L-Galactose as a natural product: Isolation from a marine octocoral of the first α-L-galactosyl saponin

Article abstract of DOI:10.1016/j.tetlet.2004.08.170

Saponine 1 (6′-O-acetyl-3β-pregna-5,20-dienyl-α-l- galactopyranoside), containing l-galactose, has been isolated from the marine octocoral Muricea c.f. purpurea. Saponine 1 (6′-O-acetyl-3β-pregna-5, 20-dienyl-α-l-galactopyranoside), that contains a l-galactose moiety linked to the aglycone through an infrequent α-glycosidic bond, has been isolated from the marine octocoral Muricea c.f. purpurea. This constitutes the first report on the occurrence of l-Gal as a nonpolymeric natural product. A CD procedure for the absolute stereochemical assignment of saponins, based on the CD analysis of its perbenzoylated derivative, is proposed.

Full text of DOI:10.1016/j.tetlet.2004.08.170

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7833–7836
L-Galactose as a natural product: isolation from a marine
octocoral of the first a-L-galactosyl saponin
a
Marcelino Gutierrez, Todd Capson, Hector M. Guzman,
b
b
´
Emilio Quin˜oa and Ricardo Riguera
´
´
a
a,
*
´
a
´
´
´
Departamento de Quımica Organica, Instituto de Acuicultura y Unidad de RMN de Biomoleculas asociada al CSIC,
Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain
bSmithsonian Tropical Research Institute, 2072 Balboa, Ancon, Republic of Panama
Received 21 July 2004; revised 30 August 2004; accepted 31 August 2004
Abstract—Saponine 1 (60-O-acetyl-3b-pregna-5,20-dienyl-a-L-galactopyranoside), that contains a L-galactose moiety linked to the
aglycone through an infrequent a-glycosidic bond, has been isolated from the marine octocoral Muricea c.f. purpurea. This consti-
tutes the first report on the occurrence of L-Gal as a nonpolymeric natural product. A CD procedure for the absolute stereochemical
assignment of saponins, based on the CD analysis of its perbenzoylated derivative, is proposed.
Ó 2004 Elsevier Ltd. All rights reserved.
The presence of L-sugars in nature is considered to be a
rare occurrence when compared to the massive presence
of D-sugars in both terrestrial and marine environments.
Nevertheless, there are some exceptions such as arabin-
ose, fucose and rhamnose that are peculiar amongst the
simple monosaccharides because they occur more com-
monly in the L- than in the D-configuration.1
organisms, particularly echinoderms.13,14 The immense
majority of the sugars found in the marine saponins
belong to the D series.13,15 Nevertheless, in a very small
number of cases, the presence of L-sugars has been
reported (e.g., L-fucose,16 L-arabinose17 and L-digital-
ose18), but up till now, no saponin—either from terres-
trial or marine sources—has ever been identified
containing L-Gal.
An extensive literature search has shown us that L-galact-
ose (L-Gal) is a rare sugar that, in terrestrial organisms,
has been found as a component of polysaccharides from
some plants2–5 and in snail galactans.6 In the marine
environment, L-Gal has been detected in red seaweeds,7
in several species of tunicates8–11 and in sulfated
glycoproteins from soft corals.12 In the case of the poly-
saccharides from red seaweeds, it has been shown that
L-Gal may appear in place of or jointly with D-Gal,7
while in tunicates, the polysaccharides that present
L-Gal are devoid of D-Gal.8–11
18
19
11
17
14
10
3
O
AcO
1'
O
OR
1 R= H
6 R= Bz
OR
RO
Apart from polysaccharides and glycoproteins, another
important class of natural products that contain sugars
are the saponins. These metabolites are widely distrib-
uted in many terrestrial plants and in certain marine
As a result of our interest on marine natural products
showing relevant bioactivities, we have recently focused
our attention on octocorals, marine invertebrates that
constitute a well-known source of acetogenins, sesqui-
terpenoids, diterpenoids and prostanoids.19 In this letter
we wish to communicate the isolation of saponin 1 (60-
O-acetyl-3b-pregna-5,20-dienyl-a-L-galactopyranoside)
from the marine octocoral Muricea c.f. purpurea. This
Keywords: L-Galactose; Saponin; Octocoral; Muricea c.f. purpurea;
Circular dichroism.
*
Corresponding author. Tel./fax: +34 981591091; e-mail: ricardo@
usc.es
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.08.170

´
M. Gutierrez et al. / Tetrahedron Letters 45 (2004) 7833–7836
7834
constitutes the first report on the occurrence of L-Gal as
a nonpolymeric natural product.
Thus, its HRESI-TOF MS showed a pseudomolecular
ion at m/z 527.29799 corresponding to
a
C29H44O7Na—[M+Na]+—formula. The overall data
from the 1D and 2D NMR experiments clearly sug-
gested the presence of a 60-O-acetylgalactopyranoside
linked to position 3 of a C21 pregnane skeleton. Conse-
quently, compound 1 was a saponin made of an acetyl-
ated galactose with a pregnane aglycone22,23 (Table 1).
In order to check if the sugar moiety was a member of
the D or L series, we resorted to the circular dichroism
(CD) study of the perbenzoylated a and b methyl glyco-
sides. These derivatives should show Davidov splitting
due to exciton coupling, characteristic for the L- and
the D-galactose, therefore allowing the safe assignment
of the absolute configuration of the sugar.
In this study, fresh specimens (0.9kg) of Muricea c.f.
purpurea, collected at Coiba National Park at the Pacific
Coast of Panama, were soaked in methanol, homo-
genized and filtered. The residue was sequentially
washed with methanol and dichloromethane. The ex-
tracts were pooled and concentrated in vacuo at 25°C
yielding 21.0g of crude extract.
This material was subjected to solvent partition as previ-
ously described20 to give a dichloromethane fraction
(2.18g) that was subjected to vacuum liquid chromato-
graphy (VLC) on silica gel. Elution with dichloro-
methane–methanol mixtures yielded eight fractions.
The third fraction (81mg) was dissolved in diethyl ether
and cooled down till a precipitate appeared. The soluble
material was concentrated and submitted to normal
phase HPLC (ethyl acetate–acetone 9:1 as mobile phase)
to give compound 1 (6mg).
Thus, 2mg of 1 were submitted to methanolysis fol-
lowed by perbenzoylation,21 affording a mixture of 2
and 3 that were separated by normal phase HPLC.
9
Mol. CD
-9
Compound 1 was obtained as a pale yellow glassy solid
with an [a]D ꢀ115 (c 0.6, CHCl3) and its structure was
established by extensive MS (LREIMS, HRESI-TOF
MS) and NMR (1H, 13C, DEPT, COSY, HMQC,
1
HMBC, NOESY and H decoupling) analysis.
R
2
R O
1
R
O
3
1
OR
OR
1
R O
1
2 (α-L) R = Bz; R = OMe; R = H
1
2
3
3 ( -L) R = Bz; R = H; R = OMe
β
1
2
3
OR
1
R O
1
210
Wavelength [nm]
260
O
R O
1
R
3
Figure 1. CD spectrum (MeCN) of methyl tetra-O-benzoyl-a-L-
galactopyranoside 2. The CD spectrum of methyl tetra-O-benzoyl-a-
D-galactopyranoside 4 has been recently published and can be found in
the Supporting Information of Ref. 21.
R O
1
R
2
α
4 ( -D) R = Bz; R = OMe; R = H
1
2
3
5 (β-D) R = Bz; R = H; R = OMe
1
2
3
Table 1. 13C and 1H NMR for compound 1 in CDCl3 at 62.5 and 750MHz, respectively
Position
1
1H NMR (m, J in Hz)
13C NMR (m)
Position
1H NMR (m, J in Hz)
13C NMR (m)
1.08 (m)
1.88 (m)
37.3 (t)
14
15
16
1.00 (m)
0.96 (m)
55.8 (d)
24.8 (t)
27.2 (t)
2
1.57 (m)
1.94 (m)
29.5 (t)
1.54 (m)
1.81 (m)
3
4
3.49 (m)
2.23 (t, 12.0)
2.34 (dd, 3.0, 12.7)
78.5 (d)
38.7 (t)
17
18
1.95 (m)
0.60 (s)
1.01 (s)
55.3 (d)
12.7 (q)
19.34 (q)
139.74 (d)
114.5 (t)
20.8 (q)
170.9 (s)
97.3 (d)
69.4 (d)
71.1 (d)
68.8 (d)
68.0 (d)
63.3 (t)
19
20
5
6
7
139.9 (s)
122.2 (d)
31.9 (t)
5.76 (ddd, 8.2, 9.7, 17.2)
4.96 (bd, 9.7)
2.07 (s)
5.36 (d, 4.5)
1.50 (m)
2.02 (m)
1.71 (m)
0.96 (m)
21
C(O)CH3
C(O)CH3
8
31.9 (d)
50.3 (d)
36.8 (s)
20.6 (t)
10
20
30
40
500
5.05 (d, 3.7)
9
3.77 (dd, 3.0, 9.8)
3.75 (dd, 2.2, 10.2)
3.96 (bs)
10
11
1.47 (m)
1.56 (m)
37.3 (t)
4.05 (t, 6.0)
4.22 (dd, 7.5, 11.2)
4.37 (dd, 2.25, 11.2)
121.71 (m)
13
6
43.3 (s)

´
M. Gutierrez et al. / Tetrahedron Letters 45 (2004) 7833–7836
7835
OAc
OH
OAc
BzO
BzO
HO
HO
HO
HO
O
O
O
i
ii
R
1
OMe
OMe
BzO
HO
HO
R
2
7 (α-D) R = H, R = OMe
1
2
8 (β-D) R = OMe, R = H
1
2
Scheme 1. Synthesis of the CD standards 6-O-acetyl-2,3,4-tri-O-benzoyl-b-D-galactopyranoside (7) and 6-O-acetyl-2,3,4-tri-O-benzoyl-b-D-
galactopyranoside (8). Reagents and conditions: (i) AcCl, sym-collidine, ꢀ40°C/25°C, 3/1h sequentially; (ii) BzCl, pyridine, 25° C, 16h.
50
237.6nm (43.5)
with no formation of secondary acetates in a detectable
amount.24 The 1H NMR data of 7 matched perfectly the
data of the sugar part of 6 confirming it as a a-
galactopyranoside.
222.4nm (14.3)
Figure 2 shows that the CD spectra of 6 is a perfect mir-
ror image of that of the a-D-derivative 7 [kmax 222 (+14)/
237 (ꢀ38)nm for 6; kmax 221 (ꢀ15)/237 (+43)nm for 7,
c = 10ꢀ4 M], thus corroborating the L stereochemistry
for the sugar and demonstrating that analysis of the
CD of the perbenzoylated saponin constitutes a very
useful tool for identification of the sugar, especially in
those cases where the amount of sample is very small.à
Mol. CD
221.2nm (-15.1)
237.8nm (-38.8)
The isolation of 1 from this octocoral is remarkable not
only because it is the first report of the presence of an L-
galactose unit in a nonpolymeric natural product but
also because the glycosidic linkage is a, being this also
a quite unusual fact in saponins. Actually, the related
saponin 9,bearing D-galactose bound through a b-gly-
cosidic linkage to the same aglycone, had been already
isolated from a gorgonian and its configuration deter-
mined by X-ray studies.25 Other three glycosides pos-
sessing the 6-O-acetyl-b-D-galactopyranoside moiety
bounded to cembrane-type diterpenes (calyculaglyco-
sides A, C and D) have also been identified.26
-50
200
Wavelength [nm]
300
Figure 2. CD spectrum (MeCN) of 60-O-acetyl-20,30,40-tri-O-benzoyl-
3b-pregna-5,20-dienyl-a-L-galactopyranoside (6) [continuous line].
Idem for 6-O-acetyl-2,3,4-tri-O-benzoyl-a-D-galactopyranoside (7)
[dashed line].
These derivatives showed retention times identical to
those of the synthetic glycosides 4 and 5, respectively
(prepared by an analogous procedure from commercial
b-D-galactose pentaacetate). When the CD spectra were
compared, those of 2 and 3 were found to be the exact
mirror images of those of the standard D-derivatives 4
and 5 [opposite signs for the absorption maxima at kmax
223 (+2)/237 (ꢀ6)nm, see Fig. 1]. These results unam-
biguously prove that 2 and 3 belong to the L series
and that the sugar present in 1 is L-Gal.
OR
HO
HO
O
O
OH
Once the absolute configuration of the sugar was
known, we decided to check if this CD procedure for
assignment could be directly applied to the intact
saponin.
9 R= H
10 R= Ac
Finally, the recent report27 of the isolation from the
octocoral Eunicea laciniata of a saponin (10) containing
a b-6-O-acetylgalactose unit is noteworthy although,
unfortunately, the chirality of the sugar was not
established.§
Thus, a small sample (2mg) of 1 was perbenzoylated as
before affording 6. Its CD spectrum presents a Davidov
splitting indicative of negative chirality and is presented
in Figure 2. For CD comparison, authentic samples of
methyl 6-O-acetyl-2,3,4-tri-O-benzoyl-a-D-galactopyr-
anoside (7), and of its b-anomer 8, were prepared as
shown in Scheme 1 from methyl a/b-D-galactopyrano-
sides. The key step in their preparation was the selective
acetylation of the primary alcohol at C-6 in the presence
of the unprotected secondary alcohols by means of AcCl
in sym-collidine (ꢀ40 and 25°C, 3 and 1h consecutively)
Our finding suggest that L-sugars, and particularly L-
Gal, may be more widespread metabolites than
previously believed and that careful examination of the
à For a collection of CD spectra of derivatized sugars, see Ref. 28.
§ The authors in Ref. 27 seem to assume a ÔDÕ configuration for the
galactose by analogy to compound 9, but they do not present any
experimental evidence.
Prepared from methanolysis of commercial b-D-galactose pentaace-
tate as previously shown.

´
M. Gutierrez et al. / Tetrahedron Letters 45 (2004) 7833–7836
7836
7. Storz, C. A.; Cerezo, A. S. Curr. Top. Phytochem. 2000, 4,
121–134.
8. Pavao, M. S. G.; Albano, R. M.; Lawson, A. M.; Mourao,
P. A. S. J. Biol. Chem. 1989, 264, 9972–9979.
9. Pavao, M. S. G.; Albano, R. M.; Mourao, P. A. S.
Carbohydr. Res. 1989, 189, 374–379.
10. Pavao, M. S. G.; Rodrigues, M. A.; Mourao, P. A. S.
Biochim. Biophys. Acta 1994, 1199, 229–237.
11. Mourao, P. A. S. Biochemistry 1991, 30, 3458–3464.
12. Molchanova, V. I.; Ovodova, R. G.; Odovov, Y. S.; Elkin,
Y. N. Carbohydr. Res. 1985, 141, 289–293.
13. Iorizzi, M.; De Marino, S.; Zollo, F. Curr. Org. Chem.
2001, 5, 951–973.
chirality of sugar units in saponins—particularly in
those from octocorals—could possibly render more
examples. CD spectroscopy of the directly derivatized
saponins may become a very useful tool for this search.
In any case, we believe this report should stimulate fur-
ther work on occurrence of L-sugars as natural
products.
Acknowledgements
´
We thank the Ministerio de Ciencia y Tecnologıa and
14. Hostettmann, K.; Marston, A. Saponins; Cambridge
University Press, 1995.
15. DÕAuria, M. V.; Minale, L.; Riccio, R. Chem. Rev. 1993,
93, 1839–1895.
16. Roussis, V.; Wu, Z.; Fenical, W. J. Org. Chem. 1990, 55,
4916–4922.
17. Takada, K.; Nakao, Y.; Matsunaga, S.; Van Soest, R. W.
M.; Fusetani, N. J. Nat. Prod. 2002, 65, 411–413.
18. Kashman, Y.; Green, D. J. Nat. Prod. 1991, 54, 1651–
1655.
the Xunta de Galicia for financial support for this
research (BQU2002-01195; SAF2003-08765-C03-01;
PGIDT02BTF20902PR, PGIDT03PXIC20908PN;
PGIDIT04PXIC20903PN) and the Centro de Super-
´
computacion de Galicia (CESGA) for the use of their
computational devices. Funding was also provided from
the Panama-ICBG program (#1 UO1 TW006634-01),
the Smithsonian Tropical Research Institute and the
´
Fundacion AVINA.
19. Rodriguez, A. Tetrahedron 1995, 51, 4571–4618.
20. Riguera, R. J. Mar. Biotechnol. 1997, 5, 187–193.
21. Costantino, V.; Fattorusso, E.; Imperatore, C.; Magnoni,
A. J. Org. Chem. 2004, 69, 1174–1179.
References and notes
22. Bandurraga, M. M.; Fenical, W. Tetrahedron 1985, 41,
1057–1065.
23. Cobar, O. M.; Rodriguez, A. D.; Padilla, O. L. J. Nat.
Prod. 1997, 60, 1186–1188.
1. Collins, P.; Ferrier, R. Monosaccharides. Their Chemistry
and Their Roles in Natural Products; Wiley: Chichester,
p 506.
2. Roberts, R. M.; Harrer, E. Phytochemistry 1973, 12, 2679–
2682.
24. Ishihara, K.; Kurihara, H.; Yamamoto, H. J. Org. Chem.
1993, 58, 3791–3793.
3. Zablackis, E.; York, W. S.; Pauly, M.; Hantus, S.; Reiter,
W. D.; Chapple, C. C. S.; Albersheim, P.; Darvill, A.
Science 1996, 272, 1808–1810.
4. Baydoun, E. A. H.; Fry, S. C. J. Plant Physiol. 1988, 132,
484–490.
5. Hantus, S.; Pauly, M.; Darvill, A. G.; Albersheim, P.;
York, W. S. Carbohydr. Res. 1997, 304, 11–20.
6. Bretting, H.; Jacobs, G. Carbohydr. Res. 1985, 139, 225–
236.
25. Wasylyk, J. M.; Martin, G. E.; Weinheimer, A. J.; Alan,
M. J. Nat. Prod. 1989, 52, 391–394.
26. Shi, Y. P.; Rodriguez, A. D.; Padilla, O. M. J. Nat. Prod.
2001, 64, 1439–1443.
27. Marville, K.; Reynolds, W. F.; Sealy, R. L.; Tinto, W. F.
Heterocycles 2004, 63, 107–113.
28. Weisler, W. T.; Berova, N.; Ojika, M.; Meyers, H. V.;
Chang, M.; Zhou, P.; Lo, L. C.; Niwa, M.; Takeda, R.;
Nakanishi, K. Helv. Chim. Acta 1990, 73, 509–551.