Journal of Medicinal Chemistry
Article
stirring was continued overnight under reduced pressure. After the
mixture was returned to room temperature, triethylamine (3 mL) was
added to the mixture. The solvent was removed, and purification of
the residue by MPLC (15:1 chloroform/methanol) afforded compound
the potential for GC 1a as an STEC treatment agent, acting via
removal of free Stx from the gut of patients.
1
EXPERIMENTAL SECTION
7 (7.8 g, 53%) as a white solid. H NMR (DMSO-d6): δ 7.37−7.27
■
(5H, m, aromatic protons), 5.79 (1H, m, CHCH2), 5.58 (1H, s,
PhCH), 5.02−4.92 (2H, m, CHCH2), 4.39 (1H, d, J = 7.7 Hz,
H-1′), 4.19 (1H, d, J = 7.8 Hz, H-1), 3.82−3.23 (14H, m, CHO of
sugar ring and OCH2CH2CH2CH2CHCH2), 2.03 (2H, m,
OCH2 CH2 CH2 CH2 CHCH2 ), 1.54 (2H, m, OCH2 -
CH2CH2CH2CHCH2), 1.41 (2H, m, OCH2CH2CH2CH2CH
CH2). MALDI-TOF MS (m/z) calcd for C25H36O11: 512.23. Found
535.48 [M + Na]+.
Materials. Chitosan was obtained from Yaizu Suisankagaku Industry
Co., Ltd. The Mw and polydispersity index (PDI = Mw/Mn) were
5000 kDa and 1.8, respectively, as measured by a GPC system
equipped with a LLS detector. The deacetylation degree was
1
80% (80% GlcN, 20% GlcNAc), as determined by the H NMR
spectrum. Compounds 3 and 6 were synthesized by methods
described previously.44,45 Stx1 and Stx2 were prepared from
Escherichia coli O157:H7 as described by Takeda et al.62,63 The purity
of the toxins was verified by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS−PAGE) and amino acid analysis (data not
shown). Recombinant C-terminal histidine-tagged Stx1B and Stx2B
were prepared from E. coli BL21(DE3) according to the published
procedures.22 Vero cells were maintained in DMEM supplemented
with 10% fetal calf serum and seeded in 96-well plastic microplates for
cytotoxicity assay. The O157:H7 E. coli N-9 strain is a clinical isolate from
patients with development of HUS. The specific pathogen free C57BL/
6 mice (female and 3-week-old) were used in the protection experiment.
All chemicals and solvents were of regent grade and used as supplied.
Synthesis of 5-Hexenyl 2,3-Di-O-acetyl-β-D-galactopyra-
nosyl-(1→4)-2,3,6-tri-O-acetyl-β-Dnoside (9). A solution of
compound 7 (6.7 g, 13.07 mmol), 4-dimethylaminopyridine
(DMAP, 35 mg, 0.29 mmol), and acetic anhydride (40 mL) in
pyridine (80 mL) was stirred at 60 °C overnight. The mixture was
allowed to return to room temperature, poured into cold water, and
then extracted with ethyl acetate. The organic layer was washed in turn
with saturated aqueous solution of sodium bicarbonate and brine,
dried (MgSO4), and concentrated to give the crude compound 8,
which was directly used in the next step without further purification.
Thus, to a stirred solution of 8 in dichloromethane (180 mL) at 0 °C
was added a 90% aqueous solution of trifluoroacetic acid (TFA, 20 mL).
The stirring was continued at 0 °C for 3 h. The mixture was di-
luted with dichloromethane, washed successively with water and
saturated aqueous solution of sodium bicarbonate, dried (MgSO4),
and evaporated. Purification of the residue by MPLC (5:1 ethyl
acetate/n-hexane) yielded compound 9 as a white powdery material
1
Measurements. H and 13C NMR spectra were recorded on a
Bruker DRX 600 spectrometer at 600 and 150 MHz, respectively. FAB
and MALDI-TOF mass spectra were obtained with a JEOL JMS-HX
110 and a Bruker BIFLEX III mass spectrometer, respectively, using
m-nitrobenzyl alcohol (NBA) or 2,5-dihydroxybenzoic acid as the
matrix. Elemental analysis was performed with a Yanaco CHN corder
MT-6. The Mw and PDI of chitosan and the conjugates were measured
by a GPC system (CTO-20A, Shimadzu Co., Ltd.) equipped with a
multiangle LLS detector (DAWN HELEOS-II, Wyatt Technology
Corp.) in acetate buffer solutions (pH 4.5). The purity of conjugates
1
(5.8 g, 70%). H NMR (CDCl3): δ 5.79 (1H, m, CHCH2), 5.21−
5.16 (2H, m, H-3 and H-2′), 5.02−4.93 (2H, m, CHCH2), 4.93−
4.90 (2H, m, H-2 and H-3′), 4.48 (1H, d, J = 7.8 Hz, H-1′), 4.47 (1H,
m, H-6a), 4.46 (1H, d, J = 7.9 Hz, H-1), 4.13 (1H, t, J = 3.7 Hz, H-4′),
4.10 (1H, dd, J = 11.9, 5.4 Hz, H-6b), 3.93 (1H, m, H-6′a), 3.84 (3H,
m, H-6′b, H-4 and OCH2CH2CH2CH2CHCH2), 3.64 (1H, m, H-5),
3.59 (1H, t, J = 5.3 Hz, H-5′), 3.49 (1H, m, OCH2CH2CH2CH2CH
CH2), 3.03 (1H, br, 4′-OH), 2.62 (1H, br, 6′-OH), 2.15-2.03 (17H, m,
COCH3 and OCH2CH2CH2CH2CHCH2), 1.58 (2H, m,
OCH2CH2CH2CH2CHCH2), 1.43 (2H, m, OCH2CH2CH2CH2CH
CH2). MALDI-TOF MS (m/z) calcd for C28H42O16: 634.25. Found
657.78 [M + Na]+.
1
(>95%) was established by the combined analyses of GPC, H NMR
spectroscopy, and CHN elemental analysis. TLC was performed on
the Merck silica gel glass plates 60F254. Optical rotation was measured
on a Perkin-Elmer polarimeter 343. Medium pressure liquid
chromatography (MPLC) was carried out using a Yamazen 700E
pump and a Yamazen model UV-10V detector with columns packed
with Merck silica gel N60 (40−63 mm). SPR analysis was performed
by a BIAcore biosensor system (BIAcore 3000, BIAcore Co., Ltd.).
Synthesis of 2,3,4,6-Tetra-O-(p-methoxybenzyl)-D-galacto-
pyranosyl Fluoride (5). To a solution of phenyl 2,3,4,6-tetra-O-(p-
methoxybenzyl)-1-thio-β-D-galactopyranoside (3, 6.0 g, 7.97 mmol) in
acetone/water (10:1, 130 mL) at 0 °C was added N-bromosuccinimide
(NBS, 5.7 g, 32.03 mmol). The mixture was allowed to return to
room temperature, stirred for 1 h, diluted with ethyl acetate, washed
successively with a 10% aqueous solution of potassium carbonate,
water, and brine, and then dried (MgSO4). The solvent was
evaporated and the resultant syrup was purified by MPLC (1:1 →
4:1 ethyl acetate/n-hexane) to give compound 4 as a white solid,
which was directly dissolved in dry tetrahydrofuran (THF, 50 mL).
The solution was cooled to −20 °C, and (diethylamino)sulfur
trifluoride (DAST, 1.2 mL, 9.36 mmol) was added. The mixture was
allowed to return to room temperature, stirred for 30 min, and then
cooled again to −20 °C. The reaction was quenched by methanol, and
the solvent was evaporated to give a residue, which was purified by
MPLC (1:2 ethyl acetate/n-hexane) to afford a light yellow syrup of
fluoride 5 (4.9 g) in 93% yield. 1H NMR (CDCl3): δ 7.33−6.87 (24H,
m, aromatic protons), 5.54 (0.5H, dd, J = 2.6 and 53.7 Hz, αH-1), 5.15
(1H, dd, J = 7.0 and 53.1 Hz, βH-1), 4.88−4.35 (12H, m, PhCH2),
4.09−3.86 (6H, m, CHO of sugar ring), 3.84−3.81 (18H, m,
PhOCH3), 3.50 (3H, m, H-6). MALDI-TOF MS (m/z) calcd for
C38H43FO9: 662.29. Found 685.28 [M + Na]+.
Synthesis of 5-Hexenyl 2,3,6-Tri-O-acetyl-β-D-galactopyra-
nosyl-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (10). To a
solution of compound 9 (5.7 g, 8.98 mmol) in dry pyridine (80 mL)
at −20 °C was added dropwise acetyl chloride (665 μL, 9.36 mmol).
The mixture was allowed to return to room temperature and stirred
for 2 h. Water (30 mL) was added to the mixture, and the solvent was
removed by evaporation. The resultant residue was dissolved in ethyl
acetate, washed in turn with water and brine, dried (MgSO4), and
evaporated. Purification of the residue by MPLC (1:1 → 4:1 ethyl
acetate/n-hexane) afforded a white powdery material of compound 10
1
(5.1 g) in 84% yield. H NMR (CDCl3): δ 5.79 (1H, m, CHCH2),
5.22−5.16 (2H, m, H-3 and H-2′), 5.03−4.95 (2H, m, CHCH2),
4.93−4.89 (2H, m, H-2 and H-3′), 4.50 (1H, m, H-6a), 4.47 (1H, d,
J = 7.8 Hz, H-1′), 4.46 (1H, d, J = 7.8 Hz, H-1), 4.30 (2H, m, H-6′a,b),
4.11 (1H, dd, J = 11.9, 5.3 Hz, H-6b), 4.01 (1H, t, J = 4.2 Hz, H-4′),
3.86 (1H, m, OCH2CH2CH2CH2CHCH2), 3.79 (1H, t, J =
9.4 Hz, H-4), 3.72 (1H, t, J = 6.5 Hz, H-5′), 3.62 (1H, m, H-5), 3.48
(1H, m, OCH2CH2CH2CH2CHCH2), 2.43 (1H, d, J = 5.1 Hz,
4′-OH), 2.14-2.04 (20H, m, COCH3 and OCH2CH2CH2CH2CH
CH2), 1.58 (2H, m, OCH2CH2CH2CH2CHCH2), 1.43 (2H, m,
OCH2CH2CH2CH2CHCH2). 13C NMR (CDCl3): δ 170.8, 170.4,
170.2, 170.1, 169.5, 169.3, 138.5, 114.7, 101.0, 100.6, 76.3, 73.3, 72.7,
72.6, 72.1, 71.7, 69.9, 69.5, 66.7, 62.2, 61.9, 33.3, 28.8, 25.1, 20.9, 20.8,
20.7, 20.6, 20.5. MALDI-TOF MS (m/z) calcd for C30H44O17: 676.26.
Found 699.41 [M + Na]+.
Synthesis of 5-Hexenyl 4,6-O-Benzylidene-β-D-galacto-
pyranosyl-(1→4)-β-D-glucopyranoside (7). A stirred mixture of
5-hexenyl β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside (6, 12.2 g,
28.74 mmol), benzaldehyde dimethyl acetal (14 mL, 93.28 mmol), and
p-toluenesulfonic acid monohydrate (250 mg, 1.32 mmol) in N,N-
dimethylformamide (DMF, 125 mL) was heated to 55 °C, and the
Synthesis of 5-Hexenyl 2,3,4,6-Tetra-O-(p-methoxybenzyl)-
α-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-β-D-galactopyr-
anosyl-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (11). To a
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dx.doi.org/10.1021/jm201570s | J. Med. Chem. 2012, 55, 2702−2710