Isolation and structures of virescenosides from the marine-derived fungus Acremonium striatisporum

Article abstract of DOI:10.1016/j.phytol.2015.11.010

Four new diterpene glycosides, virescenosides R₁ (1), R₂ (2), R₃ (3) and Z (4) were isolated from a marine strain of Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix. Structures of 1-3 were determined as the corresponding biosides having isopimaradiene-type aglycons with additional oxidation and unsaturation patterns. Virescenosides Z (4) was structurally identified as altroside, containing 7-en-6-one group in the tricyclic aglycon moiety. The compounds 1-4 were examined for inhibition of non-specific esterase activity in mouse lymphocytes.

Full text of DOI:10.1016/j.phytol.2015.11.010

Phytochemistry Letters 15 (2016) 66–71
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Isolation and structures of virescenosides from the marine-derived
fungus Acremonium striatisporum
a,*, Anatoly I. Kalinovsky A.
k46a,
Shamil Sh. Afiyatullov
Alexandr S. Antonov A.
Yuliya V. Khudyakova
Anton N. Yurchenko , Mikhail V. Pivkin
a
S
.
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. AL4bH
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3.
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, Olesya I. Zhuravleva
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.
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a
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a , Dmitry L. Aminin
.
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Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Prospect 100 let Vladivostoku, 159, Vladivostok 690022,
Russian Federation
b
Far Eastern Federal University, Sukhanova Road 8, Vladivostok 690000, Russian Federation
A R T I C L E I N F O
A B S T R A C T
Article history:
Four new diterpene glycosides, virescenosides R (1), R (2), R (3) and Z (4) were isolated from a marine
1
2
3
Received 11 August 2015
Received in revised form 6 November 2015
Accepted 17 November 2015
Available online xxx
strain of Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix.
Structures of 1–3 were determined as the corresponding biosides having isopimaradiene-type aglycons
with additional oxidation and unsaturation patterns. Virescenosides Z (4) was structurally identified as
altroside, containing 7-en-6-one group in the tricyclic aglycon moiety. The compounds 1–4 were
examined for inhibition of non-specific esterase activity in mouse lymphocytes.
Keywords:
Virescenosides
ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Acremonium striatisporum
Diterpene glycosides
Esterase activity
1. Introduction
2. Result and discussion
Marine-derived microbes, fungi in particular are recognized as
an important source of structurally novel and biologically active
secondary metabolites (Rateb and Ebel, 2011; Debbab et al., 2012;
Blunt et al., 2014). As a part of our search for secondary metabolites
from marine fungi with cytotoxity and/or novel chemical
structures we have investigated the chemical constituents of the
extracts obtained from cultures of the marine-derived fungus
Acremonium striatisporum KMM 4401. Twelve new diterpene
altrosides, virescenosides M-X have previously isolated from this
strain under cultivation on specially modified rice medium
3
The CHCl –MeOH (2:1, v/v) extract of the culture of A.
striatisporum was separated by low-pressure reversed-phase
column chromatography on Teflon powder Polycrome-1 followed
by Si gel flash column chromatography and then by RP HPLC to
yield individual glycosides 1–4 as colorless, amorphous solids.
5 5
The NMR spectra of glycosides (1) and (2) (in C D N) indicated
that both compounds contained closed carbohydrate moieties
(Table 1). Initial examination of the 1-D proton and one bond
correlation NMR data suggested the presence of two sugars
(anomeric signals at
5.28, 100.7 and
H– H COSY spectrum gave rise to spin system for these
monosaccharides, which were assigned as -altropyranose (Alt)
d
H
5.41,
d
C
99.9 and
H C
d 5.45, d 100.6 for 1 and
(
Afiyatullov et al., 2006). Further investigation for metabolites of
this fungal strain under fermentation on wort agar medium
Artemchuk, 1981) has now led to the isolation of four new
diterpene glycosides, virescenosides R –R (1–3) and Z (4). We
d
H
d
C
d
H
5.46,
C
d 100.7 for 2). Interpretation of the
1
1
(
b
13
1
3
and
a-glucopyranose (Glc) by analysis of C NMR, HSQC, HMBC,
3
report herein the isolation and structural elucidation of com-
and NOESY data as well as by
JH–H coupling constant of ring
pounds 1–4.
protons (King-Morris and Serianni, 1987; Podlasek et al., 1995;
Perlin et al., 1970). The acid hydrolysis of the sum of these
glycosides gave
capillary GC of the corresponding acetylated (ꢀ)- and (+)-2-octyl
glycosides using authentic samples prepared from - and -glucose
and -altrose (Leontein et al., 1978) The arrangement of the sugar
units was determined by HMBC and NOESY experiments. A long-
D-glucose and D-altrose which were identified by
D
L
D
*
Corresponding author.
E-mail address: afiyat@piboc.dvo.ru (S.S. Afiyatullov).
http://dx.doi.org/10.1016/j.phytol.2015.11.010
874-3900/ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
1

S.S. Afiyatullov et al. / Phytochemistry Letters 15 (2016) 66–71
67
range correlation (H1-Alt/C-19) as well as the NOESY cross-
peak between H1-Alt and H-19a (Experimental Section)
revealed a linkage between the altrose and aglycone.
Similary, a long-range correlation H1-Glc/C6-Alt and the
NOESY cross-peak between H1-Glc and H6a-Alt assigned the
linkage between these two sugar units.
Taking into consideration that one bioside, namely,
virescenoside
et al., 2004) we have designated glycosides 1 and 2 as
virescenosides R and R
The molecular formula of virescenoside R
determined as C32 13 by pseudomolecular ion at m/z
65.3113 [M + Na] in HRMALDIMS and was in accordance
R was previously described (Afiyatullov
1
2
.
1
(1) was
50
H O
+
6
1
3
with C NMR data (Fig. 1).
1
The H NMR spectrum of the aglycon part of 1 (Table 2)
showed signals due to three methyl groups, appearing as
singlets at
.05 and 4.45 (each 1H, each d, 10.2 Hz) due to a primary
alcohol on quaternary center and an ABX system
corresponding to a vinyl moiety on a quaternary carbon at
d 0.99, 1.06 and 1.50, an AB system coupling at d
4
a
d
5
5.86 (1H, dd, 17.5, 10.6 Hz), 4.98 (1H, dd, 10.6, 1.5 Hz) and
.02 (1H, dd,17.5,1.5 Hz). The C NMR spectra of the aglycon
13
part of 1 (Table 3) showed signals characteristic two
secondary alcohol function at 68.4 (CH) and 83.9 (CH).
The remaining functionality, corresponding to the carbon
signals at 128.0 (CH), 129.9 (CH), 132.7 (CH), and 135.3 (C),
suggested the presence of the diene system. The UV
spectrum exhibits a max at 240 nm (log 3.61) consistent
d
d
l
e
with the diene chromophore in structure 1. Based on this
information, together with the data obtained from the
HMBC spectrum (Table 2) a diterpene bioside with a tricyclic
aglycon structure was indicated for 1.
A direct comparison of H and 13C NMR spectra of 1 with
1
those of the virescenosides R-X (Afiyatullov et al., 2006)
suggests that virescenoside R
isopimaratrienic bioside. The position of the C-17 methyl (
.06, s) and of the exo-vinyl groups at C-13 were argued by
1
has the structure of an
d
1
HMBC and NOEs measurements (Table 2, Experimental). The
stereochemistry at C-13 was assigned to be the same as
sandaracopimaradienic derivatives on the basis of the
similarity of C-15–C-17 chemical shifts for these compounds
(Wenkert and Buckwalter, 1972; De Kimpe et al., 1982; San
Feliciano et al., 1988). Localization of the diene system at the
C-6 and C-8(14) position was evident from the COSY-45
spectrum and from HMBC correlations H-5 (
29.9); H-7 ( 6.12)/C-5 ( 55.2) and C-8 ( 135.3); H-14 (
.35)/C-7; H-17 1.06)/C-14 132.7). The relative
stereochemistry of the protons at C-2 and C-3 was defined
d 2.26)/C-7 (d
1
d
d
d
d
5
(d
(d
1
1
based on the H– H coupling constant (J = 9.5 Hz) and
assigned as axial. The position and stereochemistry of the
methyl (
C-4 and methyl group (
the basis of NOESY and HMBC data. The observed NOE
correlations H -20/H-2 ( 4.48) and H-19a,b ( 4.05, 4.45),
-18/H-3 ( 3.50) and H-5 along with long range COSY
correlations from H -20 to H-1 1.39) and H-5 indicated a
trans ring fusion between rings A and B. On the basis of all
the above data, the structure of virescenoside R (1) was
established as 19-O-[ -altro-
-glucopyranosyl-(1 !6)-
pyranosyl]-isopimara-6,8(14), 15-trien-2 ,3 ,19-triol.
HRMALDIMS of virescenoside R (2) gave a pseudomo-
d
2
1.50, s) and hydroxymethyl (71.5, CH ) groups at
d
0.99, s) at C-10 were established on
3
d
d
H
3
d
3
a (d
1
a-D
b-D
a
b
2
+
lecular ion at m/z 681.3152 [M + Na] . These data, coupled
13
with C NMR spectral data, established the molecular
1
13
formula of 2 as C32
Tables 2,3) indicated the presence of a
aglycon possessing primary alcohol on a quaternary carbon
50
H O14. H and C NMR spectra of the 2
15
(
D -pimarene-type

6
8
S.S. Afiyatullov et al. / Phytochemistry Letters 15 (2016) 66–71
Fig. 1. Structures of virescenosides 1–4.
(
AB system, coupling at
two secondary alcohol function at
remaining functionality, corresponding to the carbon signals at
99.9 (C), 164.4 (C), and 128.4 (C), suggested the presence of the
trisubstituted enone chromophore. The UV spectrum showed a
max at 248 nm (log 4.05) consistent with enone system in
structure 2. The HMBC correlations from the methyl proton signal
at 1.45 (H -20) to the carbon signal at 164.4 (C-9) and from H-5
1.90) and H -6 ( 2.89, 3.37) to C-7 ( 199.9) indicated the 8(9)-
d
4.05, d, 10.6 Hz and 4.68, d, 10.6 Hz) and
observed for the aglycon part of 2 matched those reported for the
virescenoside M (Afiyatullov et al., 2000). Thus, the structure of
virescenoside R (2) was represented as 19-O-[ -glucopyrano-
syl-(1 !6)- -altropyranosyl]-7-oxo-isopimara-8(9), 15-dien-
,3 ,19-triol.
d
68.0 (CH) and 83.1 (CH). The
d
2
a-D
1
b-D
2
a b
l
e
The molecular formula of virescenoside R
as C32 13 on the basis of a high resolution MALDIMS peak at m/z
67.3261 [M + Na] and was in accordance with C NMR data. The
3
(3) was determined
52
H O
d
3
d
d
+
13
6
(
d
2
d
13
disaccharide nature of 3 was evident from its C and DEPT spectra
en-7-one position for the enone chromophore in 2. The NMR data
(Table 1), which exhibited two signals for anomeric carbons at
Table 2
1
H NMR spectral data (C
5
D
5
N, 500 MHz) for the aglycon moiety of virescenosides R
1
–R
3
(1–3) and Z (4).
Virescenoside R
1
(1)
Virescenoside R
2
(2)
Virescenoside R
3
(3)
Virescenoside Z (4)
H
1
d
H
(J,Hz)
HMBCa
d
H
(J, Hz)
HMBCa
d
H
(J,Hz)
HMBCa
d
H
(J,Hz)
HMBCa
a
b
: 1.39 m
: 2.24 m
2,10,20
2,10
a
: 1.51 m
2,3,10,20
2,3,5,10,20
a
: 1.51 m
a
: 1.59 t (12.5)
2,3,9,10,20
b: 2.40 dd (12.5, 4.5)
b: 2.35 dd (12.9, 3.9)
b: 2.29 dd (12.5, 4.5) 2,3,5,10,20
2
3
5
6
4.48 m
4.37 m
3.40 d (9.7)
4,7,10,19,20 1.90 dd (14.5, 3.5)
4.20 m
3.46 d (9.7)
11,4,6,7,10,18,19,20 1.40 m
4.46 m
3.46 d (10.0)
2.54 s
3.50 d (9.5)
2.26 brs
6.07 m
2,4,18,19
2,4,5,18,19
2,4,18,19
1,2,4,18,19
4,6,9,10,18,19,20
5,8
2.89 dd (18.0, 3.2)
5,7,8,10
a
b
: 2.00 m
: 2.22 m
3
.37 dd (18.0, 14.5) 18.0) 5,7,10
7
9
11
6.12 dd (10.0, 3.0)
1.98 m
5,8,9
5.32 m
1.68 m
5.84 t (2.3)
2.15 m
5,9,14
8,13
9
a: 1.56 m
b: 1.33 m
a: 1.45 m
b: 1.50 m
2.14 m
a
b
a
b
a
b
: 1.50 m
: 1.30 m
: 1.30 m
: 1.40 m
: 2.03 brd (14.7)
: 1.94 brd (14.7)
a: 1.63 m
b: 1.33 m
a: 1.38 m
b: 1.44 m
a: 2.14 m
b: 2.08 m
12
a
b
a
b
: 1.46 m
: 1.16 m
: 2.52 brd (17.0)
: 2.06 brd (17.0)
11,13
14
5.35 brs
7,9,12,13,15
8,9,12,13,15
8,9,13,15,17
7,8,13,17
9,12
1
16
5
5.86 dd (17.5,10.6)
a: 4.98 dd (10.6, 1.5) 13
b: 5.02 dd(17.5, 1.5) 13
1.06 s
1.50 s
a: 4.05 d (10.2)
b: 4.45 d (10.2)
0.99 s
12,13,14,17 5.70 dd (17.5, 10.8)
5.85 dd (17.5, 10.8)
a: 4.95 dd (10.8, 1.5)
b: 5.05 dd (17.5, 1.5)
0.87 s
13,14,17
13
13
12,13,14,15 0.82 s
3,4,5,19
3,4,5,18
5.81 dd (17.5, 10.7)
4.98 m
12,13,14,17
13,15
a: 4.89 dd (17.5, 1.5)
b: 4.92 dd (10.7, 1.5)
12,13,14,15 0.92 s
13
13
1
7
12,13,14,15
3,4,5,19
3,4,5,18,1-Alt
3,4,5,18
1,5,9,10
12,13,14,15
3,4,5,19
1
8
3,4,5,19
4,5
3,4,5,18
1,5,9,10
1.37 s
1.41 s
1.79 s
19
a: 4.05 d (10.6)
b: 4.68 d (10.6)
1.45 s
a: 4.14 d (10.0)
b: 4.51 d (10.0)
0.94 s
a: 4.51 d (9.3)
b: 4.96 d (9.3)
1.01 s
2
0
1,5,9,10
1,5,9,10
a
HMBC correlations, optimized for 8 Hz, are from proton(s) stated to the indicated carbon.

S.S. Afiyatullov et al. / Phytochemistry Letters 15 (2016) 66–71
69
Table 3
chemical shift of C-5 indicated the 7-ene-6-one position for the
13
E
5 5
NMR spectroscopic data (500 MHz, C D N) for the aglycon moiety of
enone chromophore in 4. The relative stereochemistry of the
virescenosides R
1
–R
3
(1–3) and Z (4).
1
1
protons at C-2 and C-3 was defined based on the H– H coupling
constant (J = 10.0 Hz) and assigned as axial. The position and
Atom
1
2
3
4
1
2
3
4
5
6
7
8
9
44.6 CH
68.4 CH
83.9 CH
43.1 C
2
43.4 CH
68.0 CH
83.1 CH
43.4 C
2
46.9 CH
67.6 CH
84.0 CH
43.3 C
2
45.8 CH
67.4 CH
84.3 CH
42.7 C
63.2 CH
198.2 C
127.4 CH
158.6 C
51.7 C
2
stereochemistry of the methyl (
CH ) groups at C-4 and methyl group (
established as for compound 1. The axial orientation of H
assignable on the basis of observed NOE correlations (Experimen-
tal) and long-range COSY correlations H -17/H-12 1.38), H-14
2.14). The stereochemistry at C-13 in 4 was assigned to be the
d
1.79, s) and hydroxymethyl (70.8,
1.01, s) at C-10 were
-17 was
2
d
3
55.2 CH
50.2 CH
51.2 CH
24.2 CH
123.5 C
135.5 C
52.1 C
128.0 CH
129.9 CH
135.3 C
50.3 CH
37.6 C
37.5 CH
199.9 C
128.4 C
164.4 C
40.7 C
2
2
3
a
(d
a
(d
7,8
same as
D -isopimaradiene and virescenols B and C on the basis
of the similarity of the C-15–C-17 chemical shift for these
compounds (Wenkert and Buckwalter, 1972; Polonsky et al.,
1972). The absolute configuration of the altrose was determined
1
0
36.2 C
40.4 C
1
1
19.0 CH
2
23.2 CH
33.7 CH
34.5 C
2
20.3 CH
36.0 CH
36.8 C
2
20.1 CH
35.1 CH
37.0 C
2
1
2
34.6 CH
38.3 C
2
2
2
2
1
3
after acid hydrolysis of 4 as for virescenosides R
unit has been confidently linked at C-19 of the aglycon based on the
HMBC correlation between the downfield shifted C-19 ( 70.8) and
H1-Alt ( 5.57) further supported by an NOE between H1-Alt and
-19. All the above data indicate virescenoside Z (4) to be 19-
-altropyranosyl-6-oxo-isopimara-7,15-diene-2 ,3 ,19-triol.
The inhibitory activities of compounds 1–4 in respect of non-
specific esterase in mouse spleen lymphocytes were determined
according to Aminin et al., (2009). Virescenosides R , R , R and Z
applied at concentration of 100 g/mL showed inhibition of
esterase activity on 56, 58, 36 and 40%, respectively. The positive
control, cucumarioside A -2 exhibited inhibition against non-
1 2
and R . The sugar
1
4
132.7 CH
148.7 CH
110.1 CH
33.9 CH
2
46.0 CH
2
45.1 CH
2
1
5
145.8 CH
111.5 CH
27.8 CH
150.0 CH
109.4 CH
149.1 CH
110.3 CH
21.7 CH
d
1
6
2
2
2
2
1
7
26.5 CH
24.6 CH
3
3
3
21.4 CH
3
3
d
1
8
23.5 CH
72.4 CH
3
24.2 CH
72.0 CH
3
25.3 CH
70.8 CH
3
H
2
1
9
71.5 CH
14.2 CH
2
2
2
2
?
-b-D
a b
2
0
3
18.5 CH
3
16.5 CH
3
16.2 C H
3
1
2
3
m
2
d
101.4 (CH) and 99.3 (CH) and those of the corresponding protons
1
specific esterase with ED50 value of 10
mg/mL.
at
d
5.41 (d, J = 1.9 Hz) and 5.67 (d, J = 4.1 Hz) in the H NMR data.
Interpretation of the 13C NMR, HSQC and COSY spectra gave rise
to spin systems for these monosaccharides, which were assigned
as gluco- - and altro- -configured residues on the basis of
3. Experimental
a
b
3.1. General experimental procedures
chemical shifts and coupling constants of ring protons (King-
Morris and Serianni, 1987; Podlasek et al., 1995; Perlin et al., 1970).
The pyranose nature of the sugars was determined on the basis of
HMBC correlations between the anomeric methines and the C5-Alt
and C5-Glc methines groups. The absolute configurations of the
glucose and altrose were determined after acid hydrolysis of 3 as
Optical rotations were measured using a PerkinElmer 343 po-
larimeter. UV spectra were recorded on a Shimadzu UV-1601PC
1
13
spectrometer in MeOH. The H and C NMR spectra were recorded
in C D N on a Bruker Avance 500 spectrometer at 500 and
5
5
125.8 MHz respectively, using TMS as an internal standard. HR
MALDI-TOF mass spectra were recorded on a Bruker Biflex III laser
desorption mass spectrometer coupled with delayed extraction
using N2 laser (337 nm) and a-cyano-4-hydroxycinnamic acid as
matrix. HR ESIMS spectra were recorded on a Q-TOF-LC/MS Agilent
6510 mass spectrometer fitted with an electrospray ion source. GC
analyses were performed on an Agilent 6850 Series GC system
equipped with a HP-5MS column using a temperature program of
for virescenosides R
moieties in 3 was established by a combination of the HMBC and
NOESY spectra (Experimental). A long-range correlation H1-Alt (
.41)/C-19 ( 72.0) as well as an NOE between H1-Alt and H-19a
revealed a linkage between the altrose and aglycone. Similarly, a
long-range correlation H1-Glc ( 5.67)/C4-Alt ( 74.3) and an NOE
between H1-Glc and H4-Alt ( 4.91), together with the downfield
1 2
and R . The arrangement of the sugar
d
5
d
d
d
d
ꢁ
ꢁ
ꢁ
ꢀ1
chemical shift of C4-Alt assigned the 1,4-linkage between glucose
100–250 C at 5 C min ; temperatures of injector and detector
ꢁ
and altrose. The structure of the aglycon moiety of 3 was found by
were 150 C and 270 C, respectively. Low pressure liquid column
chromatography was performed using Polychrome-1 (powder
Teflon, Biolar, Latvia), Si gel L (40/100 mm, Chemapol, Praha, Czech
Republic). Glass plates (4.5 ꢂ 6.0 cm) precoated with Si gel
1
13
extensive NMR spectroscopy ( H and C NMR, COSY, HSQC, HMBC,
and NOESY) (Tables 2 and 3) to be the same that of virescenoside R
(
Afiyatullov et al., 2004). All the above data confirmed the
structure of virescenoside R (3) as 19-O-[ -glucopyranosyl-
-altropyranosyl]-isopimara-7,15-dien-2 ,3 ,19-triol.
3
a-D
(5–17 mm, Sorbfil, Russia) were used for TLC. Preparative HPLC
(
1 !4)-
b
-D
a
b
was carried out on a Beckman–Altex chromatograph, using
Diasphere-110-C18 (5
Virescenoside Z (4) exhibited the molecular formula C26
H
40
13
O
C
9
m
m, 4.0 ꢂ 250 mm) and Zorbax NH columns
2
ꢀ
as deduced from its HR (ꢀ) ESIMS [M ꢀ H] ,m/z 495.2556 and
with an RIDK-102 refractometer detector.
1
13
NMR spectra. A comparison of the H and C NMR spectroscopic
data of 4 (Tables 1–3) with those of the virescenosides R-X
3.2. Cultivation of A. striatisporum
(
Afiyatullov et al., 2006) revealed the presence of an altroside with
15
ꢁ
D
-pimarene-type aglycon possessing primary alcohol on a
4.51, d, 9.3 Hz and
.96, d, 9.3 Hz) and two secondary alcohol function at 67.4 (CH)
and 84.3 (CH). The remaining functionality, corresponding to the
carbon signals at 198.2 (C), 127.4 (CH), and 158.6 (C), suggested
the presence of the trisubstituted enone chromophore. The UV
spectrum exhibits a max at 241 nm (log 3.61), consistent with the
enone system in structure 4. The long-range correlations H -17(
51.7), H-5 (
5.84)/C-5, C-9, C-14 and the down
The fungus was grown stationary at 22 C for 14 days on 6
flasks (1 L) (medium: wort—200 mL, sodium tartrate—0.05 g/L,
agar—20 g/L, seawater—800 mL).
quaternary carbon (AB system, coupling at
d
4
d
d
3.3. Extraction and isolation
l
e
At the end of the incubation period, the mycelium and medium
were homogenized and extracted three times with a mixture of
CHCl –MeOH (2:1, v/v, 2 L). The combined extracts (4.2 g) were
3
d
0
2
.82)/C-14 (
d
45.1), H
3
-20 (
d
1.01)/C-5 (
d
63.2), C-9 (
d
d
3
.54)/C-6 (
d
198.2), H-7 (
d
concentrated to dryness and separated by low pressure RP CC (the

7
0
S.S. Afiyatullov et al. / Phytochemistry Letters 15 (2016) 66–71
_
column 20 ꢂ 8 cm) on Polychrome-1 Teflon powder in H
EtOH. After elution of inorganic salts and highly polar compounds
by H O, 50% EtOH was used to obtain the fraction of amphiphylic
2
O and 50%
L
-altrose was determined for ( )-2-octyl glycoside of the corre-
sponding -sugar according to Leontein et al. (1978).
D
2
compounds, including the virescenosides. After evaporation of the
3
3.5. Acidic hydrolysis of virescenosides R and Z (3, 4)
solvent, the residual material (2.3 g) was subjected to Si gel flash CC
(
7 ꢂ13 cm) chromatography with a solvent gradient system of
Compound 3 (4.0 mg) and 4 (2.0 mg) were hydrolyzed as
described above for virescenosides R and R . The absolute
increasing polarity from 5% to 60% EtOH in CHCl (total volume 3 L).
3
1
2
Fractions of 10 mL were collected and combined by TLC examina-
tion to obtain 2 subfractions, containing the desired compounds.
Subfraction I (240 mg) was further purified and separated by RP
configurations of the monosaccharides were determined by GC
of the acetylated (+)- and ( )-2-octyl glycosides according to
Leontein et al. (1978).
_
2
HPLC on a Diasphere-110-C18 column eluting with MeOH - H O
(
70:30) to yield virescenosides R
1
(1) (2.7 mg), R
2
(2) (3.0 mg), R
3
3.6. Esterase activity
(
3) (5.1 mg) and Z (4) (3.0 mg).
Female CD-1 mice weighing 18–20 g were purchased from the
nursery RAMS «Stolbovaya» (Russia), and kept at the animal facility
under standard conditions. All experiments were conducted in
compliance with all rules and international recommendations of
the European Convention for the Protection of Vertebrate Animals
used for experimental studies.
3
2
.3.1. 19-O-{
,3 ,19-triol (1)
Colorless amorphous solid; [
MeOH) max (log ) 237 (3.97) nm; H and C NMR data (C
a
-
D
-Glcp(1 !6)-
b-D-Altp}-isopimara-6,8(14), 15-triene-
a
b
20
a
]
D
ꢀ4.0 (c 0.09, MeOH); UV
13
1
(
l
e
5
D
5
N),
see Tables 1–3; HMBC correlations H1-Alt/C5-Alt, C-19, H6a,b-
Alt/C1-Glc, H1-Glc/C5-Glc, C6-Alt; selected NOESY correlations (H/
Mouse lymphocytes (splenocytes) were obtained from a mouse
spleen. For this purpose a spleen was isolated and cut with scissors
into small-sized slices in PBS (pH 7.4) and then pressed through
nylon gauze (280 mesh). The obtained suspension was washed
twice in PBS by centrifugation (2000 rpm, 10 min). The final
H) 1
6b/17, 19b/20, 1-Alt/5-Alt,19a, 6a-Alt/1-Glc, 1-Glc/6a-Alt; long-
range correlations observed in COSY-45 (H/H) 17/12 , 20/1 ,5, 7/
4, 9/14; HR MALDI TOF MS m/z 665.3113 [M + Na] , calcd for
13Na 665.3149.
a/5,9, 1b/20, 2/20, 3/1a,5,18, 5/9,18, 9/11a, 11b/17,20, 15/14,17,
1
a
a
+
1
6
C
32
H
50
O
concentration of cells in the incubation medium was (2–5) ꢂ 10
cells/mL. To study cytoplasmic non-specific esterase activity
3
2
.3.2. 19-O-{
,3 ,19-triol (2)
Colorless amorphous solid; [
MeOH) max (log ) 248 (4.05) nm; H and C NMR data (C
a
-
D
-Glcp(1 !6)-
b
-
D
-Altp}-7-oxo-isopimara-8,15-diene-
200 mL of the cell suspension (the final cell concentration 2–
6
a
b
5 ꢂ10 cells/mL) was placed into wells of 96-well microplate
20
a
]
D
1
+ 24.5 (c 0.1, MeOH); UV
containing 20
was conducted within 1 h at 37
diacetate solution in DMSO (the final concentration 50
m
L solutions of tested compounds. The incubation
13
ꢁ
(
l
e
5
D
5
N),
E. Then 10
m
L fluorescein
g/mL)
see Tables 1–3; HMBC correlations H1-Alt/C5-Alt, C-19, H6a,b-Alt/
m
C1-Glc, H1-Glc/C5-Glc, C6-Alt; selected NOESY correlations (H/H)
was added to each well and the microplate was incubated
ꢁ
1
-Alt/5-Alt, 19a, 6a-Alt/1-Glc, 1-Glc/6a,b-Alt; HR MALDI TOF MS m/
additionally 15 min at 37
measured at ex = 485 nm,
E. Intensity of fluorescence was
em = 518 nm.
+
z 681.3152 [M + Na] , calcd for C32
H
50
O
14Na 681.3098.
l
l
3
2
.3.3. 19-O-{
,3 ,19-triol (3)
Colorless amorphous solid; [
a
-
D
-Glcp(1 !4)-
b
-
D
-Altp}-isopimara-7,15-diene-
4. Conclusions
a
b
20
1
a
]
D
+ 40.0 (c 0.05, MeOH); H and
N), see Tables 1–3; HMBC correlations H1-Alt/
C5-Alt, C-19, H1-Glc/C5-Glc, C4-Alt; selected NOESY correlations
H/H) 1-Alt/5-Alt, 19a, 4-Alt/1-Glc, 1-Glc/5-Glc, 4-Alt; HR MALDI
Virescenosides R –R (1–3), Z (4) complement the set of twelve
1 3
13
C NMR data (C
5
D
5
new glycosides named virescenosides M-X from the same fungus
A. striatiporum (Afiyatullov et al., 2006). Structures of 1–3 were
determined as the corresponding biosides having isopimaradiene-
type aglycons with additional oxidation and unsaturation patterns.
Glycosides 1–4 contain altrose in their carbohydrate chains. The
presence of this sugar is extremely rare in natural glycosides and
was found only for the previously described virescenosides A–C
(Cagnolli-Bellavita et al., 1969,1970,1980).
(
+
52
TOF MS m/z 667.3261 [M + Na] , calcd for C32H O13Na 667.3306.
3
2
.3.4. 19-
,3 ,19-triol (4)
Colorless amorphous solid; [
MeOH) max (log ) 241 (3.61) nm; H and C NMR data (C
see Tables 1–3; HMBC correlations H1-Alt/C5-Alt, C-19; selected
NOESY correlations (H/H) 1 /3,5,9, 2/20, 3/5,18, 5/9, 11 /17,20, 17/
,14
, 19a,b/20, 1-Alt/5-Alt, 19a,b; HR (ꢀ) ESIMS m/z 495.2556
?-b-D-altropyranosyl-6-oxo-isopimara-7,15-diene-
a
b
2
0
a]
D
ꢀ 23.0 (c 0.15, MeOH); UV
1 13
(
l
e
5 5
D N),
Acknowledgment
a
b
1
2
b
b
The study was supported by the grant Russian Science
Foundation No. 14-14-00030.
ꢀ
[M ꢀ H] , calcd for C26
H
39
O
9
495.2594.
3
.4. Acidic hydrolysis of virescenosides R and R (1, 2)
1 2
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