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U.R. Mughal et al.
showed a broad signal for the protons of four
methylene groups in the range of d 1.48–
1.96, whereas rest of the methylene protons
resonated at d1.18–1.22 (br. s, 28 £ CH2). A
triplet for the terminal methyl groups was
observed at d 0.82 (6H, t, J ¼ 6.4 Hz). The
anomeric proton showed the signal at d 4.22
(1H, d, J ¼ 7.5 Hz), the oxymethine protons
of the hexose unit resonated in the range of d
3.36–3.17, and the oxymethylene protons
appeared at d 3.77 (1H, d, J ¼ 11.2 Hz) and
3.68 (1H, d, J ¼ 11.2 Hz). Two oxymethy-
lene protons resonated at d 4.01 (dd, J ¼ 4.6,
10.7 Hz) and 3.74 (dd, J ¼ 7.8, 10.7 Hz), and
three oxymethine protons were observed at d
3.95 (dd, J ¼ 3.6, 7.6 Hz), 3.48 (dd, J ¼ 5.0,
7.2 Hz), and 3.45–3.46 (1H, m), confirming
that compound 1 is a glycoside of sphingo-
lipid [4]. The 13C NMR (BB and DEPT)
spectrum showed the signal of an amide
carbonyl carbon at d 175.7 whereas the
olefinic methines resonated at d 130.2 and
129.2. The azomethine signal, characteristic
of sphingolipid, appeared at d 50.1, whereas
an oxymethylene carbon resonated at d 68.6
along with three resonances of oxymethine
carbons at d 74.2, 72.1, and 72.0. The
anomeric carbon showed signal at d 102.8
whereas the oxymethine and oxymethylene
carbons of the hexose unit appeared in the
range of d 76.0–61.0. The methylenes of the
aliphatic chains resonated in the range of d
22.6–34.2 with the two terminal methyl
carbons at d 14.0.
ton H-40 at d 3.45–3.46, revealing the
position of two hydroxyl groups at C-30
and C-40, respectively. Methanolysis of
compound 1 provided the glycone, which
could be identified as a mixture of a- and
b-anomers of methyl D-glucoside. The
fatty acid methyl ester was characterized
by mass spectrometry as methyl 2-hydro-
xyicosanoate (m/z: 342 [M]þ) [6]. Thus,
the length of sphingosine base chain was
of 22 carbons with double bond located in
the base chain. In HMBC experiment, the
oxymethine proton at C-40 (d 3.45–3.46)
showed 2J and 3J correlations with
methylene carbons at d 32.3 and 27.2,
allowing us to assign these to C-50 and C-
60, respectively. The methylenic protons of
2
C-60 (d 1.18–1.22) showed J correlation
with C-70 (d 26.0) and 3J correlation with
C-80 (d 130.3). The olefinic protons at d
5.27 and 5.30 showed 3J correlations with
C-70 (d 26.0) and C-100 (d 25.3), respect-
ively, allowing us to assign the double
bond to C-80. The fragmentation peaks in
the EI-MS at m/z 471, 209, and 183 were
due to the cleavage of vinylic bond by
McLafferty rearrangement, further con-
firming the position of double bond at C-80
(Figure 2). The attachment of O-b-D-
glucose moiety was confirmed at C-10 by
downfield shift of C-10 and confirmed
through HMBC experiment; the anomeric
proton at d 4.22 shows 3J correlation with
C-10 at d 68.6. The chemical shifts of
proton at d 4.14–4.17 (H-20) and the
carbon signals at d 68.6 (C-10), 50.1 (C-20),
74.2 (C-30), 72.1 (C-40), 175.7 (C-1), and
72.0 (C-2) were very close to the
phytoceramides having 2R, 20S, 30S, and
40R-stereochemistry [7], revealing the
same configuration at C-20, C-30, C-40,
and C-2 in 1, which was further supported
by NOESY spectrum; the azomethine
proton at d 4.15–4.17 (H-20) showed
correlations with H-2 at d 3.95, H-40 at d
3.45–3.46, and Hb-10 at d 4.01. On the
other hand, the H-30 at d 3.48 showed
correlations with Ha-10 at 3.74 as well as
the amide proton at d 8.47. On the basis of
The (Z)-configuration of the double
bondwasassignedbasedonthe significantly
upfieldshifted13CNMRsignalsforC-70 and
C-100 at d 26.0 and 25.3, respectively, and
due to the relatively small coupling constant
between H-80 and H-90 (W1/2 ¼ 3.5 Hz) [5].
The larger coupling constant of the anome-
ric proton allowed us to assign b-configur-
ation to the hexose unit.
I
In the H-IH-COSY spectrum, azo-
methine proton (d 4.14–4.17) showed
correlations with oxymethylene protons
H-10 at d 4.01 and 3.74 and oxymethine
proton H-30 at d 3.48 which further
correlated with another oxymethine pro-
Mughal, Uzma Rasheed
