, 2.12 (12H, 2s, 4×CH
3); 2.25 (t,
J 2.3 Hz, ≡CH); 2.91 (2H, dd,
J 5.6 Hz, 13.8 Hz, H-1a, H-6a); 2.99 (2H, dd,
J 4.4 Hz, 13.8 Hz, H-1b, H-6b); 3.39 (1H, dd,
J 2.3 Hz, 17.3 Hz, H-7a); 3.45 (1H, dd,
J 2.3 Hz, 17.3 Hz, H-7b); 5.40 (2H, dt,
J 1.2 Hz, 4.6 Hz, H-2, H-5); 5.48 (2H, t,
J 1.2 Hz, H-3, H-4).
13C NMR (100 MHz, CDCl
3): δ 20.8; 21.0 (CH
3); 47.9 (C-7); 54.1 (C-1, C-6); 69.9 (C-2, C-5); 70.9 (C-3 e C-4); 77.2 (≡CH); 169.9; 170.1 (C=O). ESI HRMS: [M+H]
+ calculated for C
17H
24NO
8 370.1496; found 370.1535.
N-Acetoxyethyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-L-gulitol (31a):1H NMR (400 MHz, CDCl3): δ 2.06; 2.08; 2.10 (18H, 3s, 5xCH3); 2.89 (1H, dd, J 5.0; 13.7 Hz, H-1a); 2.96 (2H, t, H-7, J 5.9 Hz, H-7); 3.04 (1H, dd, J 2.6; 13.7 Hz, H-1b); 3.45 (1H, dd, J 4.8; 11.4 Hz, H-5); 4.05 (1H, dd, J 5.6; 11.3 Hz, H-6a); 4.10–4.21 (2H, m, 2xH-8); 4.37 (1H, dd, J 7.0; 11.8 Hz, H-6b); 5.15 (1H, dd, J 3.3; 9.0 Hz, H-3); 5.20–5.25 (1H, m, H-2); 5.20–5.25 (1H, m, H-2); 5.28 (1H, dd, J 4.9; 9.0 Hz, H-4). 13C NMR (100 MHz, CDCl3): δ 20.8, 20.9, 21.0 (5xCH3); 48.9 (C-1); 52.7 (C-7); 58.2 (C-5); 59.9 (C-6); 68.0 (C-3); 68.1, 68.3 (C-2, C-4).
N-Acetoxyethyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-D-mannitol (31b):1H NMR (400 MHz, CDCl3): δ 2.03; 2.07; 2.10 (18H, 3s, 5xCH3); 2.85 (2H, t, J 5.8 Hz, H-4, H-4′); 2.91 (2H, dd, J 5.6; 14.0 Hz, H-1a, H-1′a); 3.04 (2H, dd, J 4.3; 14.0 Hz, H-1b, H-1′b); 4.11 (2H, dd, J 5.7; 9.1 Hz, H-5, H-5′); 5.29-5.37 (2H, m, H-2, H-2′); 5.43-5.47 (2H, m, H-3, H-3′). 13C NMR (100 MHz, CDCl3): δ 20.8; 20.9; 21.0 (5xCH3); 54.8 (C-1, C-1′); 56.4 (C-4); 62.2 (C-5); 70.2 (C-2, C-2′); 70.6 (C-3, C-3′).
N-Propynyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol (36a):1H NMR (400 MHz, CDCl3): δ 2.27 (1H, t, J 2.2 Hz, ≡CH); 2.50 (1H, d, J 9.5 Hz, H-5); 2.60 (1H, t, J 10.7 Hz, H-1a); 2.92 (1H, dd, J 4.9; 10.9 Hz, H-1b); 3.31–3.44 (2H, m, 2xH-7); 3.62–3.91 (5H, m, H-2, H-3, H-4, 2xH-6); 4.77 (2H, dd, J 3.8; 11.2 Hz, CH2Ph); 4.98 (2H, dd, J 11.2; 13.4 Hz, CH2Ph); 7.29–7.45 (10H, m, H-Ph). 13C RMN (100 MHz, CDCl3): δ 42.1 (C-1); 56.1 (C-7); 57.3 (C-6); 63.1 (C-2 or C-5); 69.4 (C-2 or C-5); 74.6 (C≡CH); 75.2 (CH2Ph); 87.3 (C-3; C-4); 127.9, 128.6, 128.7 (Ar); 138.1 (Cq). ESI HRMS: [M+H]+ calculated for C23H28NO4 382.2013; found 382.2002.
N-Propynyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-L-iditol (36b):1H NMR (400 MHz, CDCl3): δ 2.31 (1H, t, J 2.3 Hz, ≡CH); 2.77 (2H, dd, J 8.2; 12.3 Hz, H-1a, H-6a); 2.98 (2H, dd, J 1.1; 12.7, H-1b, H6b); 3.40–3.56 (2H, m, 2xH-7); 3.63–3.70 (2H, m, H-3, H-4); 3.80–3.93 (2H, m, H-2, H-5); 4.67 (2H, d, J 11.2 Hz, CH2Ph); 4.81 (2H, d, J 11.2 Hz, CH2Ph); 7.29–7.42 (10H, m, H-Ph). 13C RMN (100 MHz, CDCl3): δ 48.8 (C-7); 56.9 (C-1, C-6); 68.0 (C-2, C-5); 73.8 (CH2Ph); 78.2 (C≡CH); 86.5 (C-3, C-4); 127.9, 128.0, 128.6 (Ar); 137.9 (Cq). ESI HRMS: [M+H]+ calculated for C23H28NO4 382.2013; found 382.2001.
N-Butyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol (37a):1H NMR (400 MHz, CDCl3): δ 0.94 (3H, t, J 6.9 Hz, 3xH-10); 1.21–1.38 (2H, m, 2xH-9); 1.39–1.56 (2H, m, 2xH-8); 2.26 (1H, t, J 10.6Hz, H-5); 2.32–2.54 (3H, m, H-1a, H-1b, H-7a); 2.70–2.82 (1H, m, H-7b); 3.13 (1H, dd, J 4.6; 11.2 Hz, H-6a); 3.38 (1H, t, J 8.8 Hz, H-3); 3.60–3.73 (2H, m, H-4, H-6b); 3.77–3.93 (2H, m, H-2, H-XX); 4.75 (2H, dd, J 5.6; 11.2 Hz, CH2Ph); 4.96 (2H, t ap., J 11.0 Hz, CH2Ph); 7.29–7.48 (10H, m, Ar). 13C RMN (100 MHz, CDCl3): δ 14.0 (C-10); 20.6 (C-9); 27.3 (C-8); 52.1 (C-1 or C-7); 55.1 (C-1 or C-7); 57.5 (C-6); 64.9 (C-2 or C-5); 69.3(C-2 or C-5); 74.9 (CH2Ph); 75.1 (CH2Ph); 78.1 (C-3 or C-4); 86.8 (C-3 or C-4); 127.8, 127.9, 128.0, 128.5, 128.7 (C-Ar); 138.1 (Cq); 138.5 (Cq). ESI HRMS: [M+H]+ calculated for C24H34NO4 400.2483; found 400.2477.
N-Butyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-L-iditol (37b):1H NMR (400 MHz, CDCl3): δ 0.93 (3H, t, J 7.2 Hz, 3xH-10); 1.25–1.40 (2H, m, 2xH-9); 1.42–1.58 (2H, m, 2xH-8); 2.52–2.69 (4H, m, H-1a, H-6a, 2xH-7); 2.92 (2H, d, J 12.5 Hz, H-1b, H-6b); 3.60–3.70 (2H, m, H-3, H-4); 3.78–3.89 (2H, m, H-2, H-5); 4.66 (2H, d, J 11.2 Hz, CH2Ph); 4.79 (2H, d, J 11.2 Hz, CH2Ph); 7.29–7.43 (10H, m, H-Ph). 13C RMN (100 MHz, CDCl3): δ 13.9 (C-10); 20.4 (C-9); 29.3 (C-8); 57.6 (C-7); 59.0 (C-1; C-6); 67.7 (C-2, C-5); 73.6 (CH2Ph); 87.0 (C-3, C-4); 127.9, 128.6 (Ar); 137.0 (Cq). ESI HRMS: [M+H]+ calculated for C24H34NO4 400.2483; found 400.2475.
N-Hydroxyethyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol (38a):1H NMR (400 MHz, CDCl3): δ 2.29 (1H, dd, J 9.7, 11.3 Hz, H-1a); 2.37–2.51 (2H, m, H-5, H-7a); 2.94–3.08 (1H, m, H-7b); 3.16 (1H, dd, J 4.4; 11.5 Hz, H-1b); 3.43 (1H, t, J 8.3 Hz, H-3); 3.54–3.77 (4H, m, H-2, H-4, H-8a, H-8b); 3.88 (2H, qd, J 2.8; 12.3 Hz, H-6a, H-6b); 4.67–4.81 (2H, m, CH2Ph); 4.84–4.97 (2H, m, CH2Ph); 7.29–7.45 (10H, m, Ph-H). 13C RMN (100 MHz, CDCl3): δ 53.2 (C-7); 55.3 (C-1); 57.8 (C-6); 59.6 (C-8); 65.7 (C-5); 69.0 (C-2 or C-4); 74.8 (CH2Ph); 78.0 (C-2 or C-4); 85.9 (C-3); 128.0, 128.6 (Ar); 138.4 (Cq). ESI HRMS: [M+H]+ calculated for C22H30NO5 388.2119; found 388.2120.
N-Hydroxyethyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-L-iditol (38b):1H NMR (400 MHz, CDCl3): δ 2.66–2.80 (4H, m, 2xH-1, 2xH-2); 2.99 (2H, dd, J 2.8; 13.1 Hz, 2xH-7); 3.62–3.72 (4H, m, H-2, H-5, 2xH-8); 3.80–3.90 (2H, m, H-3, H-4); 4.65 (2H, d, J 11.3 Hz, CH2Ph); 4.84 (2H, d, J 11.3 Hz, CH2Ph); 7.28–7.42 (10H, m, H-Ph). 13C RMN (100 MHz, CDCl3): δ 59.0 (C-7); 59.7 (C-8); 60.7 (C-1; C-6); 70.0 (C-3; C-4); 74.2 (CH2Ph); 85.4 (C-2; C-5); 127.9, 128.0, 128.6, 137.9 (Ar). ESI HRMS: [M+H]+ calculated for C22H30NO5 388.2119; found 388.2102.
N-Propynyl-1,5-dideoxy-1,5-imino-D-glucitol (39a) [
47]
N-Propynyl-1,6-dideoxy-1,6-imino-L-iditol (39b) [
47]
N-Butyl-1,5-dideoxy-1,5-imino-D-glucitol (40a) [
55]
N-Butyl-1,6-dideoxy-1,6-imino-L-iditol (40b): 1.3 (c 1.1, MeOH), 1H NMR (400 MHz, D2O): δ 0.86 (3H, t, J 8.0 Hz, 3xH-10); 1.24–1.36 (2H, m, 2xH-9); 1.56–1.74 (2H, m, 2xH-8); 3.14–3.23 (2H, m, H-7); 3.26–3.37 (4H, m, 2xH-1, 2xH-6); 3.58–3.67 (2H, m, H-2, H-5); 4.01–4.08 (2H, m, H-3, H-4). 13C RMN (100 MHz, D2O): δ 12.9 (C-10); 19.3 (C-9); 25.6 (C-8); 58.6 (C-1; C-6; C-7) 67.1 (C-3 or C-4); 67.7 (C-3 or C-4); (C-2; C-5). ESI HRMS: [M+H]+ calculated for C10H22NO4 220.1544; found 220.1545.
N-Hydroxyethyl-1,5-dideoxy-1,5-imino-D-glucitol (41a) [
55]
N-Hydroxyethyl-1,6-dideoxy-1,6-imino-L-iditol (41b): −9.1 (c 0.6, MeOH), 1H NMR (400 MHz, D2O): δ 2.62–2.77 (4H, m, H-1a; H-1b; H-6a; H-6b); 2.91 (2H, dd, J 3.8;13.7 Hz, H-7a, H-7b); 3.40–3.48 (2H, m, H-2, H-3); 3.69 (4H, m, H-4, H-5, H-8a, H-8b,). 13C RMN (100 MHz, D2O): δ 58.4 (C-7), 59.3 (C-1, C-6, C-8), 70.8 (C-4, C-5), 75.6 (C-2, C-3). ESI HRMS: [M+H]+ calculated for C8H18NO5 208.1185; found 208.1176.
Biological assays [43]: Yeast α-glucosidase (EC 3.2.1.20) and almond β-glucosidase (EC 3.2.1.21) activity was assessed using a 96-well plate assay. Assays contained 20 mM NaOAc at pH 6.8 (α-glucosidase) and pH 6.2 (β-glucosidase), 10 mM PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid), 0.1 mM EDTA, α-glucosidase (5 μg/mL), β-glucosidase (6 μg/mL), and inhibitor (0.1–2 mM). Enzyme and inhibitor were equilibrated at 37 °C for 30 min. The reaction was initiated by the addition of
p-nitrophenyl α-D-glucopyranoside (200 μM) or
p-nitrophenyl β-D-glucopyranoside (200 μM), and then it was quenched with 100 μL of sodium carbonate 3.0 M after 25 min incubation at 37 °C. Assays were repeated in duplicate and data averaged.