Gracilarioside and gracilamides from the Red alga Gracilaria asiatica

Article abstract of DOI:10.1021/np060043e

One gracilarioside and two gracilamides with unusual cyclopropane- containing alkyl chains were isolated from the red alga Gracilaria asiatica. Their structures were determined by spectroscopic methods and microscale chemical degradation. To our knowledge, no ceramides with a cyclopropane ring have been reported from marine organisms. These three compounds were mildly cytotoxic to the human A375-S2 melanoma cell line.

Full text of DOI:10.1021/np060043e

1488
J. Nat. Prod. 2006, 69, 1488-1491
Gracilarioside and Gracilamides from the Red Alga Gracilaria asiatica
Yi Sun,† Youjun Xu,‡ Kai Liu,‡ Huiming Hua,† He Zhu,† and Yuehu Pei*,†
School of Traditional Chinese Materia Medica and School of Pharmaceutical Engineering, Shenyang Pharmaceutical UniVersity,
Shenyang 110016, People’s Republic of China
ReceiVed January 26, 2006
One gracilarioside and two gracilamides with unusual cyclopropane-containing alkyl chains were isolated from the red
alga Gracilaria asiatica. Their structures were determined by spectroscopic methods and microscale chemical degradation.
To our knowledge, no ceramides with a cyclopropane ring have been reported from marine organisms. These three
compounds were mildly cytotoxic to the human A375-S2 melanoma cell line.
Many highly bioactive sphingolipids have been discovered from
marine organisms able to act as biomediators of activation, cell
agglutination, intracellular communication, and cell development.1
Moreover, an agelasphin derivative, a glycosphingolipid isolated
from the sea sponge Agelas mauritiamus, has been in phase I clinical
trials for the treatment of cancer.2 In a search for novel bioactive
constituents from the marine red alga Gracilaria asiatica (C. F.
Chang et B. M. Xia), one gracilarioside (1) and two gracilamides
(2, 3) bearing an unusual cyclopropane ring were isolated. Their
structures were established by spectroscopic methods and chemical
degradation. Herein, we describe the isolation, structure elucidation,
and biological activities of these three compounds, named gracilari-
oside and gracilamides A and B. Cerebrosides with cyclopropane-
containing chains are rare in nature. Although Fattorusso reported
four cyclopropane-containing cerebrosides from two marine
sponges,3,4 no other such cerebrosides or ceramides have been
discovered. In this paper, gracilamides A and B are reported here
as the first ceramides from marine organisms with cyclopropane-
containing alkyl chains.
addition, signals corresponding to a glucopyranose moiety were
also identified from 1H and 13C NMR experiments (Table 1).5 The
anomeric proton doublet (δ 4.86, J ) 7.7 Hz) indicated the
â-orientation of the proton, and it was identified from its correlation
peak with the relevant carbon C-1′′ in the HMQC spectrum.
Examination of the HMBC spectrum showed that the amide
methine C-2 was coupled with the NH doublet at δ 8.51, the
oxymethylene protons at δ 4.74 and 4.48 (H-1), and the oxymethine
proton at δ 4.30 (H-3). The HMBC correlations between H-3 and
the carbon at δ 72.8 and 51.7 also showed that there was another
hydroxyl function at C-4. In fact, HMBC correlations of the
carbonyl at 175.7 to H-2′ confirmed the presence of a side chain
of an R-hydroxyl fatty acid. The double bond in 1 was assigned at
C-6 and C-7 in the sphingoid long chain base through which
correlations in the HMBC were found between H-5 and δ 72.8,
132.8, and 128.3. The geometry of the C-6/C-7 double bond was
determined to be trans by the large vicinal coupling constant (J )
14.8 Hz) displayed between H-6 and H-7.
In addition, the three upfield shifted multiplets at -0.21 (1H),
0.66 (1H), and 0.74 (2H) in the 1H NMR and the relevant carbons
assigned by the HMQC spectrum showed that 1 contained an
uncommon cyclopropane ring (Table 1). Therefore, to confirm the
location of the cyclopropane ring and establish the length of the
alkyl chains, microscale chemical degradation of 1 was carried out
using part of the method that Fattorusso reported.3 We were
fortunate to obtain the corresponding methyl fatty ester by starting
from a trace amount of 1 (1 mg) via successive reduction,
methanolysis, Lemieux oxidation, and methylation. The ester was
submitted for GC-MS analysis (Scheme 1) to determine the correct
position of the cyclopropane ring and the length of each chain.
Thus, 1 mg of 1 was subjected to hydrogen reduction catalyzed by
PtO2 using acetic acid as a solvent at room temperature.6 After the
reduction of the double bond and the cleavage of the cyclopropane
ring, the mixture of two methyl-branched alkyl chains and one
unbranched chain obtained was treated with aqueous HCl/MeOH
to produce methanolysis, giving the products R-hydroxyacid methyl
ester (4), phytosphingosine (5), and a methyl glycoside, respec-
tively.7 Furthermore, the D configuration of the glucose unit was
determined by HPLC with D- and L-glucose standard samples, which
was derived from sugars and chiral R-methylbenzylamine (MBA)
in the presence of sodium cyanoborohydride.8 In order to identify
the location of the cyclopropane ring, 4 and 5 were then determined
on the basis of the 1H NMR analysis. Two obvious additional
branched methyl signals at δ 0.92 (d, J ) 6.7 Hz) and 0.98 (d, J
) 6.7 Hz) were observed on the long-chain bases (5), but there
were no branched methyl signals of 4 in the 1H NMR. Nevertheless,
it was difficult to confirm the length of the chain, and the correct
position of the branched methyl could only be obtained by 1H NMR
analysis. Hence, similar chemical degradations of 4 and 5 were
then carried out. Following cleavage of the bonds C-3-C-4 of 5
Gracilarioside (1), white amorphous powder, had a molecular
formula of C49H93NO10 obtained from its HRESIMS at m/z
856.6898 ([M + H]+, calcd 856.6878), indicating four degrees of
1
unsaturation. The H NMR spectrum for 1 displayed resonances
typical of an aliphatic chain (δ 1.26) and several multiplets from δ
3.98 to 5.24, which are reminiscent of a phytosphingosine-type
cerebroside. Moreover, an anomeric carbon (δ 106.2) and a number
of aliphatic carbons together with an amide function (δ 175.7 and
51.7) were deduced from 13C NMR spectroscopic data, suggesting
that 1 was a monocerebroside (Table 1).
The structure of 1 was assigned by the analysis of 2D NMR
data, including HMQC and HMBC experimental methods. In
* Corresponding author. Tel/Fax: +86 24 23986485. E-mail: peiyueh@
vip.163.com.
† School of Traditional Chinese Materia Medica.
‡School of Pharmaceutical Engineering.
10.1021/np060043e CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/12/2006

Notes
Journal of Natural Products, 2006, Vol. 69, No. 10 1489
Table 1. NMR Spectroscopic Data for Gracilarioside (1) and Two Gracilamides (2, 3) (C5D5N)a
gracilarioside (1)
H (m, J, Hz)
gracilamide A (2)
H (m, J, Hz)
gracilamide B (3)
H (m, J, Hz)
pos.
δ
δC
δ
δC
pos.
δ
δC
62.0
1
4.74 (dd, 10.6, 6.4)
4.48 (dd, 10.6, 4.2)
5.24 (m)
4.30 (m)
4.23 (m)
2.99 (m)
2.67 (m)
5.95 (ddd, 14.8, 8.0, 6.9)
5.72 (ddd, 14.8, 7.6, 6.6,)
2.05 (m)
70.6
4.62 (dd, 10.5, 4.0)
4.50 (dd, 10.5, 6.2)
5.10 (m)
4.33 (m)
4.30 (m)
3.06 (m)
2.72 (m)
5.98 (ddd, 14.5, 8.1, 6.8)
61.8
1
4.52 (dd, 10.7, 4.6)
4.43 (dd, 10.7, 5.0)
5.14 (m)
4.35 (m)
4.28 (m)
1.93 (m)
2.24 (m)
1.91 (m)
1.26
2
3
4
5
51.7
75.4
72.8
37.5
52.7
76.1
73.1
37.5
2
3
4
5
53.0
76.8
73.0
34.1
6
7
8
132.8
128.3
33.2
29.6
30.6
29.0
16.2
32.1
22.9
14.3
11.4
132.6
128.1
33.0
29.4
30.4
28.8
16.0
31.9
22.7
14.0
11.1
6
26.7
29.9, 30.4
32.2
5.73 (ddd, 14.5, 8.3, 7.1)
2.04 (m)
7-23, 5′-9′
24, 14′
25, 15′
1.26
1.26
9-10
4′-22′
11, 14
12, 13
15, 23′
16, 24′
17, 25′
18
1.26
1.46
1.46
1.26
1.44
1.44
23.0
26, 16′
0.86
14.3
175.2
72.5
0.74 (m)
0.72 (m)
1′
2′
3′
1.26
1.26
1.26
1.26
0.85
4.62 (m)
2.05 (m)
2.21 (m)
1.69 (m)
1.78 (m)
35.7
0.88 (t, 6.8)
-0.21 (ddd, 5.3, 5.3, 4.3)
0.66 (ddd, 4.3, 3.9, 3.9)
-0.20 (ddd, 5.1, 5.1, 3.8)
0.65 (ddd, 3.8, 3.3, 3.3)
4′
25.8
1′
2′
3′
175.7
72.4
35.6
175.0
72.2
35.5
10′, 13′
11′, 12′
17′
1.43
0.73 (m)
-0.22 (ddd, 5.0, 5.0, 4.5)
29.0
16.2
11.4
4.54 (m)
2.17 (m)
1.96 (m)
4.53 (m)
2.22 (m)
1.94 (m)
1′′
2′′
3′′
4′′
5′′
6′′
4.86 (d, 7.7)
4.41 (t, 7.8)
4.10 (m)
4.50 (m)
3.98 (m)
4.43 (dd, 12.6, 5.0)
4.40 (dd, 12.6, 5.0)
8.50
106.2
72.6
75.2
70.3
77.1
62.4
0.68 (ddd, 4.5, 3.5, 3.5)
NH
8.58
NH
8.60
1
a 1H was recorded at 600 MHz; 13C was recorded at 125 MHz; H was recorded at 300 MHz; 13C was recorded at 75 MHz.
Scheme 1. Reagents and Conditions for the Microscale Degradation of Gracilarioside
and C-1′-C-2′ of 4 via oxidation using KMnO4 and NaIO4,3,9 the
resulting fatty acids were then methylated with CH2N2 to give four
methyl esters (6, 7). Furthermore, it was concluded that the
cyclopropane ring was on the phytosphingosines (5) by GC-MS
degradation,6 whereas 7 was a methyl lignocerate. Strong fragment
ion peaks at m/z 185, 157, and 71 showed R-cleavage from either
side of the tertiary carbon of one methyl-branched methyl myristate.
Moreover, rearrangement of the peak at m/z 157 resulted in the
diagnostic peaks at 158 and 159. The subsequent loss of methanol
and H2O at m/z 185 also showed characteristic peaks at m/z 153
and 135. Similarly, the other methyl-branched ester gave peaks at
m/z 199, 173, 172, 171, 167, 149, and 57, respectively. Therefore,
1
analysis, in agreement with the H NMR results. It was analyzed
by examining the retention time and mass spectrum compared with
standard samples, and 6 was a mixture of methyl pentadecanoate
and two inseparable methyl-branched methyl myristates following

1490 Journal of Natural Products, 2006, Vol. 69, No. 10
Notes
carried out using Si gel GF254 (Qingdao Haiyang Chemical Factory)
plates. HPLC was performed using a YMC Chromatorex C18 10 µm
preparative column (22 × 250 mm). The identification of fatty acid
methyl esters was based on the GC-MS retention times and GC-MS
spectra using a DB-1MS column (0.25 × 0.25 × 30 mm). After 3
min, the column temperature was increased from 80 °C to 250 °C at a
gradient of 10 °C/min. Quantitative determination was based on the
area of the GC peaks.
Collection. The red alga Gracilaria asiatica was collected in May
2004 in Indonesia and identified by Prof. Xiao Fan (Institute of
Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China).
A voucher specimen (No. 200405) is deposited at the Herbarium of
Shenyang Pharmaceutical University.
Extraction and Isolation. The collected red alga (dry weight 4.5
kg) was soaked in MeOH at rt for 3 × 72 h and filtered. After the
extracts were concentrated in vacuo at 40 °C, the residue was suspended
in H2O and then successively partitioned between EtOAc and n-BuOH.
The EtOAc-soluble fraction (8.6 g) was subjected to Si gel (200-300
mesh) CC using gradients of increasing acetone (0-100%) in CHCl3
and separated into 10 fractions (I-X) on the basis of TLC analyses.
Fraction VII was separated by CC over Sephadex LH-20 using CHCl3/
MeOH (1:1) as the eluent to give three corresponding subfractions.
The last two subfractions of VII were then purified by reversed-phase
preparative HPLC (RP-C18, MeOH/H2O, 97:3) to give 1 (3 mg) and
2 (2.5 mg). Fraction VI was purified by Sephadex LH20 (MeOH) to
afford 3 (4 mg).
it was concluded that the cyclopropane ring was located on the
sphingosine chain at positions 9 and 10.
Gracilamide A (2) was concluded to have the formula C43H83-
NO5 from the HRESIMS ([M + H+] m/z 694.6361), indicating three
1
degrees of unsaturation. By comparing the H NMR, 13C NMR,
and 2D NMR data, and the HRMS data of 2 with the data derived
from 1, it was found that both structures were almost identical
except for the absent glucose of 2 (Table 1). The similar chemical
degradation of 2 (also starting from 1 mg) further confirmed that
the length of the chain and the location of the cyclopropane ring
were the same as in 1.
The HRESIMS spectrum of gracilamide B (3) also gave an ion
peak at 696.6490 ([M + H]+) corresponding to a molecular formula
of C43H85NO5. There was no double bond in the structure of 3
compared with the NMR of the above two compounds (Table 1).
Chemical degradation and GC-MS analysis were again used to
determine the location of its cyclopropane ring. After reduction
and methanolysis, the products R-hydroxyacid methyl ester (8) and
the long-chain base (9) produced were also analyzed by 1H NMR.
Gracilarioside (1): white, amorphous powder; [R]20D +12.1 (c 0.005,
MeOH); CD (R-hydroxy acid methyl esters, MeOH) λmax 211.3 nm
(∆ꢀ -0.88); UV (MeOH) λmax (log ꢀ) 205.0 (0.94); 1H and 13C NMR,
see Table 1; ESIMS (pos) m/z 856 [M + H+]; HRESIMS (pos) m/z
856.6898 [M + H]+ (calcd for C49H93NO10, 856.6878).
Gracilamide A (2): white, amorphous powder; [R]20D +8.5 (c 0.002,
MeOH); CD (R-hydroxy acid methyl esters, MeOH) λmax 213.6 nm
(∆ꢀ -0.73); UV (MeOH) λmax (log ꢀ) 196.6 (0.31); 1H and 13C NMR,
see Table 1; ESIMS (pos) m/z 694 [M + H+]; HRESIMS (pos) m/z
694.6361 [M + H]+ (calcd for C43H83NO5, 694.6350).
Gracilamide B (3): white, amorphous powder; [R]20 +16.7 (c
D
There were two obvious additional branched methyl signals at 1.01
(d, J ) 7.2 Hz) and 0.93 (d, J ) 7.2 Hz) for the fatty acid methyl
esters (8), but no additional signals for the long-chain base.
Moreover, the ring of 3 was located on the methyl-branched fatty
acid on the basis of MS, and two methyl-branched fatty acid methyl
esters were also eluted together and identified from their mass
spectra with peaks at m/z 213, 187, 186, 185, 181, 163, 57 and
199, 173, 172, 171, 167, 149, 71, respectively. Therefore, it was
concluded that the cyclopropane ring was located at positions 10′
and 11′ on the fatty acid chain.
The absolute configuration at C-2′ of 1-3 was determined as R
because the CD spectrum of the R-hydroxyacid methyl esters
exhibited a negative Cotton effect.10 Also the stereochemistry of
the ceramide moieties was inferred by comparison of the 1H NMR
and the physical data of 1-3 with the analogues as reported in the
literature in terms of the signals due to 1-H to 4-H, which were
2S, 3S, 4R.11
0.008, MeOH); CD (R-hydroxy acid methyl esters, MeOH) λmax 210.5
1
nm (∆ꢀ -0.96); UV (MeOH) λmax (log ꢀ) 203.6 (0.70); H and 13C
NMR, see Table 1; ESIMS (pos) m/z 696 [M + H+]; HRESIMS (pos)
m/z 696.6490 [M + H]+ (calcd for C43H85NO5, 696.6506).
Mixture (5) of long-chain bases after methanolysis of 1: [R]20
D
+10.4 (c 0.004, MeOH); 1H NMR (300 MHz, CDCl3) δ 4.10 (m), 3.85
(m), 3.50 (m), 3.45 (m), 2.35 (m), 1.63 (m), 1.26 (m), 0.98 (d, J ) 6.7
Hz), 0.92 (d, J ) 6.7 Hz), 0.85 (t).
Mixture (8) of fatty acid methyl ester after methanolysis of 3:
1H NMR (300 MHz, CDCl3) δ 4.11 (m), 3.51 (s), 2.07 (m), 1.27 (m),
1.01 (d, J ) 7.2 Hz), 0.93 (d, J ) 7.2 Hz), 0.86 (t).
GC-MS Analysis of Methyl-Branched Fatty Acid Methyl Esters.
Gracilarioside (1): Methyl-branched methyl myristates: tR ) 16.73
min; MS m/z (relative intensities) 256 (15), 199 (10), 185 (9), 173 (1),
172 (2), 171 (6), 167 (6), 159 (2), 158 (3), 157 (12), 153 (5), 149 (3),
135 (4), 71 (15), 57 (23).
Gracilamide A (2): Methyl-branched methyl myristates: tR ) 16.67
min; MS m/z (relative intensities) 256 (14), 199 (9), 185 (9), 173 (1),
172 (1), 171 (5), 167 (4), 159 (1), 158 (1), 157 (10), 153 (4), 149 (2),
135 (2), 71 (14), 57 (25).
Gracilamide B (3): Methyl-branched methyl pentadecanoate: tR )
17.74 min; MS m/z (relative intensities) 270 (16), 213 (15), 199 (10),
187 (2), 186 (3), 185 (11), 173 (2), 172 (2), 171 (7), 167 (6), 163 (3),
149 (2), 135 (2), 71 (14), 57 (26).
The cytotoxicities of these three compounds were evaluated
against the A375-S2 melanoma cell line using the MTT assay.
Gracilarioside showed moderate cytotoxicity and induced 18.2%
cell death at 20.0 µg/mL. The other two compounds exhibited a
weak cytotoxicity of 11.7% cell death at 30.0 µg/mL.
General Procedure for Chemical Degradation. Commercially
available reagents were used without further purification. Gracilarioside
(1) or gracilamide A or B (2, 3) (1.0 mg) was hydrogenated in acetic
acid (1.0 mL) in the presence of PtO2 (7.0 mg) under atmospheric H2
for 14 h. The reaction mixture was filtered and evaporated to dryness.
The residue was treated with 0.9 mol/L HCl in 82% aqueous MeOH
(2.0 mL) at 80 °C for 18 h. It was concentrated and separated by flash
chromatography (Si gel, 200-300 mesh) eluted with CHCl3 to give
the fatty acid methyl esters (4), and with MeOH to give the sphingosines
(5) and a methyl glycoside. The mixture of 5 (gracilarioside) and the
methyl glycoside were partitioned between CHCl3 and H2O for further
separation. Then, 4 and 5 were separately subjected to Lemieux
Experimental Section
General Experimental Procedures. The optical rotation was
measured with a Perkin-Elmer 241MC polarimeter. CD spectra were
determined on a Jasco J-810 CD spectropolarimeter. UV spectra were
measured on a Shimadzu UV-1601. 1D and 2D NMR spectra were
recorded on a Bruker-ARX-600 and ARX-300 spectrometer. The
chemical shifts were quoted relative to TMS, and the coupling constants
were in Hz. HRESIMS spectra were recorded with a Q-trap LC-MS-
MS system using a Turbo ionspray source. Column chromatography
was performed on Si gel G (200-300 mesh, Qingdao Haiyang Chemical
Factory) and Sephadex LH-20 (Pharmacia Co.) columns. TLC was

Notes
Journal of Natural Products, 2006, Vol. 69, No. 10 1491
oxidation. Hence, 0.023 mol/L aqueous KMnO4 and 0.09 mol/L NaIO4
(2.0 mL) were slowly added to a mixture containing 4 or 5, t-BuOH
(1.0 mL), and 0.04 mol/L aqueous K2CO3 (0.5 mL). Then, the mixtures
were stirred for 18 h at 37 °C, quenched with 2.5 mol/L H2SO4 (0.1-
0.3 mL) and saturated aqueous Na2SO3, and then extracted with Et2O.
The organic layer was dried over Na2SO4. Finally, the concentrated,
dried residue was esterified with excess CH2N2 in Et2O overnight. The
resulting esters were used for GC-MS analysis.
Acknowledgment. This work was supported by 863 Hi-Tech
Research and Development Program of China (Grant No.
2003AA620403) and the fund for the 973-project from Ministry of
Science and Technology (Grant No. G1998051100), China.
Supporting Information Available: 1H NMR, 13C NMR, and
HRESIMS spectra of compounds 1-3. This material is available free
of charge via the Internet at http://pubs.acs.org.
Cytotoxicity Assay. The cytotoxic effect of gracilarioside (1) and
two gracilamides (2, 3) on A375-S2 cells was measured by MTT assay
as described. All the cells were cultured at 5 × 104 cells/well in 96-
well plates (NUNC, Roskilde, Denmark). Four hours before the end
of incubation, 20 µL of MTT solutions (5.0 mg/L) was added to each
well. Resulting crystals were dissolved in DMSO. The result was
measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT) assay using a plate microreader (TECAN SPECTRA, Wetzlar,
Germany). The percentage of cell growth inhibition was calculated as
follows: cell death (%) ) [A492 (control) - A492 (drug)]/A492
(control) × 100.
References and Notes
(1) Tan, R. X.; Chen, J. H. Nat. Prod. Rep. 2003, 20, 509-534.
(2) Salomon C. E.; Magarvey N. A.; Sherman D. H. Nat. Prod. Rep.
2004, 21, 105-121.
(3) Costantino, V.; Fattorusso, E.; Mangoni, A.; Rosa, M. D.; Ianaro,
A. J. Am. Chem. Soc. 1997, 119, 12465-12470.
(4) Costantino, V.; Fattorusso, E.; Mangoni, A. Tetrahedron 2000, 56,
5953-5957.
(5) Agrawal, P. K. Phytochemistry 1992, 31, 3307-3330.
(6) McCloskey, J. A.; Law, H. L. Lipids 1967, 2, 225-233.
(7) Natori, T.; Morita, M.; Akimoto, K. Tetrahedron 1994, 50, 2771-
2784.
(8) Oshima, R.; Kumanotani, J. Chem. Lett. 1981, 943-946.
(9) Pelletier, S. W. J. Org. Chem. 1970, 35, 3535-3538.
(10) Cymerman, C. J.; Pereira, W. E. Tetrahedron 1970, 26, 3457-3460.
(11) Sugiyama, S.; Honda, M.; Higuchi, R.; Komori, T. Liebigs Ann.
Chem. 1991, 349-356.
Absolute Configuration of Glucose. According to the reported
method,8 the absolute configuration of glucose was determined by
comparison of the retention time of its derivative obtained from the
methanolysis with that of standard samples using HPLC (column SiO2;
eluent n-hexane/ethanol (95:5); flow rate 1 mL/min; detection at 230
nm). The retention times of the derivatives of the sugars were as
follows: D-glucose 42.56 min, L-glucose 40.10 min.
NP060043E