Avrainvilloside, a 6-deoxy-6-aminoglucoglycerolipid from the green alga Avrainvillea nigricans

Article abstract of DOI:10.1021/np050161m

Fractionation of the organic extract obtained from the Dominican green alga Avrainvillea nigricans led to the isolation of avrainvilloside (2), a new glycoglycerolipid bearing the extremely rare 6-deoxy-6-aminoglucose moiety. The structure of avrainvilloside has been established on the basis of spectroscopic data and methanolysis/GC-MS analysis.

Full text of DOI:10.1021/np050161m

1428
J. Nat. Prod. 2005, 68, 1428-1430
Avrainvilloside, a 6-Deoxy-6-aminoglucoglycerolipid from the Green Alga
Avrainvillea nigricans
Raymond J. Andersen† and Orazio Taglialatela-Scafati*,‡
Departments of Chemistry and Earth and Ocean Sciences, University of British Columbia, Vancouver,
British Columbia, Canada V6T 1Z1, and Dipartimento di Chimica delle Sostanze Naturali,
Universita` degli Studi di Napoli “Federico II”, Via D. Montesano 49, I-80131 Napoli, Italy
Received May 12, 2005
Fractionation of the organic extract obtained from the Dominican green alga Avrainvillea nigricans led
to the isolation of avrainvilloside (2), a new glycoglycerolipid bearing the extremely rare 6-deoxy-6-
aminoglucose moiety. The structure of avrainvilloside has been established on the basis of spectroscopic
data and methanolysis/GC-MS analysis.
Marine Chlorophytes (green algae) are well known for
the production of linear oxygenated terpenes; however,
members of the genus Avrainvillea (Udoteaceae) have been
shown to elaborate characteristic feeding-deterrent1 bro-
minated diphenylmethane derivatives endowed with HMG-
CoA reductase (potential cholesterol-lowering)2 and IMP
dehydrogenase (potential anticancer and immunosuppres-
sive)3 inhibiting activities. Recently, the parent compound
of this class, avrainvilleol, has been shown to possess also
a strong antioxidant activity.4
In the course of our chemical screening of Caribbean
marine organisms, we have undertaken the analysis of the
green alga Avrainvillea nigricans Decaisne, for which a
single report in the literature described the isolation of 5′-
hydroxyisoavrainvilleol (1),5 a member of the diphenyl-
methane class. The present note deals with the isolation
and the structural elucidation of a major constituent of the
organic extract, named avrainvilloside (2), a glycoglycero-
lipid bearing the extremely rare 6-deoxy-6-aminoglucose.
During the isolation procedure, the carotenoid siphona-
xanthin (3), which is considered a taxonomic marker of
siphonaceous algae (as A. nigricans),6 was also obtained.
Specimens of A. nigricans were collected along the coasts
of Dominica, frozen on site, and transported to Vancouver
over dry ice. Thawed samples (700 g wet wt) were cut in
small pieces and extracted multiple times with fresh MeOH
at room temperature. The combined MeOH extracts were
concentrated in vacuo to a brown-colored gum, which was
successively partitioned against n-hexane, CCl4, CHCl3,
and n-butanol, following a modified Kupchan procedure.7
The combined CCl4 and CHCl3 fractions were chromato-
graphed over a Sephadex LH-20 column eluted with
MeOH; selected fractions were fractionated by sequential
application of reversed-phase flash (gradient elution from
7:3 CH3CN/H2O to CH3CN in 5% increments) and reverse-
phase high-performance liquid chromatographies to give
pure samples of avrainvilloside (2, 16 mg) and siphona-
xanthin (3, 5 mg). This carotenoid was identified by
comparison of its spectroscopic data with those reported
in the literature.8
808 in the ESI (positive ion) mass spectrum. The HR-
FABMS of 2 also presented a pseudomolecular ion peak at
m/z 786.6469 [M + H]+ (calcd 786.6459), which was
consistent with the molecular formula C45H87NO9. Analysis
1
of the H and 13C NMR data (in DMSO) of 2, guided by
1H-1H COSY and HMQC spectra, allowed the assignment
1
of all the 13C and H NMR signals for three spin systems,
as shown in Table 1. The first spin system was assigned
to a glycerol moiety (δH 4.34 and 4.12, δC 62.3; δH 5.10, δC
69.9; δH 3.87 and 3.40, δC 65.4); the second group of signals
were attributable to two long-chain saturated fatty acids,
while the third spin system indicated the presence of a
glycosyl moiety. Hence, when the anomeric proton at δH
4.56 (δC 98.3) was used as a starting point, a sequence of
four oxymethines and one deshielded methylene was
identified. The large coupling constants observed for H-2′/
H-3′, H-3′/H-4′, and H-4′/H-5′ vicinal couplings (J ) 9.6 Hz)
and the relatively small coupling constant of H-1′/H-2′ (J
) 3.6 Hz) indicated the R-glucopyranose nature of this
1
sugar. The relatively upfield shifted H NMR resonances
of H-6′a and H-6′b (δH 2.87 and 2.56) and the 13C NMR
resonance of C-6′ (δC 54.5) were strongly indicative of the
presence of an amino group at this position. The complete
pattern of 13C NMR resonances (Table 1) confirmed the
assignment of the sugar unit as 6-deoxy-6-amino-R-glu-
copyranose. This residue should be linked to the C-3 of the
glycerol, as indicated by the HMBC correlation between
the anomeric proton H-1′ and C-3, while the HMBC cross-
peaks H-2/C-1′′′, H2-2′′′/C-1′′′, H2-1/C-1′′, and H2-2′′/C-1′′
25
Avrainvilloside (2), a white amorphous solid with [R]D
+25° (c 0.3, MeOH), has a molecular weight of 785, as
indicated by the pseudomolecular ion (M + Na)+ at m/z
* To whom correspondence should be addressed. Tel: 0039 081 678 509.
Fax: 0039 081 678 552. E-mail: scatagli@unina.it.
† University of British Columbia, Vancouver.
‡ Universita` degli Studi di Napoli “Federico II”.
10.1021/np050161m CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/12/2005

Notes
Journal of Natural Products, 2005, Vol. 68, No. 9 1429
Table 1. 13C and 1H NMR Data for Avrainvilloside (2)a
fibrosarcoma); however, its biological activity profile is
worthy of further investigation.
pos.
δC
δH (int., mult., J in Hz)
1a
1b
2
62.3, CH2
4.34 (1H, dd, 10.0, 2.0)
4.12 (1H, dd, 10.0, 7.4)
5.10 (1H, m)
3.87 (1H, dd, 10.0, 5.8)
3.40 (1H, dd, 10.0, 3.5)
4.56 (1H, d, 3.6)
3.18 (1H, dd, 9.6, 3.6)
3.35 (1H, t, 9.6)
2.91 (1H, t, 9.6)
Experimental Section
69.9, CH
65.4, CH2
General Experimental Procedures. Optical rotations
were measured in MeOH on a Perkin-Elmer 192 polarimeter
equipped with a sodium lamp (λmax ) 589 nm) and a 10 cm
microcell. CD spectra (MeOH) were recorded on a JASCO 500A
polarimeter. UV spectra were obtained in MeOH using a
Beckman DU70 spectrophotometer. 1H (500 MHz) and 13C (100
MHz) NMR spectra were recorded on Bruker AMX-500 and
AM-400 spectrometers, respectively; chemical shifts were
referenced to residual solvent signals (DMSO-d6: δΗ ) 2.50,
δC ) 40.0; CDCl3: δΗ ) 7.26). 13C NMR resonances were
assigned to CH3, CH2 or CH by using DEPT experiments.
Homonuclear 1H connectivities were determined by using
3a
3b
1′
98.3, CH
72.0, CH
72.6, CH
74.1, CH
68.2, CH
54.5, CH2
2′
3′
4′
5′
3.77 (1H, ddd, 9.6, 6.2, 4.0
2.87 (1H, dd, 14.0, 4.0)
2.56 (1H, dd, 14.0, 6.2)
6′a
6′b
1′′
1′′′
172.4, C
173.0, C
40.3, CH2
29.5, CH2
2′′-2′′′
3′′-3′′′
4′′ to 17′′
4′′′ to 17′′′
18′′-18′′′
2.28 (4H, t, 6.5)
1.49 (4H, t, 6.5)
1.12-1.30 (56H, m)
COSY experiments. One-bond heteronuclear H-13C connec-
1
tivities were determined with the HMQC experiment using a
BIRD pulse of 0.50 s (interpulse delay set for 1JCH ) 135 Hz).
19.2, CH3
0.90 (6H, t, 7.3)
1
Two- and three-bond H-13C connectivities were determined
a Recorded in DMSO-d6.
by HMBC experiments optimized for a 2,3J of 10 Hz. Low-
resolution electrospray (positive ions) mass spectra were
performed with a LCQ Finnigan MAT mass spectrometer; low-
and high-resolution FAB mass spectra (CsI ions, glycerol
matrix) were performed on a VG Prospec Fisons mass spec-
trometer. Column chromatography was performed using Sigma-
Lipophilic Sephadex LH-20 or Merck RP18 as stationary
phase. High-performance liquid chromatography (HPLC) sepa-
rations in isocratic mode were achieved using a Waters 600
pump and Alltech columns (250 µ 4.6 mm) and monitored by
a RI detector (Waters 486 tunable absorbance detector). GC-
MS experiments were performed on a Hewlett-Packard 5890
gas chromatograph with a MSD HP 5790 MS mass-selective
detector. A fused-silica column (25 m × 0.20 mm HP-5; cross-
linked 25% Ph-Me-silicone, 0.33 mm film thickness) was used
with a helium carrier flow of 10 mL/min and a temperature
gradient of 100 to 300 °C, 3 °C/min.
Plant Material, Extraction, and Isolation. Specimens
of Avrainvillea nigricans Ducaisne were collected in summer
2001 in Prince Rupert Bay of Portsmouth (Dominica) at a
depth of 25 m, immediately frozen, and transported to Van-
couver at 0 °C. A voucher specimen (ref. no. 01-101f) is
deposited at the Department of Earth and Ocean Sciences,
University of British Columbia, Vancouver. Thawed samples
(700 g wet wt) were cut in small pieces and extracted multiple
times with fresh MeOH at room temperature. The obtained
extract was dissolved in 9:1 MeOH-H2O and then partitioned
against n-hexane (3 × 500 mL) to yield an apolar extract
weighing 800 mg. Successively, the water content of the
hydromethanolic phase was adjusted to 20% (v/v) and 40% (v/
v), and the solutions were partitioned against CCl4 (3 × 500
mL) and CHCl3 (3 × 500 mL), respectively, affording a carbon
tetrachloride (1.2 g) and a chloroform (1.2 g) extract. Finally,
all the MeOH was evaporated from the hydromethanolic layer,
and the water solution thus obtained was partitioned against
n-BuOH. The combined CCl4 and CHCl3 fractions were chro-
matographed over a Sephadex LH-20 column eluted with
MeOH, obtaining 200 fractions of 20 mL. Fractions 40 to 60
were combined (75 mg) and fractionated by reversed-phase
flash chromatography (gradient elution from 7:3 CH3CN/H2O
to CH3CN in 5% increments), yielding pure siphonaxanthin
(3, 5 mg) and a crude fraction that was further purified by
reversed-phase HPLC, using 1:1 CH3CN/H2O as eluant, to
obtain pure avrainvilloside (2, 16 mg).
indicated the attachment of acyl groups at the positions 1
and 2 of the glycerol moiety.
Standard acetylation of avrainvilloside gave the tetra-
acetylated derivative 2a, thus giving support to the 1,2-
diacyl-3-O-(6-deoxy-6-amino-R-glucopyranosyl)glycerol struc-
ture deduced for 2. According to the molecular formula, the
acyl moieties of 2 should account for 36 carbon atoms. To
infer the exact nature of the fatty acids, avrainvilloside (2)
was treated with NaOMe/MeOH. After partitioning, the
apolar organic extract was analyzed by GC-MS and only
methyl stearate could be detected. This result indicates
that the two acylating fatty acids at C-1 and C-2 of the
glycerol are both stearic acid (C18). The MeOH phase was
subjected to acid methanolysis, and after HPLC purifica-
tion, 1.4 mg of methyl 6-deoxy-6-amino-R-glucopyranoside
was obtained. The D configuration of this sugar was
25
deduced by comparing its optical rotation ([R]D +131.0°
in H2O) with that reported in the literature for an authentic
sample of methyl 6-deoxy-6-amino-R-D-glucopyranoside
25
([R]D +147.0° in H2O).9 The CD spectrum of avrainvillo-
side (2) exhibited a negative Cotton effect at λmax 228 nm,
and this is considered to be indicative of sn-1, sn-2
diacylglycosylglycerolipid;10 thus, the stereostructure of
avrainvilloside was completely defined as reported in 2.
Glycoglycerolipids are common in red, brown, and green
algae,11 and also their analogues containing 2-deoxy-2-
amino (or 2-acetamido) sugars have wide distribution
among marine organisms. On the other hand, the 6-deoxy-
6-amino glycosides are extremely rare. To our knowledge,
avrainvilloside (2) represents only the third example in
nature of the very small class of 6-deoxy-6-amino glycoglyc-
erolipids, which apparently seem to be restricted to plants
and algae. The first members of this class were strangu-
latosides, isolated in 2001 from rhizomes of the terrestrial
plant Serratula strangulata (Compositae)12 and reported
to possess mild antibacterial activity. The second example,
which differs from 2 in bearing an additional acyl group
at C-6 of the sugar, has been very recently isolated from
an unidentified marine alga,13 and remarkably, it has been
shown to potently and selectively inhibit Myt1 kinase, an
enzyme involved in the cell cycle, whose inhibitors are
expected to kill rapidly proliferating cells and abrogate
normal cell cycle checkpoints.
Avrainvilloside (2): white amorphous solid; [R]D25 +25.0°
(c 0.3, MeOH); CD (MeOH) λmax 228 nm (∆ꢀ - 6.5); 1H (DMSO-
d6, 500 MHz) and 13C (DMSO-d6, 100 MHz) NMR spectra, see
Table 1; ESIMS (positive ion) m/z 808 [M + Na]+; FABMS
(positive ion) m/z 786 [M + H]+; HRFABMS m/z 786.6469 [M
+ H]+ (calcd for C45H88NO9, 786.6459, ∆µ 1.3 ppm).
Avrainvilloside (2) was inactive in preliminary cytotox-
icity assays (IC50 >10 µg/mL on WEHI 164 cells, murine
Acetylation of Avrainvilloside. Compound 2 (3.8 mg) was
dissolved in dry pyridine (0.6 mL) and treated with Ac2O (0.6

1430 Journal of Natural Products, 2005, Vol. 68, No. 9
Notes
mL). After standing overnight, the reaction was worked up
by addition of a few drops of methanol to destroy the excess
Ac2O, water (ca. 1 mL), and EtOAc (ca. 3 mL). The organic
phase was washed sequentially with 2 N H2SO4, saturated
NaHCO3, and brine. After drying (Na2SO4) and removal of the
solvent, the residue was purified by HPLC (n-hexanes/EtOAc,
8:2) to afford 3.3 mg of the tetraacetate 2a.
fuged, and the supernatant was evaporated to dryness under
N2. This was then purified by HPLC (MeOH/H2O, 8:2) to afford
25
1.4 mg of methyl 6-deoxy-6-amino-R-D-glucopyranoside ([R]D
+131.0° in H2O).
Acknowledgment. Financial support was provided by the
Natural Sciences and Engineering Research Council of Canada
(R.J.A.) and from MIUR (Italy), PRIN 2003 “Sostanze Naturali
ed Analoghi Sintetici ad Attivita` Antitumorale” (O.T.S.). The
authors thank M. LeBlanc, D. Williams, and the Fisheries
Development Division, Dominica, for assistance with collecting
A. nigricans.
25
Avrainvilloside tetraacetate (2a): amorphous solid; [R]D
1
+14.5° (c 0.1, MeOH); H NMR (DMSO-d6, 500 MHz) δ 5.21
(1H, t, J ) 9.6 Hz, H-3′), 5.13 (1H, m, H-2), 4.86 (1H, d, J )
3.6 Hz, H-1′), 4.78 (1H, dd, J ) 9.6, 3.6 Hz, H-2′), 4.71 (1H, t,
J ) 9.6 Hz, H-4′), 4.34 (1H, dd, J ) 10.0, 2.0 Hz, H-1a), 4.12
(1H, overlapped, H-1b), 4.09 (1H, overlapped, H-5′), 4.02 (1H,
dd, J ) 10.0, 6.0 Hz, H-3a), 3.50 (1H, dd, J ) 14.0, 4.0 Hz,
H-6′a), 3.48 (1H, dd, J ) 10.0, 4.0 Hz, H-3b), 3.33 (1H,
submerged by residual water signal, H-6′b), 2.28 (4H, t, J )
6.5 Hz, H2-2′′ and H2-2′′′), 1.99 (3H, s, OCOCH3), 1.98 (3H, s,
OCOCH3), 1.97 (3H, s, OCOCH3), 1.95 (3H, s, OCOCH3), 1.49
(4H, m, H2-3′′ and H2-3′′′), 1.28-1.12 (56 H, m, H2-4′′ to H2-
17′′ and H2-4′′′ to H2-17′′′), 0.90 (6H, t, J ) 7.3 Hz, H3-18′′ and
H3-18′′′); FABMS (positive ion) m/z 954 [M + H]+.
References and Notes
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Methanolysis of Avrainvilloside. Avrainvilloside (2, 12.0
mg) was dissolved in 1.5 mL of methanol and treated with 2%
NaOMe/MeOH solution (1.5 mL) under stirring at room
temperature for 4 h. The reaction mixture was neutralized
with AG 50W-X8 (BIO-RAD, H+ form), the resin removed by
filtration, and the filtrate dried under reduced pressure and
partitioned between n-hexane and MeOH. The n-hexane phase
was then subjected to GC-MS. Methyl stearate was detected
on the basis of its retention time, MS data [EIMS m/z 298 (M)+,
255, 213, 199, 0143, 87, 74], and comparison with an authentic
sample of methyl stearate. The MeOH phase was dissolved in
1 N HCl in 85% MeOH (3.0 mL), and the solution was kept at
70 °C in a sealed tube for about 8 h. After being cooled, the
reaction mixture was neutralized with Ag2CO3 and centri-
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Inouye, I. J. Phycol. 2004, 40, 1170-1177.
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(8) Ricketts, T. R. Phytochemistry 1971, 10, 161-164.
(9) Cramer, F.; Otterbach, H.; Springmann, H. Chem. Ber. 1959, 92, 384-
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(10) Michelsen, P. Chem. Scr. 1985, 25, 217-218.
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NP050161M