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X. Zeng et al. / Fitoterapia 83 (2012) 609–616
(1H, dd, J=11.2, 6.4 Hz, H-1b), 4.47 (1H, m, H-2), 4.15 (2H, br
d, J=5.6 Hz, H-3), 3.90 (2H, t, J=6.4 Hz, H-34′), 2.37 (2H, t,
J=7.6 Hz, H-2′),1.77 (2H, m, H-3′), 1.65 (2H, m, H-33′), 1.53
(2H, m, H-32′), 1.22–1.34 (methylene band); 13C NMR data
(C5D5N, 100 MHz) δ 175.0 (C, C-1′), 72.2 (CH, C-2), 68.0 (CH2,
C-1), 65.6 (CH2, C-3), 63.3 (CH2, C-34′), 35.7 (CH2, C-2′), 35.1
(CH2, C-33′), 30.7–31.3 (methylenes), 27.8 (CH2, C-32′), 26.6
14.1 (CH3, C-17′ or C-18″); ESI-MS m/z 601 [M+H]+; HR-
ESI-MS m/z 623.3705 (calcd. for C38H64O5Na, 623.3761).
1-octadecanoyl-2-nonadecanoyl-3-O-(6-amino-6-deoxy)-
β-D-glucopyranosyl-sn-glycerol (13) was a white amorphous
powder; C46H89NO9; [α]25D +35.1 (c 0.20, CHCl3); 1H NMR
(DMSO-d6, 400 MHz) δ 5.42 (1H, d, J=4.0 Hz, -NH), 5.13
(1H, m, H-2), 4.77 (1H, d, J=4.4 Hz, –OH), 4.66 (1H, d,
J=6.4 Hz, –OH), 4.57 (1H, d, J=3.2 Hz, H-1‴), 4.35 (1H, dd,
J=12, 2.8 Hz, H-1a), 4.14 (1H, dd, J=11.8, 7.4 Hz, H-1b),
3.89 (1H, dd, J=10.4, 6.0 Hz, H-3a), 3.77 (1H, m, H-5‴),
3.39 (2H, m, H-3b and H-3‴), 3.18 (1H, m, H-2‴), 2.91 (2H,
m, H-6‴a and H-4‴), 2.53 (1H, m, H-6‴b), 2.27 (4H, m, H-2′
and H-2″), 1.50 (4H, m, H-3′ and H-3″), 1.23 (methylene
band), 0.85 (6H, t, J=6.8 Hz, H-18′ and H-19″); 13C NMR
data (DMSO-d6, 100 MHz) δ 173.0 (C, C-1′ or C-1″), 172.8
(C, C-1′ or C-1″), 98.8 (CH, C-1‴), 74.9 (CH, C-4‴), 73.4 (CH,
C-3‴), 72.1 (CH, C-2‴), 70.2 (CH, C-2), 69.0 (CH, C-5‴), 65.2
(CH2, C-3), 63.0 (CH2, C-1), 55.2 (CH2, C-6‴), 34.0 (CH2, C-2′
or C-2″), 33.9 (CH2, C-2′ or C-2″), 31.8 (CH2, C-16′ and
C-17″), 28.9–29.5 (methylenes), 24.9 (CH2, C-3′ and C-3″),
22.6 (CH2, C-17′ and C-18″), 14.4 (CH3, C-18′ and C-19″);
ESI-MS m/z 834 [M+Cl]−; HR-ESI-MS m/z 834.6171 (calcd.
for C46H89NO9Cl, 834.6143).
(CH2, C-3′); ESI-MS m/z 597 [M–H]−
; HR-ESI-MS m/z
599.5617 (calcd. for C37H75O5, 599.5589).
1-[nonadeca-(9Z,12Z)-dienoyl]-sn-glycerol (10) was a col-
orless oil; [α]25D +3.1 (c 0.20, CHCl3); 1H NMR (CDCl3,
400 MHz) δ 5.28 (4H, m, H-9′, H-10′, H-12′ and H-13′), 4.11
(2H, m, H-1), 3.98 (1H, t, J=7.0 Hz, H-2), 3.57 (2H, t,
J=6.6 Hz, H-3), 2.70 (2H, t, J=6.6 Hz, H-11′), 1.99 (2H, m,
H-2′), 1.93–2.01 (4H, m, H-8′ and H-14′), 1.47–1.57 (6H, m,
H-3′, H-18′ and H-17′), 1.18–1.23 (methylene band), 0.81
(3H, t, J=6.8 Hz, H-19′); 13C NMR data (CDCl3, 100 MHz) δ
174.0 (C, C-1′), 130.2 (CH CH, C-9′, C-10′, C-12′ or C-13′),
130.0 (CH CH, C-9′, C-10′, C-12′ or C-13′), 128.1 (CH CH,
C-9′, C-10′, C-12′ or C-13′), 127.9 (CH CH, C-9′, C-10′, C-12′
or C-13′), 68.3 (CH, C-2), 64.9 (CH2, C-1), 63.0 (CH2, C-3),
34.1 (CH2, C-2′), 31.9 (CH2, C-17′), 29.1–29.7 (methylenes),
28.6 (CH2, C-11′), 27.2 (CH2, C-8′and C-14′), 24.9 (CH2,
C-3′), 22.7 (CH2, C-18′), 14.1 (CH2, C-19′); ESI-MS m/z 391
[M+Na]+
22H40O4Na, 391.3513).
1-[12-hydroxypentatriaconta-(13E,15Z)-dienoyl]-sn-glyc-
erol (11) was a colorless oil; [α]25D +10.1 (c 0.20, CHCl3); 1
;
HR-ESI-MS m/z 391.3589 (calcd. for
2.4. Methanolysis of compounds (1–4 and 13)
C
Each compound (10 mg) was added to a mixture of HCl
(10 mL, 1 N) and MeOH (30 mL), with this solution refluxed
for 10 h under magnetic stirring. TLC was developed in
order to judge whether the reaction was complete. Then,
50 mL H2O were added to the refluxed mixture, which was
extracted with n-hexane (3×50 mL). One fatty acid methyl
ester or two were afforded from the n-hexane extract. The
MeOH/H2O phase was evaporated under reduced pressure
to yield a sphingosine or a mixture of glycerol and 1-[(6-
amino-6-deoxy)-β-D-glucopyranosyl]-methyl ester.
H
NMR (CDCl3, 400 MHz) δ 6.42 (1H, dd, J=15.0, 11.0 Hz, H-
14′), 5.90 (1H, t, J=10.8 Hz, H-15′), 5.59 (1H, dd, J=15.8,
6.6 Hz, H-13′), 5.39 (1H, dd, J=18.6, 7.8 Hz, H-16′), 4.12
(2H, m, H-1), 4.09 (1H, m, H-12′), 3.87 (1H, m, H-2), 3.61
(1H, m, H-3), 3.55 (1H, m, H-3), 2.25–2.33 (2H, m, H-2′),
2.11 (2H, m, H-17′), 1.97 (2H, m, H-3′), 1.47–1.57 (4H, m,
H-11′ and H-34′), 1.18–1.25 (methylene band), 0.81 (3H, t,
J=6.8 Hz, H-35′); 13C NMR data (CDCl3, 100 MHz) δ 173.4
(C, C-1′), 134.7 (CH CH, C-13′), 132.1 (CH CH, C-16′), 126.7
(CH CH, C-15′), 124.9 (CH CH, C-14′), 71.8 (CH, C-12′), 69.3
(CH, C-2), 64.1 (CH2, C-1), 62.1 (CH2, C-3), 36.3 (CH2,
C-11′), 33.2 (CH2, C-2′), 32.7 (CH2, C-18′), 30.9 (CH2, C-19′),
30.5 (CH2, C-20′), 27.9–28.7 (methylenes), 26.7 (CH2,
C-17′), 24.3 (CH2, C-3′), 23.9 (CH2, C-10′), 21.5 (CH2, C-34′),
13.1 (CH3, C-35′); ESI-MS m/z 607 [M–H]−; HR-ESI-MS m/z
631.4552 (calcd. for C38H72O5Na, 631.4603).
1-(heptadeca-6Z,9Z-dienoyl)-3-(octadeca-6Z,9Z,12Z-trien-
oyl)-sn-glycerol (12) was a colorless oil; [α]25D+1.3 (c 0.20,
CHCl3); 1H NMR (CDCl3, 400 MHz) δ 5.23–5.33 (10H, m, H-6′,
H-7′, H-9′, H-10′, H-6″, H-7″, H-9″, H-10″, H-12″ and H-13″),
5.19 (1H, m, H-2), 4.22 (2H, dd, J=8.2, 3.8 Hz, H-1), 4.08 (2H,
dd, J=11.8, 5.8 Hz, H-3), 2.72 (6H, m, H-8′, H-8″ and H-11″),
2.25 (4H, m, H-2′ and H-2″), 1.93 (8H, m, H-5′, H-11′, H-5″
and H-14″), 1.54 (8H, m, H-3′, H-14′, H-3″ and H-18″),
1.18–1.23 (methylene band), 0.81 (6H, m, H-15′ and H-18′);
13C NMR data (CDCl3, 100 MHz) δ 173.2 (C, C-1′ or C-1″),
172.8 (C, C-1′ or C-1″), 127.1–132.0 (CH CH, C-6′, C-7′, C-9′,
C-10′, C-6′, C-7″, C-9″, C-10″, C-12″ and C-13″), 68.9 (CH,
C-2), 62.1 (CH2, C-1 and C-3), 34.2 (CH2, C-2′ or C-2″), 34.0
(CH2, C-2′ or C-2″), 31.5 (CH2, C-13′ and C-16″), 27.9–28.7
(methylenes), 27.2 (CH2, C-5′, C-11′, C-5″ and C-14″), 25.6
(CH2, C-8′, C-8″ and C-11″), 24.8 (CH2, C-3′ and C-3″), 22.6
(CH2, C-16′), 20.6 (CH2, C-17″), 14.2 (CH3, C-17′ or C-18″),
2.5. Epoxidation of compouds (2a, 3a, 7, 10, 11 and 12)
A solution of substrate (10 mg) in 3 mL CH2Cl2 was stirred
and cooled in an ice bath as a solution of m-CPBA (50 mg) in
CH2Cl2 (5 mL) was added dropwise. The resulting mixture was
stirred in the ice bath for an additional 30 min. TLC was devel-
oped to judge whether the reaction was complete. The mixture
was washed with 10% Na2CO3 (5×6 mL) and saturated NaCl so-
lution (10 mL). The organic layer was dried (Na2SO4) and the
solvent was removed on a rotary evaporator to give a viscous
oil, and GC–MS was performed to determine the position of
the double bond.
2.6. Enzymatic hydrolysis of 13
Compound 13 (10 mg) were dissolved in 10 mL of dioxane-
H2O (1:1) and treated with lipase enzyme type III (2 mg, 46 U,
from a Pseudomonas species, lot 093 K0698, Sigma-Aldrich) at
37 °C, with shaking for 8 h. TLC was developed in order to
judge whether the reaction was complete. The reaction mix-
tures were quenched with 5% acetic acid (2.5 mL), and the prod-
uct was dried under reduced pressure. The crude residues were
dissolved in water and extracted with CHCl3, concentrated
under reduced pressure, and analyzed by ESI-MS.