New glycosides of the fungus Acremonium striatisporum isolated from a sea cucumber

Article abstract of DOI:10.1021/np0305324

Four new diterpene glycosides, virescenosides R (1), S (2), T (3), and U (4), have been isolated from a marine strain of Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix. Their structures have been elucidated on the basis of HRFABMS, 1D and 2D NMR (¹H, ¹³C, DEPT, COSY-45, COSY-RCT, HSQC, HMBC, and NOESY spectra), and the results of acidic hydrolysis as 19-O-{β-D-glucopyranosyl(1→6)-β- D-altropyranosyl}-isopimara-7,15-diene-2α,3β-diol (1), 19-O-β-D-altropyranosyl-3-oxo-isopimara-8(14),15-diene-7α-ol (2), 19-O-β-o-altropyranosyl-3,7-dioxo-isopimara-8,-15-diene (3), and 19-O-β-o-altropyranosyl-3,7-dioxo-isopimara-8(14),15-diene (4). The cytotoxic activity of the virescenosides was examined.

Full text of DOI:10.1021/np0305324

J . Nat. Prod. 2004, 67, 1047-1051
1047
New Glycosid es of th e F u n gu s Acr em on iu m str ia tispor u m Isola ted fr om a Sea
Cu cu m ber
Shamil Sh. Afiyatullov, Anatoly I. Kalinovsky, Tatyana A. Kuznetsova,* Mikhail V. Pivkin, Nina G. Prokof’eva,
Pavel S. Dmitrenok, and George B. Elyakov
Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences,
Vladivostok 22, Russian Federation
Received December 18, 2003
Four new diterpene glycosides, virescenosides R (1), S (2), T (3), and U (4), have been isolated from a
marine strain of Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta
fraudatrix. Their structures have been elucidated on the basis of HRFABMS, 1D and 2D NMR (1H, 13C,
DEPT, COSY-45, COSY-RCT, HSQC, HMBC, and NOESY spectra), and the results of acidic hydrolysis
as 19-O-{â-D-glucopyranosyl(1f6)-â-D-altropyranosyl}-isopimara-7,15-diene-2R,3â-diol (1), 19-O-â-D-
altropyranosyl-3-oxo-isopimara-8(14),15-diene-7R-ol (2), 19-O-â-D-altropyranosyl-3,7-dioxo-isopimara-8,-
15-diene (3), and 19-O-â-D-altropyranosyl-3,7-dioxo-isopimara-8(14),15-diene (4). The cytotoxic activity
of the virescenosides was examined.
In our search for secondary metabolites from marine
fungi with cytotoxity and/or novel chemical structures, we
have previously isolated five new diterpene altrosides,
virescenosides M-Q, from a marine strain of Acremonium
striatisporum originally separated from the holothurian
Eupentacta fraudatrix.1,2 Further investigation for metabo-
lites of this fungal strain has now led to the isolation of
four new cytotoxic glycosides, virescenosides R-U (1-4).
We report herein the isolation and structures of compounds
1-4 and their cytotoxic activity.
The fungus was cultured for 21 days on specially modi-
fied rice medium.1 The CHCl3-MeOH (2:1, v/v) extract of
the culture of A. striatisporum was fractionated by Si gel
column chromatography followed by reversed-phase and
normal-phase HPLC to yield individual glycosides 1-4. The
structures of new compounds 1-4 were established by the
interpretation of spectral data (NMR and HRFABMS), as
well as by comparison of their spectra with those of related
compounds.
The FAB mass spectrum of virescenoside R (1) exhibited
a quasimolecular ion peak at m/z 643 [M - H]- in the
negative ion mode and at m/z 645 [M + H]+ and 667 [M +
Na]+ in the positive ion mode. The molecular formula of 1
was determined as C32H52O13 on the basis of a high-
resolution FABMS (positive ions) peak at m/z 667.3312
(C32H52O13Na requires m/z 667.3306) and was in ac-
cordance with 13C NMR data. Initial examination of the
1-D proton and one-bond 1H-13C correlation NMR data
suggested the presence of two sugars (anomeric signals at
δ1H 5.46 f δ13C 99.2 and δ1H 5.41 f 100.2). This was
supported by fragment ions in the positive ion mass
spectrum which were consistent with the loss of a C6-
terminal sugar (m/z 483 [M + H - sugar]+) and with the
loss of both C6-sugars (m/z 321 [M + H - 2 sugars]+).
The aglycon moiety of glycoside 1 was found by extensive
NMR spectroscopy (1H, 13C, DEPT, COSY-45, COSY-RCT,
the presence of trifluoroacetic acid and then with pyridine-
HSQC, HBMC, and NOESY spectra) (Table 1) to be the
same as that of virescenoside A (5), isolated earlier from
the terrestrial fungus Acremonium luzulae.3-6 Acid hy-
drolysis of virescenoside R gave 1a , which was identical to
virescenol A by NMR and optical rotation. The sugar part
of the hydrolysate was treated first with (+)-2-octanol in
acetic anhydride. Analysis of the resulting acetylated (+)-
2-octyl glycosides by capillary GC led to the identification
of D-altrose and D-glucose.7 The identification of each sugar
as well as their sequence, interglycosidic linkage, and
configuration of glycosidic bonds in 1 were determined by
1D and 2D NMR including HBMC and NOESY (Table 1)
1
1
and various H-1H COSY experiments. A long-range H-
* Corresponding author. Fax: 7(4232)314050. E-mail: kuzta@piboc.dvo.ru.
13C correlation (δ1H 5.46 f δ13C 70.0) revealed a linkage
10.1021/np0305324 CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/20/2004

1048 J ournal of Natural Products, 2004, Vol. 67, No. 6
Notes
Ta ble 1. 1H and 13C NMR Data of Virescenoside R (1) in C5D5N (J , Hz)
atom
1
δC
δH
R: 1.35 m
HMBC
NOESY
46.5 CH2
3
20
â: 2.28 dd (4.1, 12.7)
4.48 m
3, 5, 10, 20
2
3
4
5
6
67.8 CH
83.7 CH
43.6 C
51.2 CH
24.4 CH2
19a,b, 20
1R, 5, 18
3.48 d (9.5)
1, 2, 4, 18, 19
1.40 m
3, 9
18
20
R: 2.00 m
â: 2.22 m
5.38 m
7
8
9
10
11
122.2 CH
135.1 C
52.4 CH
36.1 C
14â
1.68 m
5, 12R,14R
20.5 CH2
R: 1.47 m
â: 1.28 m
R: 1.27 m
â: 1.37 m
17
17
12
36.2 CH2
13
14
36.9 C
46.1 CH2
R: 2.03 brd (14.5)
â: 1.94 brd (14.5)
5.85 dd (10.8, 17.5)
a: 4.95 dd (1.5, 10.8)
b: 5.00 dd (1.5, 17.5)
0.88 s
7, 8, 9, 12, 13
12, 13, 14, 16, 17
13, 15
13, 15
12, 13, 14, 15
7, 17
15
16
150.5 CH
109.4 CH2
12R, 14R, 17
12R, 14â, 17
11â, 12â, 14â, 15, 16b
3, 6R, 19a,b
6â, 18, 20, 1-Alt
6â, 18, 20, 1-Alt
1â, 2, 6â, 19a,b
17
18
19
21.4 CH3
25.7 CH3
70.0 CH2
1.29 s
a: 3.85 d (10.0)
b: 4.44 d (10.0)
1.05 s
3, 4, 5, 18, 1-Alt
1, 5, 9, 10
20
15.7 CH3
Alt (1fC-19)
1
2
3
4
5
6
99.2 CH
71.1 CH
72.1 CH
65.1 CH
74.2 CH
67.6 CH2
5.46 d (1.2)
19
19a,b, 5-Alt
4.53 dd (1.2, 4.4)
4.70 dd (3.3, 4.4)
4.86 dd (3.3, 9.4)
4.49 m
a: 4.09 dd (4.1, 10.5)
b: 4.63 dd (3.2, 10.5)
Glc (1f6Alt)
3,4-Alt
1,2,4,5-Alt
5-Alt
1-Glc
1-Glc
1
2
3
4
5
6
100.2 CH
73.4 CH
74.8 CH
71.2 CH
74.3 CH
62.5 CH2
5.41 d (3.7)
4.10 dd (3.7, 9.5)
4.70 t (9.5)
4.22 t (9.7)
4.47 m
5,6-Alt
3-Glc
1,2,4-Glc
6a,b-Alt
a: 4.35 dd (5.6, 12.2)
b: 4.46 dd (2.6, 12.2)
between H1-Alt and C-19 of the aglycon. This interpreta-
tion was confirmed by a strong NOESY cross-peak between
H-19a,b and H1-Alt. Similarly, a long-range correlation
(δ1H 5.41 f δ13C 67.6) and the downfield chemical shift of
C6-Alt (67.6), and the NOESY cross-peak between H1-Glc
and H6a,b-Alt, assigned the linkage between these two
sugar units.
sugar portion of virescenosides S, T, and U and NOESY
correlations (Table 2 and the Experimental Section) elu-
cidated the presence of a â-D-altropyranoside unit of C1
form in these glycosides.
In HRFABMS virescenoside S (2) gave a quasimolecular
ion at m/z 503.2707 [M + Na]+. These data, coupled with
13C NMR spectral data (DEPT), established the molecular
formula of 2 as C26H40O8. The 13C NMR spectra of the
aglycon part of 2 (Table 2) showed signals characteristic
of a saturated ketone group, one secondary alcohol function,
and two double bonds at 213.6 (C), 71.8 (CH), 148.3 (CH),
139.6 (C), 132.6 (CH), and 110.6 (CH2).
The general features of the 1H and 13C NMR spectra
(Table 2 and the Experimental Section) of the aglycon part
of 2 closely resembled those of virescenoside O (6) with the
exception of proton and carbon signals belonging to the A
ring.2 The correlation observed in the COSY-45, COSY-
RCT, and HSQC spectra of 2 indicated the presence of the
following isolated spin systems: - -CH2- -CH2- - (C-1- -C-
2) and - -CH2-O- - (C-19). In the HBMC experiment the
methyl singlet at δ 1.41 (H3-18) showed long-range cor-
relations with C-3 (213.6), C-4 (52.5), C-5 (49.7), and C-19
(73.1). These data and correlations observed in the COSY-
45 and NOESY spectra indicated the position and stereo-
chemistry of the methyl and hydroxymethyl groups at C-4
and the position of the carbonyl group at C-3. The strong
NOEs from H1-Alt to H-19a,b and the downfield chemical
A comparison of the 13C NMR spectrum of 1 with
published data for R- and â-D-altropyranoses and R-methyl-
D-altropyranoside together with magnitudes of 1H-1H and
C1-Alt/H1-Alt (162.6 Hz) spin-coupling constants in the
NMR spectra of 1 elucidated the presence of a â-D-
altropyranoside unit of C1 form in 1.8-10 Similarly, the
terminal sugar was determined as R-D-glucopyranose of C1
1
form based on 13C chemical shifts and H-1H and C1-Glc/
H1-Glc (167.5 Hz) spin-coupling constants. Thus, the
structure of virescenoside R (1) was determined as 19-O-
{R-D-Glcp(1f6)-â-D-Altp}-isopimara-7,15-diene-2R,3â-di-
ol.
The 13C and 1H NMR spectra of the sugar moieties of
virescenosides S, T, and U showed a close similarity of all
proton and carbon chemical shifts and proton multiplicities
(Table 2). The acid hydrolysis of the sum of these viresceno-
sides gave D-altrose as one of the sugars which was
identified by GLC of the corresponding acetylated (+)-2-
octyl glycoside, using authentic samples prepared from D-
and L-altrose. Proton NMR coupling constant values for the

Notes
J ournal of Natural Products, 2004, Vol. 67, No. 6 1049
Ta ble 2. 1H and 13C NMR Data of Virescenosides S (2), T (3), and U (4) in C5D5N (J , Hz)
2
3
4
δC
δH
δC
δH
δC
δH
1
2
38.3 CH2
1R: 1.50 m
1â: 1.92 m
2R: 2.39 m
34.9 CH2
R: 1.55 m
37.6 CH2
R: 1.43 m
â: 1.92 ddd (2.9, 5.7, 13.0)
R: 2.42 ddd (3.0, 4.5, 11.4)
â: 2.95 td (5.6, 11.4)
â: 1.92 m
36.4 CH2
35.9 CH2
36.2 CH2
R: 2.38 dt (4.6, 13.4)
â: 2.97 td (5.3, 13.6)
2â: 2.94 td (5.1, 14.4)
3
4
5
6
213.6 C
52.5 C
49.7 CH
31.3 CH2
211.6 C
52.1 C
50.9 CH
36.2 CH2
212.7 C
52.1 C
52.0 CH
37.9 CH2
2.53 dd (2.7, 13.0)
R: 1.97 dt (2.7, 13.3)
â: 1.75 td (3.2, 13.0, 13.3)
4.39 brt
2.23 dd (3.7, 14.3)
R: 2.68 dd (3.7, 17.4)
â: 2.89 dd (14.3, 17.4)
2.00 dd (7.8, 11.1)
R,â: 2.70 m
7
8
9
10
11
71.8 CH
139.6 C
45.2 CH
38.3 C
197.6 C
128.9 C
163.1 C
39.2 C
197.6 C
134.5 C
50.0 CH
35.7 C
2.40 m
1.94 m
18.9 CH2
R: 1.54 m
â: 1.45 m
R,â: 1.42 m
23.2 CH2
R,â: 2.07 m
19.1 CH2
R: 1.36 m
â: 1.53 m
R,â: 1.46 m, 1.56 m
12
34.1 CH2
33.4 CH2
R: 1.50 m
â: 1.21 m
33.9 CH2
13
14
37.5 C
132.6 CH
34.3 C
33.7 CH2
38.7 C
143.9 CH
5.61 d (1.9)
R: 2.55 brd (17.7)
â: 2.14 brd (17.7)
5.73 dd (10.8, 17.4)
a: 4.92 dd (1.5, 17.4)
b: 4.97 dd (1.5, 10.8)
0.96 s
6.96 dd (1.2, 2.9)
15
16
148.3 CH
110.6 CH2
5.76 dd (10.6, 17.4)
a: 4.92 dd (1.5, 10.6)
b: 4.99 dd (1.5, 17.4)
1.09 s
145.6 CH
111.5 CH2
146.6 CH
111.7 CH2
5.85 dd (10.5, 17.5)
a: 5.01 dd (1.2, 10.5)
b: 5.07 dd (1.2, 17.4)
1.05 s
17
18
19
25.7 CH3
21.4 CH3
73.1 CH2
27.5 CH3
20.7 CH3
72.3 CH2
25.6 CH3
20.8 CH3
72.8 CH2
1.41 s
1.24 s
1.28 s
a: 4.17 d (10.0)
b: 4.25 d (10.0)
1.08 s
a: 4.05 d (10.0)
b: 4.33 d (10.0)
1.37 s
a: 4.07 d (9.8)
b: 4.27 d (9.8)
1.10 s
20
14.8 CH3
17.7 CH3
14.1 CH3
Alt (1fC-19)
5.48 d (1.3)
4.51 dd (1.2, 3.6)
4.74 dd (2.7, 3.6)
4.76 dd (3.3, 7.5)
4.55 m
1
2
3
4
5
6
100.4 CH
72.0 CH
72.6 CH
66.3 CH
76.6 CH
63.3 CH2
100.4 CH
71.9 CH
72.6 CH
66.3 CH
76.7 CH
63.3 CH2
5.45 d (1.3)
4.51 dd (1.3, 3.3)
4.74 t (3.3)
4.78 dd (3.3, 10.8)
4.55 m
a: 4.37 dd (6.3, 12.3)
b: 4.52 m
100.4 CH
72.0 CH
72.6 CH
66.2 CH
76.6 CH
63.3 CH2
5.43 d (1.4)
4.49 dd (1.4, 3.7)
4.74 m
4.78 dd (3.1, 7.8)
4.53 m
a: 4.34 dd (6.3, 12.3)
b: 4.52 m
a: 4.38 dd (6.7, 12.2)
b: 4.56 m
shift of C-19 (δ 73.1) indicated that the sugar moiety was
linked at C-19. On the basis of the above data, the structure
of virescenoside S was established as 19-O-â-D-altropyra-
nosyl-3-oxo-isopimara-8(14),15-diene-7R-ol (2). This agrees
with the molecular mass difference of 2 mass units between
virescenoside O and 2.
The molecular formula of virescenoside U (4) was
determined as C26H38O8 by pseudomolecular ions at m/z
479.2625 [M + H]+ and 501.2474 [M + Na]+ in the
HRFABMS (positive mode) and 13C NMR analyses. A close
1
inspection of the H and 13C NMR spectral data (Table 2)
of the aglycon part of virescenoside T by DEPT and HSQC
revealed the presence of three quaternary methyls (δ 25.6,
C-17; 20.8, C-18; 14.1, C-20); six methylenes (δ 37.6, C-1;
36.2, C-2; 37.9, C-6; 19.1, C-11; 33.9, C-12; 72.8, C-19),
including one oxygen-bearing methylene; two tertiary (δ
52.0, C-5; 50.0, C-9) and three saturated quaternary
carbons (δ 52.1, C-4; 35.7, C-10; 38.7, C-13); one carbonyl
group (δ 212.7, C-3); and a monosubstituted double bond
(146.6, C-15; 111.7, C-16). The remaining functionality,
corresponding to the carbon signals at δ 197.6 (C), 143.9
(CH), and 134.5 (C), suggested the presence of the trisub-
stituted enone chromophore. The UV spectrum showed a
The molecular formula of virescenoside T (3) was deter-
mined as C26H38O8 by pseudomolecular ions at m/z 479.2672
[M + H]+ and 501.2476 [M + Na]+ in HRFABMS (positive
mode) and 13C NMR analyses. The aglycon part of 3 was
found by extensive NMR (1H and 13C NMR, COSY-45,
COSY-RCT, HBMC, and NOESY) and UV spectroscopy
(Table 2 and Experimental Section) to closely resemble that
of virescenoside P (7) with the exception of proton and
carbon signals belonging to the A ring.2 The 13C NMR
spectrum of 3 showed a peak at δ 211.6, consistent with a
saturated ketone. Correlations observed in the COSY-45
and HSQC spectra of 3 indicated the presence of an isolated
spin system corresponding to the sequence -CH2-CH2- -
(C-1- -C-2). The long-range correlation of the methyl proton
signal at δ 1.24 (H3-18) with the carbon signal at δ 211.6
and the downfield chemical shift of C-4 (δ 52.1) placed a
ketone group at C-3 of ring A. The position and stereo-
chemistry of the methyl (1.24, s) and hydroxymethyl (72.3,
CH2) groups at C-4 were established on the basis of NOEs
and HBMC data. The attachment of the sugar moiety to
C-19 was established by the correlation of H1-Alt and
H-19a,b in the NOESY spectrum and the downfield chemi-
cal shift of C-19 (δ 72.3). The above data showed that the
structure of 3 is 19-O-â-D-altropyranosyl-3,7-dioxo-isopi-
mara-8,15-diene.
λmax at 247 nm (log ꢀ 3.4), consistent with the enone system
in structure 4.
The correlations observed in the COSY-45, COSY-RCT,
and HSQC spectra of the aglycon part of 4 indicated the
presence of the following isolated spin systems: - -CH2-
CH2- (C-1- -C-2), >CH-CH2- (C-5-C-6), - -CHdCH2- (C-
15-C-16), - -CH2-O- (C-19). Furthermore, the COSY-45
spectrum of 4 contained a cross-peak attributed to allylic
coupling between the olefinic proton at δ 6.96 (H-14) and
a signal at 1.94 (H-9), which was in turn coupled with two
signals at δ 1.36 (H-11R) and 1.53 (H-11â). On the basis of
this information together with the data obtained from the

1050 J ournal of Natural Products, 2004, Vol. 67, No. 6
Notes
Extr a ction a n d Isola tion . At the end of the incubation
period, the mycelium and medium were homogenized and
extracted three times with a mixture of CHCl3-MeOH (2:1,
v/v, ca. 2 L). After evaporation of the solvent, the residual
material (4 g) was passed over normal-phase silica, which was
eluted first with CHCl3 (500 mL) followed by a step gradient
from 5% to 40% MeOH in CHCl3 (total volume 3 L). Fractions
of 10 mL were collected and combined by TLC examination.
Fractions containing the desired compounds were further
purified by reversed-phase HPLC on a Diasphere-110-C18
column eluting with a step gradient from 45% to 75% MeOH
in H2O and then by normal-phase HPLC on a Zorbax SIL
column with EtOAC-(CH3)2CO-EtOH (100:20:15) to give 1
(15 mg) and EtOAC-(CH3)2CO (70:30) to yield 2 (4.5 mg), 3
(5 mg), and 4 (3.0 mg).
HBMC spectrum (Experimental Section) a diterpene al-
troside with a tricyclic aglycon structure was indicated for
4.
A direct comparison of 1H and 13C NMR spectra of 4 with
those of the glycosides M-Q,1,2 and virescenosides A-C,
obtained earlier from the terrestrial fungus Acremonium
luzulae6,11-13 suggests that virescenoside U has the struc-
ture of an isopimaradienic altroside. The proton signals of
a typical ABX system of a vinyl group at δ 5.85 (1H, dd,
10.5, 17.5 Hz), 5.07 (1H,dd, 1.2, 17.4 Hz), and 5.01 (1H,
dd, 1.2, 10.5 Hz) indicated a monosubstituted double bond
located at the C-15, C-16 position of this double bond.4,14-16
Furthermore, the positions of the C-17 methyl group (δ
1.05, s) and of the exo-vinyl group at C-13 were confirmed
by HBMC and NOE measurements (Experimental Section).
The long-range correlation of the methyl proton signal at
δ 1.05 (H3-17) with the carbon signal at 143.9 (C-14) and
the downfield chemical shift of H-14 (δ 6.96) indicated the
8(14)-en-7-one position for the trisubstituted enone chro-
mophore in 4. The stereochemistry at C-13 in 4 was
assigned to be the same as sandaracopimaradienic deriva-
tives on the basis of the similarity of the C-15-C-17
chemical shifts for these compounds.17-20
The NMR spectrum of 4 showed two signals correspond-
ing to an AB system coupling at δ 4.07 and 4.27 (each 1H,
d, 9.8 Hz), which was consistent with the presence of a
CH2O- - group linked to a quaternary sp3 carbon. The
position and stereochemistry of the methyl (1.28, s) and
hydroxymethyl (72.8, CH2) groups at C-4 and methyl group
(1.10, s) at C-10 were established as for compound 3. The
location of the carbonyl group at C-3 was evident from the
COSY-45 spectra and downfield chemical shift of C-4 (δ
52.1). The NOE correlation between H-2â and H3-20 and
H-19a as well as between H3-18 and H-5 indicated a trans
ring fusion between rings A and B. All of these data are
consistent with a ∆8(14),15-isopimaradienic skeleton with a
carbonyl function at C-3, an axial hydroxymethyl group at
C-4, and a 7-keto group conjugated with a C-8/C-14 double
bond in 4. The strong NOEs from H1-Alt to H-19a,b and
the downfield chemical shift of C-19 (δ 72.8) indicated that
the sugar moiety was linked at C-19. On the basis of these
data, the structure of virescenoside U was established as
19-O-â-D-altropyranosyl-3,7-dioxo-isopimara-8(14),15-di-
ene.
19-O -{r-D -G lc p (1f6)-â-D -Alt p }-is o p im a r a -7,15-d i-
en e-2r,3â-d iol (1): colorless amorphous solid; [R]20 +12° (c
D
0.6, MeOH); H and 13C NMR spectra (C5D5N), see Table 1;
1
HRFABMS (positive ion) m/z 667.3312 [M + Na]+ (calcd for
C
32H52O13Na, 667.3306).
19-O-â-D-Altr opyr an osyl-3-oxo-isopim ar a-8(14),15-dien e-
7r-ol (2): colorless amorphous solid; [R]20 -42.5° (c 0.4,
D
MeOH); 1H and 13C NMR spectra (C5D5N), see Table 2; HBMC
correlation (H/C) H-16b/C-13; Me-17/C-12, C-13, C-14, C-15;
Me-18/C-3, C-4, C-5, C-19; M-20/C-1, C-5, C-9, C-10; H-11/C-
21; NOESY correlation (H/H) 1R/9, 1â/11R,20, 2â/20,
5/1R,9,18, 6R/18, 6â/19b,20, 7/14, 9/1R,5, 9/1R, 11R/1â, 11â/20,
14/7,17, 15/17, 16b/17, 17/14,15,16b, 18/5,6R,19b, 19a/11,20,
19b/6â,18, 20/1â,2â,6â,11â,19a, 11/19a,b,51; HRFABMS (posi-
tive ion) m/z 503.2617 [M + Na]+ (calcd for C26H40O8Na,
503.2621).
19-O-â-D-Alt r op yr a n osyl-3,7-d ioxo-isop im a r a -8,15-d i-
en e (3): colorless amorphous solid; [R]20D +23° (c 0.48, MeOH);
UV (MeOH) λmax (log ꢀ) 248 (3.7) nm; 1H and 13C NMR spectra
(C5D5N), see Table 2; HBMC correlation (H/C) H-15/C-13;
H-16a,b/C-13; Me-17/C-12, C-13, C-14, C-15; Me-18/C-3, C-4,
C-5, C-19; Me-20/C-1, C-5, C-9, C-10; H-19b/C-3; H1-Altr/C2-
Altr; NOESY correlation (H/H) 1R/5, 2â/19a,20, 5/1R,18, 6R/
18, 6â/19a,b,20, 11â/20, 12R/17, 14R/16a,17, 14â/17, 15/12R,17,
16a/14R,17, 17/12R,14R,â,15,16a, 18/5,6R,19a,b, 19a/2â,18,20,1-
Altr, 19b/6â,18,20,1-Altr, 20/1â,2â,6â,11â, 1-Altr/19a,b,5-Altr;
HRFABMS (positive ions) m/z 479.2672 [M + H]+ (calcd for
C
26H39O8, 479.2645), 501.2476 [M + Na]+.
19-O-â-D-Altr op yr a n osyl-3,7-d ioxo-isop im a r a -8(14),15-
d ien e (4): colorless amorphous solid; [R]20 -30° (c 0.33,
D
MeOH); UV (MeOH) λmax (log ꢀ) 247 (3.4) nm; 1H and 13C NMR
spectra (C5D5N), see Table 2; HBMC correlation (H/C) Me-17/
C-12, C-13, C-14, C-15; Me-18/C-4, C-5, C-19; Me-20/C-1, C-5,
C-9, C-10; H-19b/C-4, C-5; NOESY correlation (H/H) 1â/20,
2â/19a,20, 5/18, 6R/18, 6â/19a,b,20, 14/17, 15/17, 16b/17,
Virescenosides R, S, T, and U exhibited cytotoxic action
against tumor cells of Ehrlich carcinoma (IC50 ) 25-60 µM)
in vitro. These glycosides showed a weak cytotoxic effect
on developing eggs of the sea urchin Strongylocentrotus
intermedius (IC50 ) 100-150 µM).
17/11â,14,15,
18/5,6R,19a,b,
19a/2â,6â,18,20,1-Altr,
19b/6â,18,20,1-Altr, 20/1â,2â,6â,19a,b, 1-Altr/19a,b,5-Altr;
HRFABMS (positive ions) m/z 479.2625 [M + H]+ (calcd for
C
26H39O8, 479.2645), 501.2474 [M + Na]+.
Acid ic Hyd r olysis of Vir escen osid e R (1). A solution of
Exp er im en ta l Section
compound 1 (9 mg) in 0.2 M TFA (1 mL) was heated in a
stoppered reaction vial for 30 min. The water layer was
extracted with CHCl3. The residue obtained after evaporation
of the extract was chromatographed on a Diasphere-110-C18
column (5 µm, 4 × 250 mm) eluting with 80% MeOH to yield
1.9 mg of 1a . The residue obtained after evaporation of the
water layer was purified on a Zorbax NH2 column (5 µm, 4.6
× 15 mm) eluting with 90% AcCN to yield 1.0 mg of glucose
and 0.8 mg of altrose. The monosaccharides were treated with
(+)-2-octanol (0.2 mL) in the presence of trifluoroacetic acid
(1 drop) in a stoppered reaction vial at 130 °C overnight.7 The
obtained mixtures were evaporated to dryness and acetylated
with Ac2O in pyridine. The acetylated (+)-2-octyl glycosides
were analyzed by GLC using the corresponding authentic
samples prepared from D- and L-glucose and d- and L-altrose.
Gen er a l Exp er im en ta l P r oced u r e. Optical rotations
were measured on a Perkin-Elmer 141 polarimeter. UV spectra
were recorded on a Specord UV-vis spectrometer in MeOH.
1H and 13C NMR spectra were recorded in both CDCl3 and
C5D5N on a Bruker DPX-300 spectrometer operating at 300
and 75.4 MHz, respectively, using TMS as an internal stan-
dard. FABMS spectra were measured in a glycerol matrix on
a AMD-604S mass spectrometer. GLC analyses were per-
formed on a Agilent 6850 Series GC system equipped with a
HP-5MS column and a temperature program of 100 to 250 °C
at 5 °C min-1. Helium was used as the carrier gas. Preparative
HPLC was carried out on a Beckman apparatus equipped with
an RIDK-22 refractometric detector, using Diasphere-110-C18
(5 µm, 4 × 250 mm) and Zorbax SIL (5 µm, 4 × 150 mm)
columns.
Isop im a r a -7,15-d ien e-2r,3â,19-tr iol (1a ): [R]20 -42° (c
D
0.2, CHCl3); 1H and 13C NMR spectra and optical rotation data
obtained for 1a were in agreement with published data3-5 for
virescenol A.
Cu ltiva tion of A. str ia tispor u m . The cultivation of the
fungus was performed as previously reported.1

Notes
Acid ic Hyd r olysis of Vir escen osid es S-U (2-4). A
solution of a mixture of compounds 2, 3, and 4 (each 1 mg) in
0.1 M TFA (1 mL) was heated in a stoppered reaction vial for
45 min. The residue obtained after evaporation of the water
layer was treated as described above for 1. The absolute
configuration of the monosaccharide was determined by GLC
of the acetylated (+)-2-octyl glycoside using the corresponding
authentic samples prepared from D- and L-altrose.
J ournal of Natural Products, 2004, Vol. 67, No. 6 1051
(6) Bellavita, N.; Bernassau, J . M.; Ceccherelli, P.; Raju, M. S.; Wenkert,
E. J . Am. Chem. Soc. 1980, 102, 17-20.
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359-362.
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3501-3508.
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2606.
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Am. Chem. Soc. 1995, 117, 8635-8644.
(11) Cagnolli-Bellavita, N.; Ceccherelli, P.; Ribaldi, M.; Polonsky, J .;
Baskevitch, Z. Gazz. Chim. Ital. 1969, 99, 1354-1363.
(12) Cagnolli-Bellavita, N.; Ceccherelli, P.; Mariani, R.; Polonsky, J .;
Baskevitch, Z. Eur. J . Biochem. 1970, 15, 356-359.
(13) Cagnolli-Bellavita, N.; Ceccherelli, P.; Ribaldi, M.; Polonsky, J .;
Baskevitch-Varon, Z.; Varenne, J . J . Chem. Soc., Perkin Trans. 1
1977, 351-354.
(14) Wenkert, E.; Beak, P. J . Am. Chem. Soc. 1961, 83, 998-1000.
(15) Borkosky, S.; Bardon, A.; Catalan, C. A. N.; Diaz, J . G.; Herz, W.
Phytochemistry 1995, 40, 1477-1479.
(16) De Pascual, J . T.; Barrero, A. F.; Muriel, L.; San Feliciano, A.; Grande,
M. Phytochemistry 1980, 19, 1153-1156.
(17) Wenkert, E.; Buckwalter, B. L. J . Am. Chem. Soc. 1972, 94, 4367-
4369.
(18) San Feliciano, A.; Medarde, M.; Lopez, J . L.; del Corral, J . M. M.;
Puebla, P.; Barrero, A. F. Phytochemistry 1988, 27, 2241-2248.
(19) Bardyshev, I. I.; Degtyarenko, A. S.; Pekhk, T. L.; Yankovskaya, G.
S. Zhur. Org. Khim. 1981, 17, 2568-2573.
(20) De Kimpe, N.; Schamp, N.; van Puyvelde, L.; Dube, S.; Chagnon-
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Ack n ow led gm en t. The study was supported by the Rus-
sian Foundation for Basic Research grants 03-04-49528, 02-
04-49517, and 02-04-49518 and grant of the President of
Russian Federation 1237.2003.4, grant FEB RAS 03-1-05-005,
and grant 03-19 from the Ministry for Industry, Science and
Technologies of the Russia Federation.
Refer en ces a n d Notes
(1) Afiyatullov, Sh. Sh.; Kuznetsova, T. A.; Isakov, V. V.; Pivkin, M. V.;
Prokof’eva, N. G.; Elyakov, G. B. J . Nat. Prod. 2000, 63, 848-850.
(2) Afiyatullov, Sh. Sh.; Kalinovsky, A. I.; Kuznetsova, T. A.; Isakov, V.
V.; Pivkin, M. V.; Dmitrenok, P. S.; Elyakov, G. B. J . Nat. Prod. 2002,
65, 641-644.
(3) Polonsky, J .; Baskevitch, Z.; Cagnolli-Bellavita, N.; Ceccherelli, P.
Chem. Commun. 1968, 1404-1405.
(4) Polonsky, J .; Baskevitch, Z.; Cagnolli-Bellavita, N.; Ceccherelli, P.
Bull. Soc. Chim. Fr. 1970, 5, 1912-1918.
(5) Polonsky, J .; Baskevitch, Z.; Cagnolli-Bellavita, N.; Ceccherelli, P.;
Buckwalter, B. L.; Wenkert, E. J . Am. Chem. Soc. 1972, 94, 4369-
4370.
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