10.1002/ejoc.201800183
European Journal of Organic Chemistry
FULL PAPER
1H, H5), 2.03 (s, 3H, CH3). 13C NMR (101 MHz, d6DMSO) δ 163.65,
100.04, 73.89, 72.65, 69.44, 68.26, 61.80, 13.69.
field used is OPLS 2005, preferred for biological systems and
organic molecules.19
At first the protein structure was processed and optimized within
the Protein Preparation Wizard, in order to be used as a docking
N-Acetylglucosamine-6-sulfate (1).13 Pyridine – sulfur trioxide in DMF
(0,1M, 20mL ) was added to a solution of GlcNAc (0,5g, 2,26mmol)
dissolved in DMF over 3h at r.t. The mixture was stirred for another hour
and concentrated in high vacuum at 40°C to remove DMF and pyridine.
The pH was adjusted to 7.5 with KOH 2N and concentrated to remove
pyridine. The mixture was purified with flash chromatography
AcOEt:MeOH:H2O 7:3:0,5:0,5 rf 0,32 (1,37 g – 31% yield). HRMS calcd
for [M - H]- 300,2636; found [M - H]- 300,2641. 1H NMR (400 MHz, D2O)
δ 5.01 (d, J = 3.3 Hz, 1H, H-1α), 4.54 (d, J = 8.4 Hz, 0.6H, H-1β), 4.15
(dd, J = 11.1, 1.6 Hz, 0.6H, H-6aβ), 4..01-4.08 (m, 2H, H-6abα), 4.03 (dd,
J = 11.1, 5.2 Hz, 1H, H-6bβ), 3.87 (dt, J = 9.9, 3.5 Hz, 1H, H-5α), 3.72
(dd, J = 10.6, 3.4 Hz, 1H, H-2α), 3.58 (bt, J = 10.0, 1H, H-3α), 3.55 –
3.46 (m, 1.2H, H-2β, 5β), 3.40 – 3.29 (m, 2.2H, H-4α, 3β, 4β), 1.86 (s, 4H,
Ac α and β). 13C NMR (101 MHz, D2O) δ 174.35 (CO), 94.87 (C1β),
90.78(C1α), 73.66, 73.60, 70.49, 69.59, 69.47, 69.36 (C3,4,5 αβ), 67.01
(C6αβ), 56.40(C2β), 53.79(C2α), 20.54 (Ac).
receptor, and a sampling box (18x18x18 Å3) centered on the
enzyme active site was considered for the following docking
studies. The docking of the potential inhibitors into the protein
has been carried out assuming protonation state compatible with
pH 7 and sampling a box (18x18x18 Å3) centered on the enzyme
active site.
All ligands were docked with the extra precision (XP) method
and explicitly taking into account the conformational flexibility of
the ligands.
Synthesis:
General Remarks. All solvents, when necessary, were dried with
molecular sieves for at least 24 h prior to use. All the reagents and
starting material were purchased from Sigma Aldrich Italy o Carbosynth
United UK. Thin layer chromatography (TLC) was performed on silica gel
60 F254 plates (Merck) with detection by using UV light when possible,
or by charring with a solution of concd. H2SO4/EtOH/H2O (5:45:45) or a
solution of (NH4)6Mo7O24 (21 g), Ce(SO4)2 (1 g), concd. H2SO4 (31 mL)
in water (500 mL). Flash column chromatography was performed on
silica gel 230–400 mesh (Merck). 1H and 13C NMR spectra were
recorded at 25 °C, unless otherwise stated, with a Varian Mercury 400
MHz instrument. Chemical shift assignments, reported in ppm, are
referenced to the corresponding solvent peaks. MS data were recorded
with a LCQ-FLEET ion trap Thermo Fischer. HRMS were recorded on a
QSTAR elite LC/MS/MS system with a nanospray ion source. Optical
rotations were measured at room temperature using an Atago Polax-2L
polarimeter and are reported in units of 10-1 deg·cm2·g-1,
concentrations are reported as g/ml.
2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-α-D-glucopyranosyl chloride
(14).22 Acetylchloride (35mL) was added drop wise to GlcNAc (10,07g,
45,52 mmol) under stirring at r.t. for 20h. At complete disappearance of
the starting material, the product was obtain by extraction with H2O-
ice/CH2Cl2 and by washing the organic phase with NaHCO3/CH2Cl2. The
purification was done by flash chromatography EP:AcOEt 3:7, rf 0,35 to
afford pure compound 14 (10,38 g – 82% yield), MS: m/z calcd for [M]+ =
20
365.8, found [M]+ = 365,1; [α]D +109 (c = 0.55 10-2, CHCl3).1H NMR
(400 MHz, CDCl3) δ 6.17 (d, J = 3.6 Hz, 1H, H-1), 5.90 (d, J = 8.6 Hz, 1H,
NH), 5.31 (t, J = 10.1 Hz, 1H, H-4), 5.20 (t, J = 9.8 Hz, 1H, H-3), 4.53
(ddd, J = 10.6, 8.8, 3.7 Hz, 1H, H-2), 4.31 – 4.22 (m, 2H, H-5, 6a), 4.12
(bd, J = 10.6 Hz, 1H, H-6b), 2.09 (s, 3H, Ac), 2.04 (s, 6H, 2Ac), 1.98 (s,
3H, Ac). 13C NMR (101 MHz, CDCl3) δ 171.47, 170.67, 170.29, 169.23,
93.76, 70.92, 70.12, 67.07, 61.19, 53.44, 23.14, 20.80, 20.75, 20.66.
2-Acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-
glucopyranose (15).23 To a suspension of 14 (10,15g 27,80mmol) in dry
toluene (108mL), under inert atmosphere, AIBN cat was added. Then
Bu3SnH (12,14g, 41,7mmol,1,5 eq) was added and the reaction was
stirred at 120°C for 12h. The reaction was quenched with KF 2M. The
product was extracted with CH2Cl2/H2O. The organic layer was dried over
anhydrous NaSO4, filtered and the organic layer removed under reduced
pressure. The purification was done with flash chromatography AcOEt, rf
0,35 to afford pure 15 (8,61 g – 93%), MS: m/z calcd for [M + H]+ = 332.3,
[M + Na]+ = 354.1; found [M + H]+ = 332.2, [M + Na]+ = 354.1. [α]D20 +7 (c =
2-Methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyranoso)-[1,2-d]-
2-oxazoline (12).14,20 Commercially available peracetylated GlcNAc
(1,55g, 3,97 mmol) was dissolved in dry CH2Cl2 (0,1M) under inert
atmosphere and the reaction was cooled to 0°C, then TMSOTf (1,6eq,
1,14mL) was added and the reaction was left stirring overnight at 65°C.
The reaction was quenched though the addition of 2 mL of triethylamine
and the solvent was removed under reduced pressure. The crude
compound was purified with flash chromatography Et/AcOEt 8:2, rf 0,31
affording 1,05 g of 12 (yield 81%), MS: m/z calcd for [M + Na]+ = 352.1, [M
+ K]+ = 368.1; found [M + Na]+ = 352.1, [M + K]+ = 368.0. [α]D20 +0 (c = 1.2
10-2 MeOH). 1H NMR (400 MHz, Chloroform-d) δ 5.95 (d, J = 7.5, 1.9 Hz,
1H, H1), 5.30 – 5.21 (m, 1H, H3), 4.92 (d, J = 9.2, 1.8 Hz, 1H, H4), 4.20 –
4.08 (m, 3H, H2, H6a, H6b), 3.64 – 3.54 (m, 1H), 2.14 – 2.02 (m, 12H, 4
CH3). 13C NMR (101 MHz, CD3OD) δ 172.20, 171.19, 170.84, 169.61,
101.11, 71.05, 69.62, 68.68, 64.94, 64.57, 20.80, 20.75, 20.63, 13.72.
20
0.7 10-2 CHCl3). Lit.24 [α]D 0 (c = 1.1, CHCl3). 1H NMR (400 MHz,
Chloroform) δ 5.78 (t, J = 6.9 Hz, 1H, NH), 5.06 (t, J = 9.6 Hz, 1H, H-4),
4.94 (t, J = 9.7 Hz, 1H, H-3), 4.23 – 4.10 (m, 4H, H-2,6a, 1a, 1b), 3.54-
3.51 (m, 1H, H-5), 3.14 (t, J = 12.0 Hz, 1H, H-6b), 2.08 (s, 3H, Ac), 2.05
(s, 3H, Ac), 2.02 (s, 3H, Ac), 1.92 (s, 3H, Ac). 13C NMR (101 MHz,
CDCl3) δ 172.22, 170.88, 170.34, 169.42, 76.63, 74.27, 68.30, 68.19,
62.43, 50.56, 23.30, 20.92, 20.88, 20.74.
2-Methyl-(1,2-dideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline (13).14,21
Compound 12 (1,05g, 3,03mmol) was dissolved in 30 mL of dry MeOH
(0,1M) under inert atmosphere, then NaOMe (6eq) was added to the
solution and the reaction was stirred for 3h. The reaction was neutralized
with Amberlite 5% HCl to pH 7. After filtration the solvent was removed
under reduced pressure affording compound 13 (0,85 g – quant), in
agreement with literature data. MS: m/z calcd for [M - H]- = 202.1; [M - H]-
= 202.14. [α]D20 +32 (c = 0.4 10-2 MeOH). 1H NMR (400 MHz, Methanol-
d4) δ 6.03 (d, J = 7.3 Hz, 1H, H1), 3.99 (bs, 1H, H2), 3.87 (t, J = 3.7 Hz,
1H, H3), 3.77 (dd, J = 12.0, 2.5 Hz, 1H, H6a), 3.67 – 3.61 (dd, J = 6.8 Hz,
1H, H6b), 3.55 – 3.50 (m, 1H, H4), 3.33 (ddd, J = 12.0 Hz, 2.4Hz, 6.4 Hz,
2-Acetamido-1,5-anhydro-2-deoxy-D-glucopyranose
(16).15
Compound 15 (4,01g, 12,12mmol) was dissolved in dry MeOH (0,1M)
under Argon atmosphere, and NaOMe (6eq) was added. After 3h the
stirring was stopped and the reaction was neutralized with Amberlite 5%
HCl to pH 6. After filtration the solvent was removed under reduced
pressure affording compound 16 (2,68 g – quant), MS: m/z calcd for [M -
20
H]- = 204.1; found [M - H]- = 204.1. [α]D +3 (c = 0.9 10-2 H2O). 1H NMR
(400 MHz, Deuterium Oxide) δ 3.73 (dd, J = 11.2, 5.2 Hz, 1H, H-1a), 3.68
(dd, J = 12.3, 1.9 Hz, 1H, H-6a), 3.64 (dt, J = 10.7, 5.6 Hz, 1H, H-2), 3.50
(dd, J = 12.3, 5.7 Hz, 1H, H-6b), 3.32 (t, J = 9.3 Hz, 1H, H-3), 3.21 (t, J =
9.1 Hz, 1H, H-4), 3.15 (m, 1H, H-5), 3.05 (t, J = 11.1 Hz, 1H, H-1b), 1.81
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