A. J. Humphrey et al. / Bioorg. Med. Chem. 10 (2002) 3175–3185
3183
formic acid (200 mL), collecting 6-mL fractions. Frac-
tions were analysed by silica thin-layer chromatography
[eluent ethyl acetate/acetic acid/water (5:2:2)], staining
with ninhydrin. Fractions 1–4 contained GlcNAc,
ManNAc and phosphate salts. Neu5Ac (Rf 0.10) was
present in fractions 17–24, which were lyophilised to
give the product N-acetyl-d-neuraminic acid as a brown
powder (48 mg, 33%), which was found to be >95%
6.4 Hz, H-90), 3.46 (1H, d, J=9.2 Hz, H-7), 2.21 (1H,
dd, J=12.7, 4.6 Hz, H-3eq), 1.78 (1H, dd, J=13.0, 11.4
Hz, H-3ax) ppm.
N-Difluoroacetyl-D-neuraminic acid 6f. 39% yield;
HPLC retention time 11.0 min; dH (300 MHz, D2O) 6.09
(1H, t, JH-F=54 Hz, CHF2–), 4.10 (1H, d, J=10.3 Hz,
H-6), 4.06 (1H, m, H-4), 3.96 (1H, t, J=10.1 Hz, H-5),
3.75 (1H, dd, J=11.8, 2.6 Hz, H-9), 3.65 (1H, m, H-8),
3.51 (1H, dd, J=12.4, 6.3 Hz, H-90), 3.44 (1H, d, J=9.2
Hz, H-7), 2.23 (1H, dd, J=13.2, 4.8 Hz, H-3eq), 1.80
(1H, dd, J=12.8, 11.2 Hz, H-3ax) ppm.
1
pure by H NMR spectroscopy.
The same procedure was used to prepare N-propionyl,
N-hexanoyl, and N-benzoyl d-neuraminic acids, from
enriched N-acyl-d-mannosamine/N-acyl-d-glucosamine
mixtures. N-Chloroacetyl, N-difluoroacetyl and N-tri-
fluoroacetyl d-neuraminic acids were prepared using the
same procedure from samples of pure N-acyl-d-manno-
samines. Isolated yields and data as follows.
N-trifluoroacetyl-D-neuraminic acid 6g. 43% yield;
HPLC retention time 11.9 min; dH (300 MHz, D2O) 4.13
(1H, d, J=10.5 Hz, H-6), 4.06 (1H, m, H-4), 3.98 (1H, t,
J=9.8 Hz, H-5), 3.76 (1H, dd, J=11.4, 2.7 Hz, H-9),
3.66 (1H, m, H-8), 3.51 (1H, dd, J=11.8, 6.4 Hz, H-90),
3.43 (1H, d, J=9.2 Hz, H-7), 2.24 (1H, dd, J=13.1, 4.8
Hz, H-3eq), 1.81 (1H, dd, J=13.1, 11.0 Hz, H-3ax) ppm.
N-Acetyl-D-neuraminic acid (6a). Yield 33%; HPLC
retention time 12.2 min; dH (300 MHz, D2O) 3.91 (1H,
d, J=8.1 Hz, H-6), 3.86 (1H, m, H-4), 3.83 (1H, t,
J=9.4 Hz, H-5), 3.76 (1H, dd, J=11.2, 2.3 Hz, H-9),
3.68 (1H, m, H-8), 3.53 (1H, dd, J=11.2, 5.9 Hz, H-90),
3.43 (1H, d, J=8.8 Hz, H-7), 2.13 (1H, dd, J=12.7, 5.4
Hz, H-3eq), 1.97 (3H, s, CH3CO–), 1.74 (1H, dd, J=12.7,
10.9 Hz, H-3ax) ppm, m/z (ES+) 310 (MH+ 69%).
13C NMR data (75 MHz, D2O) for 6a-g (ND=not
detected).
6a
6b
6c
6d
6e
6f
6g
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
177.4 181.5 180.8 203.4 173.0 188.2 200.6
97.9 98.0 98.0 98.0 98.0 98.0 98.0
41.4 41.5 41.6 41.7 41.5 41.5 41.5
69.3 69.2 69.2 69.3 69.1 69.0 68.8
54.6 54.5 54.5 55.3 55.1 54.7 55.2
72.7 72.7 72.8 72.8 72.7 72.4 72.2
70.8 70.8 71.0 71.1 70.8 70.8 70.8
73.0 73.0 73.0 73.0 72.8 72.8 72.7
65.7 65.7 65.8 65.8 65.7 65.7 65.7
N-Propionyl-D-neuraminic acid (6b). Yield 38%; HPLC
retention time 12.0 min; dH (300 MHz, D2O) 4.00 (1H,
m, H-4), 3.88 (1H, d, J=11.1 Hz, H-6), 3.84 (1H, t,
J=9.6 Hz, H-5), 3.75 (1H, dd, J=11.8, 2.2 Hz, H-9),
3.66 (1H, ddd, J=9.6, 5.9, 2.2 Hz, H-8), 3.52 (1H, dd,
J=11.8, 5.9 Hz, H-90), 3.44 (1H, d, J=9.6 Hz, H-7),
2.27–2.17 (1H, m, H-3eq), 2.23 (2H, q, J=7.4 Hz,
CH2CO–), 1.79 (1H, dd, J=13.2, 11.8 Hz, H-3ax), 1.04
(3H, t, J=7.4 Hz, CH3CH2CO–) ppm, m/z (ES+) 324.2
(MH+ 100%).
Acyl C¼O 176.0 176.0 176.1 ND176.3 176.2 176.0
C-2
C-3
C-4
C-5
C-6
24.7 31.9 38.6 ND44.9 110.9 138.4
12.2 33.2 129.8
27.7 131.4
24.3 134.9
15.8
N-Hexanoyl-D-neuraminic acid (6c). Yield 5%; HPLC
retention time 15.2 min; dH (300 MHz, D2O) 4.00 (1H,
m, H-4), 3.94 (1H, d, J=10.3 Hz, H-6), 3.82 (1H, t,
J=10.3 Hz, H-5), 3.73 (1H, dd, J=11.8, 2.2 Hz, H-9),
3.64 (1H, ddd, J=8.8, 5.9, 2.2 Hz, H-8), 3.49 (1H, dd,
J=11.8, 5.9 Hz, H-90), 3.43 (1H, d, J=8.8 Hz, H-7),
2.23–2.16 (1H, m, H-3eq), 2.19 (2H, t, J=6.6 Hz,
CH2CO–), 1.76 (1H, t, J=12.5 Hz, H-3ax), 1.50 (2H,
qui, J=6.6 Hz, CH2CH2CO–), 1.19 (4H, m), 0.76 (3H,
t, J=6.6 Hz, CH3) ppm, m/z (ES+) 366.3 (MH+ 43%).
Preparation of the sialoglycolipid neomembrane
One track at a time of an HPA sensor chip was
impregnated with bovine brain lipid (Type VII brain
extract from Sigma, containing bovine brain phospholi-
pids and glycolipids) in a BIAcore 2000 instrument at
30 ꢁC. A concentration of lipid (16.6 mg/10 mL) was
suspended in 0.01 M HEPES, 0.15 M NaCl pH 7.4 and
homogenized thoroughly at 30ꢁC before injection (2Â325
mL) onto a sensor chip at a flow rate of 5 mL/min. The
tracks were washed between injections with a fast flow of
buffer to remove excess layers of lipid and with 50 mM
NaOH (2Â15 mL) to produce a stable neomembrane.
N-Benzoyl-D-neuraminic acid (6d). Yield 11%; HPLC
retention time 14.3 min; dH (300 MHz, D2O) 7.7–7.35
(5H, m, Ar), 4.2–3.8 (3H, m, H-4, H-5, H-6), 3.72 (1H,
dd, J=11.7, 2.2 Hz, H-9), 3.67 (1H, ddd, J=8.8, 5.9, 2.2
Hz, H-8), 3.51 (1H, d, J=8.8 Hz, H-7), 3.48 (1H, dd,
J=11.1, 5.9 Hz, H-90), 2.24 (1H, dd, J=13.2, 3.7 Hz, H-
3eq), 1.82 (1H, dd, J=12.9, 11.8 Hz, H-3ax) ppm, m/z
(ES+) 372.3 (MH+ 16%).
N-Chloroacetyl-D-neuraminic acid 6e. 10% yield; HPLC
retention time 11.6 min; dH (300 MHz, D2O) 4.08 (2H, s,
CH2Cl-), 4.04 (1H, d, J=9.0 Hz, H-6), 4.00 (1H, m, H-
4, 3.89 (1H, t, J=10.1 Hz, H-5), 3.73 (1H, dd, J=11.9,
2.6 Hz, H-9), 3.66 (1H, m, H-8), 3.50 (1H, dd, J=11.5,
Interaction of influenza virus particles to the
neomembrane by surface plasmon resonance
Human influenza A virus strain A/PR/8/34 (PR8;
H1N1) (harvested from the allantoic fluid of 10-day-old