Article
Macromolecules, Vol. 43, No. 8, 2010 3611
under a nitrogen or argon atmosphere was dissolved at 0 °C using
dry N,N-dimethylformamide (20 mL) and suspended using
lithium hydride (1 equiv). The mixture was stirred for 1 h at
0 °C underreducedpressure(∼22mbar) until hydrogenevolution
ceased. The vacuum was released, and the flask flushed with
nitrogen, followed by stirring for 2 h at 40 °C. The solution was
neutralized using a mixed bead ion exchanger (Ion exchanger V,
Merck) and filtered, and the solvent was removed under vacuum
at 40 °C. The raw product was dissolved in deionized water and
lyophilized. A small portion of the sample was homogenized by
spatula. The substance was permethylated according toGM3 and
MALDI-TOF-MS analysis performed.
acid was neutralized by addition of sodium carbonate until no
gas evolution was visible, and the pH was adjusted slightly
above pH ∼7. The organic phase was separated, the aqueous
layer was extracted three times using dichloromethane, and the
combined organic phases were dried using sodium sulfate and
filtered, and the solvent was removed under vacuum. The raw
product was purified by flash column chromatography (ethyl
acetate/petrol ether 1:2).
GM5. Preparation of Glycopyranosyl Fluorides. The perace-
ꢀ
tylated product was deacetylated under Zemplen conditions,
using a solution of methanolate in methanol (pH 8.5) at 0 °C
while stirring for 2 h under TLC control (ethyl acetate/petrol
ether 3:1). After completion the solution was neutralized using
ion-exchange resin (Dowex 50, Hþ-form) and filtered, and the
solvent was removed under vacuum. No further purification
was done.
MALDI-TOF-MS. Matrix-assisted laser desorption ioniza-
tion time-of-flight mass spectrometric analysis (MALDI-TOF
MS) were performed on a Bruker Biflex III, positive mode
(matrix: 2,5-dihydroxybenzoic acid (DHB).
GM2. Peracetylation Using Pyridine and Acetic Acid Anhy-
dride. The starting material was dissolved in an excess of
pyridine approximately 20-fold and then cooled to 0 °C prior
to the addition of acetic acid anhydride (5 equiv) for each
hydroxyl group while stirring. After the entire volume of acetic
acid anhydride was added the cooling was removed. After
completion of the reaction, the solvent was removed under
vacuum, and the raw product was codistilled with toluene to
remove pyridine. Purification was done either by flash column
chromatographic purification or recrystallization from ethanol.
GM3. Methylation Analysis. The peracetylated saccharide
mixture (20 mg) was dissolved in dry methanol (10 mL) and
suspended with a spatula of sodium methanolate to adjust pH
∼8. The solution was stirred overnight, followed by neutraliza-
tion using Dowex 50 (Hþ-form) and then filtered, and the
solvent was removed under vacuum. The residue was taken up
in DMSO (20 mL) and methyl iodide (1 mL) added, followed by
sodium hydroxide (50%, 4 mL) and stirred at rt. After 30 min
the solution was diluted with deionized water (20 mL) and
extracted three times using dichloromethane. The combined
organic phases were washed with water and then dried using
sodium sulfate. Under mild vacuum the majority of the solvent
was removed and carefully dried using a blown nitrogen stream.
A portion of 10 mg of the residue is treated with trifluoro-
acetic acid (5 mL, 2.5 N) and heated for 1 h at 120 °C using a
microwave reactor. The solution is dried in nitrogen air stream
and codistilled twice using dry acetonitrile. The residue was
taken up into sodium borodeuteriohydride (40 mg), ammonia
solution (0.27 mL, 25%), and deionized water (1.73 mL of 0.5 M
NaBD4 in 2 M NH3 solution).
The solution was stirred for 1 h at 60 °C, followed by an
addition of acetone (2 mL) and stirring for an additional 20 min.
To the solution acetonitrile (5 mL) was added, and the mixture
was dried in a nitrogen air stream and this step repeated once.
The residue was dissolved in glacial acetic acid (2 mL) and was
suspended with ethyl acetate (1 mL) and acetic acid anhydride
(3 mL) and shaken, and perchloric acid (0.1 mL) was added. The
solution was stirred for 5 min and then cooled to 0 °C, and
deionized water (10 mL) and 1-methylimidazole (0.2 mL) were
added and stirred for an additional 5 min at this temperature.
The phase was extracted by addition of dichloromethane
(1 mL), and the flask was shaken vigorously. After phase
separation, the dichloromethane layer was carefully pipetted
off and was kept at -26 °C for GC/MS analyses.
Gel Permeation Chromatography (GPC). GPC was done at
room temperature using a multiangle light scattering and con-
centration detection TSK-Gel PWXL G3000, G4000, G5000,
and G6000 columns (Toso Haas, Stuttgart, Germany) in de-
creasing pore size were coupled online to a multiangle light
scattering (MALS) detector Dawn DSP (Wyatt Technology,
Santa Barbara, CA) and a differential refractive index detector
(dRI) Optilab DSP (Wyatt Technology). Data acquisition and
processing was performed using Astra 4 software (Wyatt Tech-
nology, Santa Barbara, CA). Flow rate was maintained at
0.5 mL/min using a Constametric 3500 pump. The GPC refrac-
togram for glucopyranosyl oligomers is plotted as molar mass
(or detector response) vs elution volume showing a distribution
of Mw ∼ 2000-3000.
2,3,4,6-Tetra-O-acetyl-R-D-glucopyranosyl Fluoride [1]. Pre-
paration according to GM4. C14H19O9F (350.30 g/mol);
[R]25046 = þ80 (c 1, CHCl3). Lit.:22 [R]D25 = þ91 (c 1.1, CHCl3);
mp 100 °C. Lit.:23 112 °C; colorless solid. Yield: 70%. 1H NMR
(400 MHz, CDCl3): δ = 5.74 (d, 1H, H-1), 5.41 (vt, 1H, H-3),
5.13 (vt, 1H, H-4), 4.96 (dd, 1H, H-2), 4.27 (vt, 1H, H-6a), 4.14
(dd, 1H, H-5, H-6b), 2.03, 2.02, 1.97, 1.92 ppm (CH3). J1,2 = 2.8,
J2,3 = 10.0, J3,4 = 10.2, J4,5 = 10.1, J5,6a = 4.6, J5,6b = 2.4,
J
6a,6b = 12.5, J1,F = 53.0, J2,F = 25.0 Hz. 13C NMR (100 MHz,
CDCl3): δ = 170.00, 170.00, 169.66, 169.84, (CO), 103.61
(C1), 70.00 (C2), 69.64 (C5), 69.57 (C3), 67.15 (C4), 61.0
(C6), 20.50, 20.55, 20.25, 20.31 ppm (CH3). JC1,F = 228, JC2,F
24 Hz.
=
2,3,4,6-Tetra-O-acetyl-R-D-galactopyranosyl Fluoride [2]. Pre-
=
paration according to GM4. C14H19O9F (350.30 g/mol); [R]D20
þ100° (c 1, CHCl3). Lit.:11,23 [R]D20 = þ96.5° (c 1, CHCl3); mp
1
62 °C. Lit.:23 67-68 °C; colorless solid. Yield: 68%. H NMR
(400 MHz, CDCl3): δ (ppm) = 5.74 (dd, 1H, H-1), 5.46 (d, 1H,
H-4), 5.29 (dd, 1H, H-3), 5.12 (ddd, 1H, H-2), 4.34 (vt, 1H, H-5),
4.08 (m, 2H, H-6a. H-6b) 2.09, 2.05, 1.99, 1.94 (CH3). J1,2 = 2.8,
J
2,3 = 11.0, J3,4 = 3.4, J5,6a = 6.6, J5,6b = 6.6, J1,F = 53.3, J2,F =
23.6 Hz. 13C NMR (100 MHz, CDCl3): δ (ppm) = 174.34,
172.98, 172.85, 171.02 (CO), 104.72 (C-1), 69.27 (C-5), 67.95
(C-3), 67.76 (C-4), 67.40 (C-2), 61.67 (C-6), 21.06, 21.03, 21.00,
20.94 (CH3). 1JC1,F = 113.6 Hz.
GC MS was done on a HP 6890, using a HP-5 column (30 m),
with an inner diameter (i.d.) of 0.32 mm and film thickness (f.th.)
of 0.25 μm with H2 as a carrier gas. Temperature program
(oven): 40 °C for 2 min, 30°/min up to 60 °C, then 5°/min up to
300 °C. Temperature programmable injector (PTV); 50 °C for
0.2 min, 300°/min up to 250 °C. Detector type: flame ionization
detector (FID) (Figure 1).
GM4. Preparation of Peracetylated Glycopyranosyl Fluorides.
The reaction was performed in a sealed plastic vial. 10 mmol of
the starting peracetylated pentaacetate was dissolved in HF/
pyridine (10 mL, 70%) solution and stirred for 4 h at rt. The
reaction was monitored by TLC (ethyl acetate/petrol ether 1:1).
After completion of the reaction, the solution was diluted with
dichloromethane (30 mL) and deionized water (30 mL). Excess
2,3,4,6-Tetra-O-acetyl-R-D-mannopyranosyl Fluoride [3]. Pre-
=
paration according to GM4. C14H19O9F (350.30 g/mol); [R]D20
20
þ7 (c 1, CHCl3); mp 115 °C. Lit.:24 67-68 °C; [R] = þ29 (c 1,
546
CHCl3). Lit.:24 [R]D20 = þ22 (c 1.68, CHCl3); colorless syrup.
Yield: 65%. 1H NMR (400 MHz, CDCl3): δ (ppm) = 5.55
(d, 1H, H-1), 5.40-5.30 (m, 3H, H-2, H-3, H-4), 4.14 (m, 2H,
H-6a, H-6b), 2.01, 1.98, 1.98, 1.97 (CH3). J1,2 = 1.7, J1,F = 48.3
Hz. 13C NMR (100 MHz, CDCl3): δ (ppm) = 104.01 (C1), 72.73
(C5), 68.37 (C3), 66.62 (C2), 66.15 (C4), 62.51 (C6), 20.58, 20.05,
19.95, 19.56 (CH3). CO signals were not resolved due to the low
signal intensity. JC1,F = 220, JC2,F = 24 Hz.