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Organic & Biomolecular Chemistry
Page 5 of 8
DOI: 10.1039/C7OB02976D
Journal Name
ARTICLE
immunogenicity of natural MUC1 glycopeptides and deserve further and washed with 1M HCl, water and brine in turn. The organic layer
research.
was dried over Na2SO4 and concentrated. The crude product was
purified by column chromatography (petroleum ether/ethyl
acetate, 5:1) to give 3 (65.92g, 92%, α:β = 3:7).
1H NMR (400 MHz, CDCl3) δ 6.27 (d, J = 3.6 Hz, 0.3H, H-1α), 5.52 (d, J
= 8.4 Hz, 0.7H, H-1β), 5.43 (d, J = 2.8 Hz, 0.3H, H-4α), 5.33 (d, J = 3.2
Hz, 0.7H, H-4β), 5.27 (dd, J = 10.8, 2.8 Hz, 0.3H, H-3α), 4.87 (dd, J =
10.8, 3.2 Hz, 0.7H, H-3β), 4.23 (t, J = 6.8 Hz, 0.3H, H-5α), 4.13 – 4.01
(m, 2H), 3.99 (t, J = 6.4 Hz, 0.7H, H-5β), 3.90 (dd, J = 10.8, 3.6 Hz,
0.3H, H-2α), 3.79 (dd, J = 10.8, 8.4 Hz, 0.7H, H-2β), 2.15 (s, 2.1H),
2.13 (s, 0.9H), 2.12 (s, 3H), 2.03 (s, 0.9H), 2.02 (s, 2.1H), 1.99 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 170.3, 170.0, 169.9, 169.8, 169.6,
168.7, 168.6, 92.8, 90.4, 71.7, 71.3, 68.7, 68.6, 66.9, 66.2, 61.1,
61.0, 59.7, 56.8, 20.9, 20.8, 20.6, 20.5, 20.5.
Experimental section
Chemical Synthesis
All reagents were purchased from commercial sources and were
used without further purification unless otherwise noted. All
solvents were available with commercially dehydrated or freshly
dehydrated and distilled prior to use. Reactions were monitored
using Thin Layer Chromatography (TLC) on silica gel GF254 plates
with detection by short wave UV fluorescence (λ = 254 nm) after
being
stained
with
p-anisaldehyde
solution
(ethanol/panisaldehyde/acetic acid/sulfuric acid 135:5:4:1.5) or
10% phosphomolybdic acid in EtOH, followed by heating on a hot
plate. Column chromatography was conducted by silica gel (200-
300 mesh) with petroleum ether and ethyl acetate as eluent. 1H and
13CNMR spectra were obtained using a Bruker AV 400 MHz
spectrometer at 400M Hz and 100M Hz respectively. Signals are
reported in terms of their chemical shift (δ in ppm) relative to CDCl3
(1H, 7.26 and 13C, 77.16), CD3OD (1H, 3.31 and 13C, 49.00), (CD3)2SO
(1H, 2.50 and 13C, 39.52). Coupling constants are reported in hertz.
High-resolution electrospray-ionization mass spectra (HRESIMS)
were obtained on a Varian QFT-ESI mass spectrometer. Matrix-
assisted laser desorption/ionizationtime of flight mass spectra
(MALDI-TOF MS) were performed using 2,5-dihydroxybenzoic acid
(DHB) or α-cyano-4-hydroxy cinnamic acid (CHCA) as matrix on
Varian 7.0T FTMS instrument. Reversed-phase HPLC separations
were performed on a Waters system 2487 using solution A (0.1%
trifluoroacetic acid in 100% acetonitrile) and solution B (0.1%
trifluoroacetic acid in 100% water) for elution. UV absorption
signals were detected with an UV detector at a wavelength of
220nm.
3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl
trichloroacetimidate (4)
To a solution of 3(65.91 g, 176.14 mmol) in THF/DCM (1:1, v/v, 150
mL) was added DMAPA (109.66 mL, 880.69 mmol). The mixture was
stirred at room temperature for 2 hours. The solution was diluted
with dichloromethane and washed with saturated ammonium
chloride solution and brine. The organic layer was dried over
Na2SO4 and concentrated. The afforded colourless oil was dissolved
in dehydrated dichloromethane (1000 mL) in an ice-water bath.
CCl3CN (176.14 mL, 1.76 mol) and DBU (17.61mL, 140.91 mmol)
were added successively under the protection of argon. The
mixture was stirred for 45 min. The solution was diluted with
dichloromethane and washed with saturated ammonium chloride
solution and brine. The organic layer was dried over Na2SO4 and
concentrated. The crude product was purified by column
chromatography (petroleum ether/ ethyl acetate, 1:1) to give
(71.24 g, 85% over two steps, α/β mixture, α>95%)
1H NMR (400 MHz, CDCl3, α) δ 8.79 (s, 1H), 6.50 (d, J = 3.6 Hz, 1H),
5.54 (d, J = 2.4 Hz, 1H), 5.37 (dd, J = 11.2, 3.2 Hz, 1H), 4.41 (t, J = 6.8
Hz, 1H), 4.14 (dd, J = 11.2, 6.8 Hz, 1H),4.07 – 4.01 (m, 2H), 2.17 (s,
3H), 2.07 (s, 3H), 2.00 (s, 3H).
4
2-azido-2-deoxy-D-galactose (2)
The spectral data were consistent with the reported results in the
literature.33
The compound was synthesized using our previously reported
method. D-galactosamine hydrochloride (46.6 g, 216 mmol) and
anhydrous potassium carbonate (44.8 g, 324 mmol) were dissolved
in methnol (350 mL). CuSO4 (568 mg, 3.20 mmol) was added and
the mixture was stirred in dark for 0.5 h. Freshly prepared
imidazole-1-sulfonyl azide31 (240 mL, 180 mmol) was added and the
mixture was stirred overnight. The mixture was filtered off through
Celite and the solvent was concentrated in vacuo. The crude
product was purified by flash chromatography (CH2Cl2: MeOH, 5:1)
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-(3,4,6-tri-O-acetyl-2-
azido-2-deoxy-α-D-galactopyranosyl)-L-threonine benzylester (
The isomers of (71.22 g, 149.43 mmol), Fmoc-Thr-COOBn (58.11 g,
5)
4
136.32 mmol), and 4 Å MS (2.0 g) in dehydrated CH2Cl2 (140 mL)
was stirred at RT under an argon atmosphere for 1 h. After being
cooled to -20℃, TMSOTf (6.99 mL, 38.62 mmol) was added, and the
reaction was stirred at RT for another 2 h. When TLC showed that
the reaction was completed, saturated aq. NaHCO3 was added to
quench the reaction, and it was then diluted with CH2Cl2. Molecular
sieves were filtered through Celite. The solution was washed with
saturated ammonium chloride solution and brine. The organic layer
was dried over Na2SO4 and concentrated. The crude product was
purified by column chromatography (petroleum ether/ ethyl
to give 2 as a light yellow solid (32.83 g, 89%, α:β = 2:3).
1H NMR (400 MHz, CD3OD) δ 5.18 (d, J = 3.5 Hz, 0.4H, H-1α), 4.43 (d,
J = 7.1 Hz, 0.6H, H-1β), 4.04 – 3.94 (m, 1H), 3.90 (d, J = 2.8 Hz, 0.4H),
3.77 (m, 0.6H), 3.75 – 3.65 (m, 2H), 3.50 – 3.42 (m, 1H), 3.39 (m,
1H).
The spectral data were consistent with the reported results in the
literature.32
acetate, 6:1) to give 5 (84.59 g, 76%,α:β=4:1).
1H NMR (400 MHz, CDCl3, α) δ 7.77 (d, J = 7.4 Hz, 2H), 7.62 (d, J =
7.3 Hz, 2H), 7.35 (m, 9H), 5.68 (d, J = 9.2 Hz, 1H), 5.44 (br s, 1H),
5.31 – 5.16 (m, 4H), 4.91 (d, J = 3.3 Hz, 1H), 4.50 – 4.35 (m, 4H), 4.29
– 4.20 (m, 2H), 4.08 (d, J = 6.3 Hz, 2H), 3.59 (dd, J = 11.2, 3.6 Hz, 1H),
2.15 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 1.35 (d, J = 5.9 Hz, 3H).
3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-galactopyranose (
3)
The compound (32.8 g, 160.05 mmol) was dissolved in pyridine
2
(300 mL). Acetic anhydride (78 mL) was added dropwise in an ice-
water bath under the protection of argon and the mixture was
stirred overnight. The solution was diluted with dichloromethane
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