molecules
Article
Three Pairs of New Isopentenyl
Dibenzo[b,e]oxepinone Enantiomers from
Talaromyces flavus, a Wetland Soil-Derived Fungus
Tian-Yu Sun 1,† , Run-Qiao Kuang 1,† , Guo-Dong Chen 1, *, Sheng-Ying Qin 2 , Chuan-Xi Wang 1 ,
Dan Hu 1 , Bing Wu 3 , Xing-Zhong Liu 3 , Xin-Sheng Yao 1 and Hao Gao 1, *
1
2
3
*
†
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University,
Guangzhou 510632, China; [email protected] (T.-Y.S.); [email protected] (R.-Q.K.);
[email protected] (C.-X.W.); [email protected] (D.H.); [email protected] (X.-S.Y.)
Clinical Experimental Center, First Affiliated Hospital of Jinan University, Guangzhou 510632, China;
[email protected]
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190,
China; [email protected] (B.W.); [email protected] (X.-Z.L.)
Correspondence: [email protected] (G.-D.C.); [email protected] (H.G.);
Tel./Fax: +86-20-8522-1559 (G.-D.C.); +86-20-8522-8369 (H.G.)
These authors contributed equally to this work.
Academic Editors: Derek J. McPhee and Antigoni Kotali
Received: 12 August 2016; Accepted: 2 September 2016; Published: 7 September 2016
Abstract: Three pairs of new isopentenyl dibenzo[b,e]oxepinone enantiomers, (+)-(5S)-arugosin K
(1a), (−)-(5R)-arugosin K (1b), (+)-(5S)-arugosin L (2a), (−)-(5R)-arugosin L (2b), (+)-(5S)-arugosin M
(3a), (−)-(5R)-arugosin M (3b), and a new isopentenyl dibenzo[b,e]oxepinone, arugosin N (4), were
isolated from a wetland soil-derived fungus Talaromyces flavus, along with two known biosyntheticallyrelated compounds 5 and 6. Among them, arugosin N (4) and 1,6,10-trihydroxy-8-methyl-2-(3-methyl2-butenyl)-dibenz[b,e]oxepin-11(6H)-one (CAS: 160585-91-1, 5) were obtained as the tautomeric
mixtures. The structures of isolated compounds were determined by detailed spectroscopic analysis.
In addition, the absolute configurations of these three pairs of new enantiomers were determined by
quantum chemical ECD calculations.
Keywords: isopentenyl dibenzo[b,e]oxepinone; wetland soil-derived fungus; Talaromyces flavus;
arugosins K–N
1. Introduction
Isopentenyl dibenzo[b,e]oxepinones possess a dibenzo[b,e]oxepin-11(6H)-one skeleton (intact 6-7-6
tricyclic system) with isopentenyl substitution. Up to now, about nine isopentenyl dibenzo[b,e] oxepinones
had been reported from fungi in Nature, including arugosins A–D from Aspergillus rugulosus [1,2]
and A. variecolor [3], arugosin G from Emericella nidulans var. acristata [4], pestalachloride B
from Pestalotiopsis adusta [5], cephalanones D–E from Graphiopsis chlorocephala [6], and 1,6,10trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one (CAS: 160585-91-1, 5) from
Penicilium sp. [7].
Talaromyces flavus, the commonest species of the genus Talaromyces, has been reported to produce
a series of bioactive compounds [8,9]. In our search for bioactive secondary metabolites from wetland
fungi [10–15], several interesting molecules also had been isolated from this species [12–15]. At present,
a chemical investigation of metabolites from Talaromyces flavus AHK07-3 was carried out, which led to
the isolation of three pairs of new isopentenyl dibenzo[b,e]oxepinone enantiomers ((+)-(5S)-arugosin K
(1a), (−)-(5R)-arugosin K (1b), (+)-(5S)-arugosin L (2a), (−)-(5R)-arugosin L (2b), (+)-(5S)-arugosin M
Molecules 2016, 21, 1184; doi:10.3390/molecules21091184
www.mdpi.com/journal/molecules
Molecules
Molecules2016,
2016,21,
21,1184
1184
22of
of12
12
(−)-(5R)-arugosin M (3b)), a new isopentenyl dibenzo[b,e]oxepinone, arugosin N (4), and two known
(3a),
(−)-(5R)-arugosin
M (3b)), a new isopentenyl dibenzo[b,e]oxepinone, arugosin N (4), and
compounds
1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one
(5) two
and
known
compounds
1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one
chrysophanol (6) (Figure 1). The absolute configurations of the three pairs of enantiomers were
(5)
and chrysophanol
(6) (Figure
1).ECD
The calculations.
absolute configurations
the threeNpairs
of enantiomers
were
determined
by quantum
chemical
In addition,ofarugosin
(4) and
1,6,10-trihydroxydetermined
by
quantum
chemical
ECD
calculations.
In
addition,
arugosin
N
(4)
and
1,6,10-trihydroxy8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one (5) were a pair of tautomers, and they
8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one
(5)elucidations
were a pair of
and
were isolated as mixtures. Detail of the isolation and structural
of tautomers,
compounds
1–6they
are
were
isolated
as
mixtures.
Detail
of
the
isolation
and
structural
elucidations
of
compounds
1–6
are
presented herein.
presented herein.
Figure
Figure 1.
1. Production
Production and
and chemical
chemicalstructures
structuresof
of1–6.
1–6.
2. Results
2.
Results and
and Discussion
Discussion
Compound11was
wasobtained
obtainedas
asaayellow
yellowsolid.
solid.The
Thequasi-molecular
quasi-molecularion
ionatatm/z
m/z355.1550
355.1550[M
[M++H]
H]++
Compound
obtained by
by HRESIMS
HRESIMS indicated
indicated the
the molecular
molecular formula
formula ofof 11was
wasCC
H2222O
O55 with 11
11 degrees
degrees of
of
obtained
2121H
13
13C-NMR spectrum combined with the DEPT-135 spectrum showed 21 signals
unsaturation.
The
unsaturation.
C-NMR spectrum combined with
spectrum showed 21 signals
(Table 1),
1),assigned
assignedtototen
tensp
sp2 2quaternary
quaternary carbons
carbons [including
[including one
one ketone
ketone carbonyl
carbonyl (δ(δCC 197.5),
197.5), nine
nine
(Table
aromatic/olefinic carbons
124.8, 116.8,
116.8, and
and 113.6),
113.6),five
fivesp
sp22
aromatic/olefinic
carbons (δCC 162.8,
162.8, 162.4,
162.4, 154.5, 147.1, 138.5, 133.2, 124.8,
3
3
methinecarbons
carbons(δ(δ
137.6,
121.8,
119.5,
116.8,
109.3),
oneoxygenated
sp oxygenated
methine
(δC
methine
137.6,
121.8,
119.5,
116.8,
andand
109.3),
one sp
methine
carboncarbon
(δC 103.4),
CC
103.4),
sp3 methylene
(δCand
27.8),
and
four methyl
C 56.9,
25.8,
21.8,
andIn17.8).
one
sp3 one
methylene
carbon carbon
(δC 27.8),
four
methyl
carbonscarbons
(δC 56.9,(δ25.8,
21.8,
and
17.8).
the
1In
the 1H-NMR
spectrum
of 1),
1 (Table
1), the characteristic
signals
of two exchangeable
protons
H-NMR
spectrum
of 1 (Table
the characteristic
signals of two
exchangeable
protons (δH 13.62
(1H,(δs)H
J = 8.3
Hz),
6.94 (1H,
(1H, s),
13.62
(1H,(1H,
s) and
(1H, s)), five
aromatic
or olefinic
protons
and
11.46
s)),11.46
five aromatic
or olefinic
protons
(δH 7.33
(1H, (δ
d,HJ 7.33
= 8.3(1H,
Hz),d,6.94
(1H,
s), 6.87
3 oxygenated
6.87(1H,
(1H,d,
s),J 6.58
J = 5.33
8.3 Hz),
(1H,
br one
t, J =sp
7.3
Hz), one sp3methine
oxygenated
methine
proton
6.58
= 8.3(1H,
Hz),d,and
(1H,and
br t,5.33
J = 7.3
Hz),
proton
(δH 5.64
(1H,
3
3 methylene (δH 3.34 (2H, d, J = 7.3 Hz)), and four methyl protons (δH 3.57 (3H, s),
(δ
H
5.64
(1H,
s)),
one
sp
s)), one sp methylene (δH 3.34 (2H, d, J = 7.3 Hz)), and four methyl protons (δH 3.57 (3H, s), 2.37 (3H,
2.37
(3H,
(3H,
br(3H,
s), and
(3H,
br s)) were
observed. The non-exchangeable
s),
1.77
(3H,s),br1.77
s), and
1.73
br s))1.73
were
observed.
The non-exchangeable
proton resonancesproton
were
resonanceswith
werethe
associated
the directly
atomsexperiment
in the HSQC
experiment
1).
associated
directlywith
attached
carbonattached
atoms incarbon
the HSQC
(Table
1). The (Table
analysis
1
1
1
1
The
of the experiment
H- H COSY
experiment
andvalues
the coupling
valuesindicated
of the protons
indicated
the
of
theanalysis
H- H COSY
and
the coupling
of the protons
the presence
of two
0 –C-20 ) as
1 H-1 H
presence (C-7–C-8
of two subunits
and
C-1′–C-2′)
as shown
in Figurewith
2. Combined
with
subunits
and C-1(C-7–C-8
shown
in Figure
2. Combined
the analysis
ofthe
theanalysis
1H-1H COSY experiment and molecular formula, the HMBC correlations from H3-1 to
of theexperiment
COSY
and molecular formula, the HMBC correlations from H3 -1 to C-2/C-3/C-15, from
C-2/C-3/C-15,
from H-3 tofrom
C-1/C-5/C-13/C-15,
from H-5 to3 , from
C-3/C-6/C-13/5-OCH
3, from
H-7H-8
to
H-3
to C-1/C-5/C-13/C-15,
H-5 to C-3/C-6/C-13/5-OCH
H-7 to C-6/C-9/C-11,
from
0 , from
0 to C-20 /C-30 /C-4
0,
C-6/C-9/C-11,
from
C-6/C-10, from
H2-1′
from
H3-4′Hto
C-2′/C-3′/C-5′,
from
to
C-6/C-10, from
H2H-8
-10 totoC-8/C-9/C-10,
from
H3to
-40 C-8/C-9/C-10,
to C-20 /C-30 /C-5
-5
3
H3-5′5-OCH
to C-2′/C-3′/C-4′,
from
5-OCH
3 to C-5, fromand
10-OH
C-9/C-10/C-11,
and from
14-OHthe
to
from
10-OH
to C-9/C-10/C-11,
fromto
14-OH
to C-13/C-14/C-15
revealed
3 to C-5, from
C-13/C-14/C-15
revealed
the2).
planer
structure
(Figureanalyses,
2). On the
of structure
the aboveofanalyses,
planer
structure of
1 (Figure
On the
basis of of
the1 above
thebasis
planar
1 (Figurethe
2)
planar
structure
of
1
(Figure
2)
was
established,
and
it
was
named
arugosin
K.
The
assignments
of
all
was established, and it was named arugosin K. The assignments of all proton and carbon resonances
proton
and carbon
resonances
are provided in Table 1.
are
provided
in Table
1.
Molecules 2016, 21, 1184
3 of 12
Table 1. The 13 C- and 1 H-NMR data of 1–3 (δ in ppm, J in Hz).
1a
No.
δC
δH
No.
δC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
10
21.8
147.1
116.8
138.5
103.4
154.5
109.3
137.6
124.8
162.8
113.6
197.5
116.8
162.4
119.5
27.8
2.37, s
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
10
21.9
147.2
116.8
138.8
102.0
154.8
109.3
137.6
124.7
162.7
113.7
197.6
116.9
162.3
119.4
27.8
20
30
40
50
5-OCH3
121.8
133.2
25.8
17.8
56.9
1.77, br s
1.73, br s
3.57, s
20
30
40
50
100
121.8
133.3
25.8
17.8
65.3
200
10-OH
14-OH
14.8
13.62, s
11.46, s
10-OH
14-OH
a
3a
2b
6.94, s
5.64, s
6.58, d (8.3)
7.33, d (8.3)
6.87, s
3.34, d (7.3)
5.33, br t (7.3)
δH
c
2.38, s
6.98, s
5.74, s
6.55, d (8.3)
7.32, d (8.3)
6.86, s
3.35, dd (15.9, 7.4), a
3.31, dd (15.9, 7.4), b
5.32
1.76, br s
1.73, br s
3.96, dq (9.6, 7.1), a
3.66, dq (9.6, 7.1), b
1.26, t (7.1)
13.63, s
11.44, s
No.
δC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
10
21.9
147.1
116.6
138.3
103.9
163.5
112.2
138.4
121.8
153.2
113.9
197.5
117.0
161.9
119.4
28.0
20
30
40
50
5-OCH3
122.1
133.2
25.7
17.8
57.3
6-OH
14-OH
δH
c
2.39, s
6.97, s
5.71, s
6.70, d (8.5)
7.35, d (8.5)
6.87, s
3.36, dd (15.6, 7.5), a
3.30, dd (15.6, 7.5), b
5.25
1.73, br s
1.71, br s
3.57, s
13.14, s
11.22, s
The data recorded in CDCl3 (1 H-NMR for 400 MHz, 13 C-NMR for 100 MHz); b The data recorded in CDCl3 (1 H-NMR for 600 MHz, 13 C-NMR for 150 MHz); c Indiscernible
signals owing to overlapping or having complex multiplicity are reported without designating multiplicity. The letters of a and b mean that these two potons on C-10 are not
chemical equivalence.
Molecules 2016, 21, 1184
Molecules 2016, 21, 1184
4 of 12
4 of 12
Molecules 2016, 21, 1184
4 of 12
11H11 H
Figure
2.
Key
HCOSY and
and HMBC
of of
1 and
3. 3.
Figure
2.
Figure
2. Key
Key
HHCOSY
HMBCcorrelations
correlations
1 and
1
1
The specific rotation value of 1 was close to zero, indicating that 1 was a racemic mixture.
The specific
specific rotation
rotation value
value of
of 11 was
was close
close to
to zero,
zero, indicating
indicating that
11 was
aa racemic
mixture.
The
thatcolumn
was(Figure
racemic
mixture.
Compound 1 displayed
two peaks
in a Phenomenex
Lux Cellulose-2 chiral
3), which
Compound
1
displayed
two
peaks
in
a
Phenomenex
Lux
Cellulose-2
chiral
column
(Figure
3),
which
Compound
1 displayed
two peaksTherefore,
in a Phenomenex
Cellulose-2
chiral
column
3), which
confirmed
the above deduction.
(+)-1a (tR = Lux
20.3 min)
and (−)-1b
(tR = 23.2
min) (Figure
were isolated
confirmed
the
above
deduction.
Therefore,
(+)-1a
(t
R = 20.3 min) and (−)-1b (tR = 23.2 min) were isolated
confirmed
deduction.
Therefore,
(+)-1a
(tR =
20.3CH
min)
and
−)-1b v/v)
(tR =as23.2
min)
from 1 the
by aabove
Phenomenex
Lux Cellulose-2
chiral
column
using
3OH/H
2O((88/12,
eluent
at awere
27
from
1
by
a
Phenomenex
Lux
Cellulose-2
chiral
column
using
CH
3OH/H
2O (c
(88/12,
v/v)
as
eluent
flow
rate
of
0.7
mL/min.
The
ECD
spectra
and
[α]
values
of
1a
([α]
+27.8
0.5,
CHCl
3
))
and
isolated from 1 by a Phenomenex Lux Cellulose-2 chiral
column using
CH3 OH/H2 O (88/12, 1b
v/v)atasa
D
D
27
27
27
flow
rate
of
0.7
mL/min.
The
ECD
spectra
and
[α]
values
of
1a
([α]
+27.8
(c
0.5,
CHCl
3
))
and 1b
−27.0
(c
0.5,
CHCl
3
))
showed
their
enantiomeric
relationship.
([α]
D and [α]
D
eluent
atDa flow rate of 0.7 mL/min. The ECD spectra
D values of 1a ([α]D +27.8 (c 0.5, CHCl3 ))
27
27
0.5,
CHCl
3)) showed their enantiomeric relationship.
([α]D1b−27.0
and
([α]D(c−
27.0
(c 0.5,
CHCl3 )) showed their enantiomeric relationship.
mAU
208nm,4nm (1.00)
3000
mAU
208nm,4nm (1.00)
30002500
25002000
20001500
1000
1500
1000
500
0
500
0
0
10
mAU
208nm,4nm (1.00)
2500
20
30
40
min
2250
0
2500
1500
2000
1250
1750
1000
1250
20
30
40
min
1750
2250
1500
10
2000
mAU
208nm,4nm (1.00)
750
500
250
1000
0
750
-250
500
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
min
250
Figure 3. HPLC0analysis of 1. a: the analysis of 1 on routine ODS HPLC; b: the analysis of 1 on chiral HPLC.
-250
The absolute configurations
of 10.0
1a and 1b
were determined
by quantum
chemical
ECD
calculations
0.0
5.0
15.0
20.0
25.0
30.0
35.0
min
at the APFD/6-311++G(2d,p) level. The predicted ECD curves of (5S)-1 and (5R)-1 (Figure 4) were in
Figure
3.3.HPLC
analysis
1.ofa:
thea:analysis
of
1 onfor
routine
ODS
HPLC;
the analysis
of analysis
1 onthe
chiral
good accordance
with of
the
experimental
ECD
1b,
respectively.
Therefore,
absolute
Figure
HPLC
analysis
1.
the analysis
of
11aonand
routine
ODSb:HPLC;
b: the
ofHPLC.
1 on
configurations
of
1a
and
1b
were
identified
as
5S
and
5R,
respectively.
chiral HPLC.
The absolute configurations of 1a and 1b were determined by quantum chemical ECD calculations
at the APFD/6-311++G(2d,p) level. The predicted ECD curves of (5S)-1 and (5R)-1 (Figure 4) were in
The absolute configurations of 1a and 1b were determined by quantum chemical ECD calculations
good accordance with the experimental ECD for 1a and 1b, respectively. Therefore, the absolute
at the APFD/6-311++G(2d,p) level. The predicted ECD curves of (5S)-1 and (5R)-1 (Figure 4) were
configurations of 1a and 1b were identified as 5S and 5R, respectively.
in good accordance with the experimental ECD for 1a and 1b, respectively. Therefore, the absolute
configurations of 1a and 1b were identified as 5S and 5R, respectively.
Molecules 2016, 21, 1184
5 of 12
Molecules
21, 1184
Molecules
2016, 2016,
21, 1184
5 of 125 of 12
15
Expt. 1a
15
Expt.
1a1b
Expt.
Expt.
1b5S-1
Calc.
Calc.
5S-1
Calc.
5R-1
Calc. 5R-1
10
10
5
0 0
Δε
Δε
5
-5 -5
-10-10
-15-15200
200
250
250
300
300
350
350
400
400
wavelength (nm)
450
450
500
500
wavelength (nm)
Figure 4. The experimental ECD spectra of 1a and 1b and calculated ECD spectra for (5S)-1 and (5R)-1
Figure
4.
The
ECD spectra
of 1a and
1b and calculated ECD spectra for (5S)-1 and (5R)-1
Figure
4. correction
The experimental
experimental
(UV
= −10 nm,ECD
bandspectra
width σof=1a
0.3and
eV).1b and calculated ECD spectra for (5S)-1 and (5R)-1
(UV
10 nm,
(UV correction
correction==−−10
nm, band
bandwidth
widthσσ==0.3
0.3eV).
eV).
Compound 2 was obtained as a yellow solid. The quasi-molecular ion at m/z 369.1695 [M + H]+
by
HRESIMS22indicated
the molecular
formula
of The
2The
was
C22H24O5 with 11ion
degrees
of369.1695
unsaturation.
Compound
wasobtained
obtained
asaayellow
yellow
solid.
quasi-molecular
ion
m/z
369.1695
[M++H]
H]++
Compound
was
as
solid.
quasi-molecular
atatm/z
[M
Except forindicated
the loss of the
an
at δof
C 56.9
(5-OCH
3) 24
and the
appearance
of an
by HRESIMS
HRESIMS
indicated
theoxygenated
molecularmethyl
formula
of
was
H
11 degrees
by
molecular
formula
22was
CC2222H
with
ofadditional
unsaturation.
24 O55 with
oxygenated
methylene
at
δ
C 65.3 (C-1′′) and methyl at δC 14.8 (C-2′′), the NMR data of 2 (Table 1) were
Except for
for the
the loss
lossof
ofan
anoxygenated
oxygenatedmethyl
methylat
atδδCC 56.9
56.9 (5-OCH
(5-OCH33) and the appearance
appearance of
of an
an additional
additional
Except
similar to
those of 1 (Table
1), which
that the δ5-OCH
3 in 1 00
was
replaced
by 5-OCH
2CH3 in 2.
00 indicated
oxygenated
methylene
atδδCC 65.3
65.3
(C-1′′)
C
14.8
(C-2′′),
the
NMR
data
of
2
(Table
1) were
were
oxygenated
methylene
at
(C-1
) and
methyl
at
δ
(C-2
),
the
NMR
1)
C
This deduction was supported by the 1H-1H COSY correlations of Ha-1′′/Hb-1′′ and H3-2′′ and the
similar to
to those
those of
of 11 (Table
(Table1),
1),which
whichindicated
indicatedthat
thatthe
the5-OCH
5-OCH33 in
in 11 was
was replaced
replaced by
by 5-OCH
5-OCH22CH33 in 2.
similar
key HMBC correlations from H-5 to C-1′′,
from Ha-1′′/Hb-1′′ to C-5/C-2′′, and
from H3-2′′ to C-1′′.
On
1 H1 1HCOSY
00 /Hb-100 and
00 and the
1HCOSYcorrelations
correlations
Ha-1′′/Hb-1′′
and H
H33-2
-2′′
the
This deduction
deduction the
wasexhaustive
supported
bythe
theof
This
supported
by
ofofHSQC,
Ha-1
the basis of was
analysis
2DH
NMR data
(1H-1H COSY,
and HMBC) (Tables
S2,
00 , from Ha-1′′/Hb-1′′
00 /Hb-100toto
00 , and
00C-1′′.
00 .
key HMBC
HMBC
correlations
C-1′′,
C-5/C-2′′,
and
from
H
3-2′′
to
On
key
correlations
from
H-5
to
C-1
Ha-1
C-5/C-2
from
H
-2
to
C-1
3
Supplementary Materials), the planar structure of 2, named arugosin L, was established (Figure
1).
1H1 COSY, HSQC, and HMBC) (Tables S2,
the the
basis
of the
exhaustive
of strain,
2D2D
NMR
(a1HOn
basis
of1the
exhaustive
analysis
of
data
(1 HH mixture,
COSY, HSQC,
and HMBC)
(Table
Since
and
2 coexist analysis
in the
same
2NMR
wasdata
also
racemic
and it displayed
two peaks
Supplementary
Materials),
theplanar
planarchiral
structure
of2,
2,named
named
arugosin
L,was
wasMaterials).
established
(Figure1).
1).
Supplementary
Materials),
the
structure
of
L,
established
(Figure
in a Phenomenex
Lux Cellulose-2
column
(Figure
S1,arugosin
Supplementary
Therefore,
(+)-2a
= 22.6
min) and
(tR = strain,
26.6
min)
werealso
isolated
from 2 mixture,
by a Phenomenex
Lux Cellulose-2
Since 1(t1Rand
and
coexist
in(−)-2b
the same
same
strain,
was
also
racemic
mixture,
and itit displayed
displayed
two peaks
peaks
Since
22 coexist
in
the
22 was
aa racemic
and
two
3OH/H2O chiral
(88/12,
v/v)
as eluent
at a S1,
flow
rate of 0.7 mL/min.
The ECD spectra
chiral
column using
CH
in aa Phenomenex
Phenomenex
Lux Cellulose-2
Cellulose-2
chiral column
column
(Figure
S1,Supplementary
Supplementary
Materials).
Therefore,
in
Lux
(Figure
Materials).
Therefore,
27
27
and
[α]
values
of
2a
([α]
+27.4
(c
0.5,
CHCl
3)) and 2b ([α]D −26.8 (c 0.5, CHCl3)) showed their
D
D
(+)-2a (t
(tRR = 22.6 min) and ((−)-2b
Lux Cellulose-2
Cellulose-2
(+)-2a
−)-2b(t(tRR==26.6
26.6min)
min)were
wereisolated
isolated from
from 22 by a Phenomenex Lux
relationship.
Since
the ECD dataasofeluent
2a were similar
to thatofof0.7
1a mL/min.
(Figure 5), the absolute
OH/H
22O
ECD spectra
spectra
chiralenantiomeric
columnusing
using
CH33OH/H
chiral
column
CH
O (88/12,
(88/12,v/v)
v/v) as eluentat
at aa flow rate of
0.7 mL/min.
The ECD
configuration of 2a was27determined
as 5S (Figure 5). The similar
observation
was
found between 2b
27
27
27
and [[α]
values of
of 2a
2a([([α]
+27.4 (c(c0.5,
0.5,CHCl
CHCl33)))) and
and 2b
2b([([α]
(c 0.5,
0.5, CHCl
CHCl3))
)) showed
showed their
their
and
α]DD values
α]DD +27.4
α] D −−26.8
26.8 (c
and
1b. Therefore, the absolute
configurations of
2a and 2b wereDassigned as 5S and 5R, 3respectively.
enantiomeric relationship.
relationship. Since the ECD data of
of 2a
2a were
were similar
similar to
to that
thatof
of1a
1a(Figure
(Figure5),
5),the
theabsolute
absolute
enantiomeric
configuration
of
2a
was
determined
as
5S
(Figure
5).
The
similar
observation
was
found
between
2b
configuration of 2a was determined
as 5S (Figure 5). The similar observation
was found between 2b
15
2a
and 1b.
1b. Therefore,
Therefore, the
the absolute
absolute configurations
configurations of
of 2a
2a and 2b were assigned
respectively.
and
as 5S and 5R, respectively.
2b
10
Δε
15
10
1a
1b
2a
2b
1a
1b
5
0
Δε
5 -5
0-10
-5-15
200
250
300
350
400
450
500
wavelength (nm)
-10
Figure 5. The experimental ECD spectra of 2a, 2b, 1a, and 1b.
-15
200
250
300
350
400
450
500
wavelength (nm)
Figure 5.
5. The
The experimental
experimental ECD
ECD spectra
spectra of
of 2a,
2a, 2b,
2b, 1a,
1a, and
and 1b.
1b.
Figure
Molecules 2016, 21, 1184
6 of 12
Compound 3 was obtained as a yellow solid. The quasi-molecular ion at m/z 355.1541 [M + H]+
Molecules indicated
2016, 21, 1184 the molecular formula of 3 was C H O with 11 degrees of unsaturation,
6 of 12
by HRESIMS
21 22 5
which indicated that 3 was the isomer of 1. Furthermore, the NMR data of 3 (Table 1) were similar
+
Compound 3 was obtained as a yellow solid. The quasi-molecular ion at m/z 355.1541 [M + H]
to thosebyofHRESIMS
1, whichindicated
suggested
that 3 and
1 hadofthe
similar
analysis
of the 1 H-1 H
the molecular
formula
3 was
C21H22skeleton.
O5 with 11The
degrees
of unsaturation,
COSY experiment
andthat
the3 was
coupling
values
the protons
presence
of two
subunits
which indicated
the isomer
of 1.of
Furthermore,
theindicated
NMR data the
of 3 (Table
1) were
similar
to
0
0
1
1
1
1
(C-7–C-8
andofC-1
–C-2suggested
) as shown
Figure
2. the
Combined
with the
theH-H–
H COSY
those
1, which
thatin
3 and
1 had
similar skeleton.
The analysis
analysis ofofthe
H COSY
experiment
and the coupling
values
the protons
indicated(Figure
the presence
of two
(C-7–C-8
experiment
and molecular
formula,
theofHMBC
correlations
2) from
H3subunits
-1 to C-2/C-3/C-15,
1H–1H COSY experiment and
and
C-1′–C-2′)
as
shown
in
Figure
2.
Combined
with
the
analysis
of
the
from H-3 to C-1/C-5/C-13/C-15, from H-5 to C-3/C-10/C-13/5-OCH3 , from H-7 to C-6/C-9/C-11,
molecular
formula,
theHa-1
HMBC
correlations
(Figure 2) from
from H
H3-1
from
H-3 to 0
0 /Hb-1
0 to C-8/C-9/C-10,
0 to C-2/C-3/C-15,
0
0
0
from H-8
to C-6/C-10,
from
3 -4 to C-2 /C-3 /C-5 , from H3 -5 to
C-1/C-5/C-13/C-15,
from H-5 to C-3/C-10/C-13/5-OCH3, from H-7 to C-6/C-9/C-11, from H-8 to
0
0
0
C-2 /C-3 /C-4 , from 5-OCH3 to C-5, from 6-OH to C-6/C-7/C-11, and from 14-OH to C-13/C-14/C-15
C-6/C-10, from Ha-1′/Hb-1′ to C-8/C-9/C-10, from H3-4′ to C-2′/C-3′/C-5′, from H3-5′ to C-2′/C-3′/C-4′,
confirmed
the planar structure of 3, which is named arugosin M.
from 5-OCH3 to C-5, from 6-OH to C-6/C-7/C-11, and from 14-OH to C-13/C-14/C-15 confirmed the
Since
1
3 coexist
the same
strain,
3 wasM.
also a racemic mixture, and it displayed two peaks
planarand
structure
of 3,in
which
is named
arugosin
in a Phenomenex
Lux
Cellulose-2
chiral
column
(Figure
S2, Supplementary
Materials).
Since 1 and 3 coexist in the same strain, 3 was also a racemic
mixture, and it displayed
twoTherefore,
peaks
Phenomenex
Cellulose-2
chiral
column
Supplementary
Materials).Lux
Therefore,
(+)-3a (tin
min) andLux
(−)-3b
(tR = 22.1
min)
were (Figure
isolatedS2,
from
3 by a Phenomenex
Cellulose-2
R =a 20.4
(+)-3a (tRusing
= 20.4 min)
(−)-3b
(tR = 22.1 min) were isolated from 3 by a Phenomenex Lux Cellulose-2
chiral column
CH3and
OH/H
2 O (88/12, v/v) as eluent at a flow rate of 0.7 mL/min. The ECD
27
3OH/H
2O (88/12, v/v) as eluent at a flow rate of
0.7 mL/min. The ECD spectra
chiral
column
using
CH
spectra and [α]D values of 3a ([27α]D +28.4 (c 0.5, CHCl3 )) and 3b
([α]27
D −27.6 (c 0.5, CHCl3 )) showed
27
and [α]D values of 3a ([α]D +28.4 (c 0.5, CHCl3)) and 3b ([α]D −27.6 (c 0.5, CHCl3)) showed their
their enantiomeric relationship. The absolute configurations of 3a and 3b were determined by quantum
enantiomeric relationship. The absolute configurations of 3a and 3b were determined by quantum
chemical
ECD calculations at the APFD/6-311++g(2d,p) level. The predicted ECD curves of (5S)-3
chemical ECD calculations at the APFD/6-311++g(2d,p) level. The predicted ECD curves of (5S)-3 and
and (5R)-3
were
in in
good
experimentalECD
ECD
and
respectively
(Figure
(5R)-3
were
goodaccordance
accordance with
with the
the experimental
forfor
3a 3a
and
3b,3b,
respectively
(Figure
6). 6).
Therefore,
the
absolute
configurations
of
3a
and
3b
were
identified
as
5S
and
5R,
respectively.
Therefore, the absolute configurations of 3a and 3b were identified as 5S and 5R, respectively.
15
Expt. 3a
Expt. 3b
Calc. 5S-3
Calc. 5R-3
10
Δε
5
0
-5
-10
-15
200
250
300
350
400
450
500
wavelength (nm)
Figure
6. The experimental
ECD spectra
3a 3b
andand
3b calculated
and calculated
spectra
(5S)-3
and
Figure 6.
The experimental
ECD spectra
of 3a of
and
ECDECD
spectra
for for
(5S)-3
and
(5R)-3
(5R)-3 (UV correction = −20 nm, band width σ = 0.3 eV).
(UV correction = −20 nm, band width σ = 0.3 eV).
Compounds 4 and 5 were obtained as mixtures of yellow solids. The quasi-molecular ion at m/z
Compounds
and+ by
5 were
obtained
as mixtures
of yellow
quasi-molecular
ion at
363.1213 [M 4+ Na]
HRESIMS
indicated
the molecular
formulassolids.
of 4 andThe
5 were
C20H20O5, with 11
+
degrees
of
unsaturation.
The
NMR
data
of
the
mixtures
presented
two
sets
of
signals,
assigned
to
m/z 363.1213 [M + Na] by HRESIMS indicated the molecular formulas of 4 and 5 were C20 H420 O5 ,
5 respectively
(Table 2).The
The NMR
molecular
of 4 was fourteen
atomic
less than
3.
with 11 and
degrees
of unsaturation.
dataformula
of the mixtures
presented
twomass
sets units
of signals,
assigned
Except for the loss of an oxygenated methyl at δC 57.3 (5-OCH3), the 1H NMR and 13C-NMR data of 4
to 4 and 5 respectively (Table 2). The molecular formula of 4 was fourteen atomic mass units less than
(Table 2) were similar to those of 3 (Table 1), which indicated that the 5-OCH3 in 3 was replaced by the
3. Except for the loss of an oxygenated methyl at δC 57.3 (5-OCH3 ), 1the1 1 H NMR and 13 C-NMR data
hydroxyl in 4. On the basis of the exhaustive analysis of 2D NMR data ( H- H COSY, HSQC, and HMBC)
of 4 (Table
2) were
similar to those
of 3 (Table
1), which
indicated
the 5-OCH
3 was replaced
3 in
(Table
S4, Supplementary
Materials),
the planar
structure
of 4 wasthat
established,
named
arugosin
N.
1 H-1 H COSY, HSQC,
by the hydroxyl
in
4.
On
the
basis
of
the
exhaustive
analysis
of
2D
NMR
data
(
The molecular formula of 5 was fourteen atomic mass units less than 1. Except for the loss of an
13C-NMR
and HMBC)
(Tablemethyl
S4, Supplementary
theand
planar
structure
was 2)
established,
oxygenated
at δC 56.9 (5-OCHMaterials),
3), the 1H-NMR
data ofof5 4
(Table
were similarnamed
to
those
arugosin
N. of 1 (Table 1), which indicated that the 5-OCH3 in 1 was replaced by the hydroxyl in 5.
Onmolecular
the basis offormula
the exhaustive
of 2D NMR
datamass
(1H-1Hunits
COSY,less
HSQC,
and
S5, loss
The
of 5 analysis
was fourteen
atomic
than
1. HMBC)
Except(Table
for the
Supplementary
Materials),
the
planar
structure
of
5
was
established
as
the
known
compound,
1
13
of an oxygenated methyl at δC 56.9 (5-OCH3 ), the H-NMR and C-NMR data of 5 (Table 2) were
1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one [7].
similar to those of 1 (Table 1), which indicated that the 5-OCH3 in 1 was replaced by the hydroxyl
in 5. On the basis of the exhaustive analysis of 2D NMR data (1 H-1 H COSY, HSQC, and HMBC)
(Table S5, Supplementary Materials), the planar structure of 5 was established as the known compound,
1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one [7].
Molecules 2016, 21, 1184
7 of 12
Table 2. The 1H-NMR and 13C-NMR data of 4 and 5 (δ in ppm, J in Hz).
Molecules 2016, 21, 1184
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1′
2′
3′
4′
5′
6-OH
14-OH
4a
5a
7 of 12
δC
δH
No.
δC
δH
1
13
Table
and
in ppm, J in Hz). 2.40, s
22.0 2. The H-NMR
2.40,
s C-NMR data of
1 4 and 5 (δ 21.9
147.6
2
147.4
4 a 7.07, s
5a
116.0
3
116.2
7.04, s
No.
δC
δH
δC
δH
139.7
4 No.
139.3
6.11, s2.40, s
5 1
96.6
1 97.1
22.0
21.9
2.40, s 6.12, s
2163.6 147.6
147.4
6 2
154.5
3112.9 116.0
7.07, s
116.2
7.04,
s d (8.4)
6.71, d (8.4)
7 3
109.6
6.53,
4
139.7
4
139.3
138.5
7.34, d (8.4)
8
137.7
7.32, d (8.4)
5
97.1
6.11, s
5
96.6
6.12, s
122.4
9
125.1
6
163.6
6
154.5
10 7
162.9
7154.0 112.9
6.71, d (8.4)
109.6
6.53, d (8.4)
8114.6 138.5
7.34, d (8.4)
8
137.7
7.32, d (8.4)
11
113.5
9197.3 122.4
9
125.1
12
197.3
10
154.0
10
162.9
116.5
13
116.7
11
114.6
11
113.5
14 12
162.7
12163.3 197.3
197.3
13119.8 116.5
116.7
6.89, s
15 13
119.7
6.89, s
1428.9
163.3
14
162.7
3.36, dd (15.4, 7.2), a
1′
27.8
3.33, d (7.5)
15
119.8
6.89, s
15
119.7
6.89, s
3.22, dd (15.4, 7.2), b
0
0
1
28.9
3.36, dd (15.4, 7.2), a
1
27.8
3.33, d (7.5)
123.1
5.24,
brdd
t (7.2)
2′
121.7
5.31, br t (7.5)
3.22,
(15.4, 7.2), b
0
0
3′ 2
133.4 5.31, br t (7.5)
2133.0 123.1
5.24, br t (7.2)
121.7
0
30 25.7
133.0
133.4
1.76, s
4′ 3
25.8
1.76, s
40
25.7
1.76, s
40
25.8
1.76, s
18.0
1.74,
s
5′
17.8
1.72, s
50
18.0
1.74, s
50
17.8
1.72, s
12.95,12.95,
br s br s
10-OH
6-OH
10-OH
13.61, 13.61,
br s br s
11.82,11.82,
br s br s
14-OH
14-OH
14-OH
11.54, 11.54,
br s br s
13C-NMR for 100 MHz). The letters of a and b
1 (1H-NMR
13 C-NMR
The
CDCl
for MHz,
400 MHz,
Thedata
datarecorded
recorded ininCDCl
for 400
for 100 MHz). The letters of a and b mean that
3 (3 H-NMR
0 are not chemical equivalence.
these
two
potons
on
C-1
mean that these two potons on C-1’ are not chemical equivalence.
a a
The C-5 positions of 4 and 5 were hemiacetal linkage, which
which were
were unstable
unstable and
and easy
easy to
to split.
split.
inseparable
tautomeric
mixtures
in
Therefore, 4 and
and 55 can
can self-interconvert
self-interconvert(Figure
(Figure7)7)and
andexist
existasas
inseparable
tautomeric
mixtures
Nature.
in
Nature.
Figure
7. The
of 44 and
and 5.
5.
Figure 7.
The interconversion
interconversion of
Compound 6 was obtained as a yellow solid. The ESI-MS (positive): m/z 255 [M + H]+; ESI-MS
Compound 6 was obtained as a yellow solid. The ESI-MS (positive): m/z 255 [M + H]+ ;
(negative): m/z 253 [M − H]− indicated the
molecular weight was 254. It was identified as chrysophanol
ESI-MS (negative): m/z1 253 [M13− H]− indicated the molecular weight was 254. It was identified as
by comparing its data ( H- and C-NMR, MS) with literature values [16].
chrysophanol by comparing its data (1 H- and 13 C-NMR, MS) with literature values [16].
Molecules 2016, 21, 1184
Molecules 2016, 21, 1184
8 of 12
8 of 12
Based on the structural features of compounds 1–6 and literature investigation, a plausible
biosynthetic pathway of them was
was proposed
proposed (Scheme
(Scheme 1).
1). Chrysophanol
Chrysophanol (6)
(6) undergoes
undergoes oxidization
oxidization[17,18],
[17,18],
ring-cleavage [17,18], reduction
reduction [17,18],
[17,18], and
and isoprentenylation
isoprentenylation to yield arugosin I [17], which may be the
precursor of arugosin N (4) and 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin11(6H)-one
Then, arugosin
arugosin K
K (1)
(1) and
and arugosin
arugosin L
L (2) may originate from 5 by
11(6H)-one (5). Then,
by methylation
methylation and
ethylation, respectively. Arugosin M (3) may originate from 4 by
by methylation.
methylation.
Scheme 1.
1. The
pathways of
of 1–6.
1–6.
Scheme
The plausible
plausible biosynthetic
biosynthetic pathways
3. Materials
3.
Materialsand
andMethods
Methods
3.1. Chemicals
Methanol (MeOH) was purchased
purchased from Yuwang Industrial Co. Ltd. (Yucheng, China).
China).
Cyclohexane, ethyl
ethylacetate
acetate(EtOAc),
(EtOAc),and
and
petroleum
ether
(PE)
were
purchased
Tianjin
Cyclohexane,
petroleum
ether
(PE)
were
purchased
fromfrom
Tianjin
FuyuFuyu
Fine
Fine
Chemical
Co.
Ltd.
(Tianjin,
China).
Chemical Co. Ltd. (Tianjin, China).
3.2. General Experimental Procedures
Optical
rotationswere
weremeasured
measured
a P1020
digital
polarimeter
International
Co.
Ltd.,
Optical rotations
onon
a P1020
digital
polarimeter
(Jasco(Jasco
International
Co. Ltd.,
Tokyo,
Tokyo,
Japan).
were using
recorded
using
a Jasco
V-550
UV/Vis spectrometer.
IR data were
Japan). UV
data UV
weredata
recorded
a Jasco
V-550
UV/Vis
spectrometer.
IR data were recorded
on a
recorded
on a Jasco
FT/IR-480 plus
spectrometer.
ECD
spectrum
recorded
on a Jasco J-810
Jasco FT/IR-480
plus spectrometer.
ECD
spectrum was
recorded
on awas
Jasco
J-810 spectrophotometer
spectrophotometer
MeOH
as the solvent.
The recorded
ESI-MS spectra
were recorded
on a Finnigan
using MeOH as the using
solvent.
The ESI-MS
spectra were
on a Finnigan
LCQ Advantage
MAX
LCQ
massFisher
spectrometer
(Thermo
Fisher Scientific,
Inc.,
Waltham,
MA,spectra
USA).
mass Advantage
spectrometerMAX
(Thermo
Scientific,
Inc., Waltham,
MA, USA).
The
HR-ESI-MS
The
spectra
were obtained
a Micromass
Q-TOF
mass spectrometer
wereHR-ESI-MS
obtained on
a Micromass
Q-TOFon
mass
spectrometer
(Waters
Corporation, (Waters
Milford,Corporation,
MA, USA).
Milford,
MA,
USA).
The
NMR
spectra
were
measured
with
Bruker
AV-400
and
Bruker
AV-600
The NMR spectra were measured with Bruker AV-400 and Bruker AV-600 spectrometers (Bruker
spectrometers
BioSpin
Group, Faellanden,
Switzerland)
using 3the
signals
BioSpin Group,(Bruker
Faellanden,
Switzerland)
using the solvent
signals (CDCl
: δHsolvent
7.26/δC 77.0)
as (CDCl
internal
3:
δstandard.
7.26/δ
77.0)
as
internal
standard.
The
analytical
HPLC
was
performed
on
a
Dionex
HPLC
system
The
analytical
HPLC
was
performed
on
a
Dionex
HPLC
system
equipped
with
an
Ultimate
H
C
equipped
with
Ultimate
3000
pump,
an Ultimate
diode
detector
anCompartment,
Ultimate 3000
3000 pump,
anan
Ultimate
3000
diode
array
detector 3000
(DAD),
an array
Ultimate
3000(DAD),
Column
Column
Compartment,
an Ultimate
3000Fisher
autosampler
(Thermo
Fishera Scientific,
Inc.)
using(4.6
a Gemini
an Ultimate
3000 autosampler
(Thermo
Scientific,
Inc.) using
Gemini C18
column
mm ×
C
column
(4.6
mm
×
250
mm,
5
µm)
(Phenomenex
Inc.,
Los
Angeles,
CA,
USA).
The
semi-preparative
250
mm,
5
μm)
(Phenomenex
Inc.,
Los
Angeles,
CA,
USA).
The
semi-preparative
HPLC
was
performed
18
HPLC
was performed
on aLiquid
Shimadzu
LC-6-AD Liquid
with an
SPD-20A
Detector
on a Shimadzu
LC-6-AD
Chromatography
withChromatography
an SPD-20A Detector
using
a Phenomenex
using
aC
Phenomenex
Gemini C18 Inc.)
column
Inc.)
(10.0
mm
× 250
mm,was
5 µm).
The chiral
Gemini
18 column (Phenomenex
(10.0(Phenomenex
mm × 250 mm,
5 μm).
The
chiral
HPLC
performed
on
HPLC
was
performed
on
a
Shimadzu
LC-6-AD
Liquid
Chromatography
with
an
SPD-20A
Detector
a Shimadzu LC-6-AD Liquid Chromatography with an SPD-20A Detector using a Phenomenex Lux
using
a Phenomenex
Lux Cellulose-2
chiral
column
Cellulose-2
chiral column
(4.6 mm × 250
mm,
3 μm).(4.6 mm × 250 mm, 3 µm).
Molecules 2016, 21, 1184
9 of 12
3.3. Fungus Material
The strain AHK07-3 was isolated from the soil, collected at the wetland of Ahongkou, Sinkiang
Province, China. The strain was identified as Talaromyces flavus based on the morphological characters
and gene sequence analysis. The ribosomal internal transcribed spacer (ITS) and the 5.8S rRNA gene
sequences (ITS1-5.8S-ITS2) of the strain have been deposited at GenBank as KX011167. The fungus
was cultured on slants of potato dextrose agar at 25 ◦ C for 5 days. Agar plugs were used to inoculate
four Erlenmeyer flasks (250 mL), each containing 100 mL of potato dextrose broth. Four flasks of the
inoculated media were incubated at 25 ◦ C on a rotary shaker at 200 rpm for 5 days to prepare the seed
culture. Fermentation was carried out in 20 Erlenmeyer flasks (500 mL), each containing 70 g of rice.
Distilled H2 O (105 mL) was added to each flask, and the rice was soaked overnight before autoclaving
at 120 ◦ C for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the
seed culture containing mycelia and incubated at room temperature for 50 days.
3.4. Extraction and Isolation
The culture was extracted with EtOAc (3 × 5000 mL). Removal of EtOAc under reduced
pressure yielded a crude extract (19.7 g) that was dissolved in 90% v/v aqueous MeOH (400 mL).
The methanolic solution was further extracted with cyclohexane (400 mL) to afford a cyclohexane
fraction C (4.6 g). Fraction C was subjected to silica gel (200–300 mesh) column chromatography
initially eluting with cyclohexane (500 mL) and then with MeOH (500 mL) to afford subfractions CC
and CM. Subfraction CC (3.3 g) was further separated by open silica gel column chromatography
eluting with cyclohexane–EtOAc (100:0 (150 mL), 99:1 (150 mL), 98:2 (150 mL), 97:3 (150 mL), 96:4
(150 mL), 95:5 (150 mL), 94:6 (150 mL), 93:7 (150 mL), 92:8 (150 mL), 91:9 (150 mL), 90:10 (150 mL),
50:50 (150 mL), and 0:100 (150 mL), v/v) to yield four sub-subfractions (CC1 to CC4). Sub-subfraction
CC2 (2.8 g) was subjected to open silica gel column chromatography eluting with PE (petroleum
ether)–EtOAc (100:0 (100 mL), 99:1 (100 mL), 98:2 (100 mL), 97:3 (100 mL), 96:4 (100 mL), 95:5 (100 mL),
94:6 (100 mL), 93:7 (100 mL), 92:8 (100 mL), 91:9 (100 mL), 90:10 (100 mL), 50:50 (100 mL) and 0:100
(100 mL), v/v) to yield four sub-subfractions (CC2a to CC2d). Sub-subfraction CC2b (1.1 g) was isolated
by semi-preparative HPLC purification using MeOH–H2 O (88:12, v/v) at a flow rate of 3 mL/min
to yield 1 (617.4 mg), 2 (2.6 mg), and 3 (24.7 mg). Sub-subfraction CC2c (0.6 g) was purified by
semi-preparative HPLC purification using MeOH–H2 O (90:10, v/v) at a flow rate of 3 mL/min to
yield 6 (1.1 mg). Sub-subfraction CC4 (220.1 mg) was isolated by semi-preparative HPLC purification
using MeOH–H2 O (80:20, v/v) at a flow rate of 3 mL/min to yield the mixtures of 4 and 5 (12.5 mg).
Enantioseparation of 1–3 was carried out on a Phenomenex Lux Cellulose-2 chiral column using
CH3 OH/H2 O (88/12, v/v) as mobile phase.
3.5. Spectroscopic Data of Isolated Compounds
(±)-Arugosin K (1): yellow solid; UV (MeOH) λmax (log ε) 205 (3.75), 221 (3.63), 304 (3.35), 359 (3.29) nm;
IR (KBr) νmax 3438, 2920, 1708, 1619, 1590, 1421, 1206, 1052 cm−1 ; ESI-MS (positive): m/z 355
[M + H]+ , ESI-MS (negative): m/z 353 [M − H]− ; HRESIMS (positive) m/z 355.1550 [M + H]+ (calcd.
for C21 H23 O5 , 355.1545); 1 H- and 13 C-NMR data see Table 1.
−5 M, MeOH) λ
(+)-S-Arugosin K (1a): [α]27
max (∆ε) 208 (+12.50),
D +27.8 (c 0.5, CHCl3 ); ECD (c 8.5 × 10
294 (−5.71), 334 (+3.76), 382 (−1.77) nm.
−5 M, MeOH) λ
(−)-R-Arugosin K (1b): [α]27
max (∆ε) 208 (−11.38),
D −27.0 (c 0.5, CHCl3 ); ECD (c 8.5 × 10
295 (+6.96), 334 (−3.88), 382 (+2.82) nm.
(±)-Arugosin L (2): yellow solid; UV (MeOH) λmax (log ε) 206 (3.70), 222 (3.57), 304 (3.30), 359 (3.23) nm;
IR (KBr) νmax 3440, 2916, 1624, 1590, 1422, 1206, 1057 cm−1 ; ESI-MS (positive): m/z 369 [M + H]+ ;
ESI-MS (negative): m/z 367 [M − H]− ; HRESIMS (positive) m/z 369.1695 [M + H] + (calcd. for
C22 H25 O5 , 369.1702); 1 H- and 13 C-NMR data see Table 1.
Molecules 2016, 21, 1184
10 of 12
−5 M, MeOH) λ
(+)-S-Arugosin L (2a): [α]27
max (∆ε) 208 (+13.90),
D +27.4 (c 0.5, CHCl3 ); ECD (c 6.8 × 10
295 (−6.07), 335 (+5.07), 382 (−1.91) nm.
−5 M, MeOH) λ
(−)-R-Arugosin L (2b): [α]27
max (∆ε) 208 (−13.79),
D −26.8 (c 0.5, CHCl3 ); ECD (c 6.8 × 10
295 (+7.98), 335 (−3.44), 382 (+3.40) nm.
(±)-Arugosin M (3): yellow solid; UV (MeOH) λmax (log ε) 206 (3.85), 221 (3.73), 299 (3.42), 357 (3.36) nm;
IR (KBr) νmax 3441, 2913, 1620, 1464, 1202, 1050 cm−1 ; ESI-MS (positive): m/z 355 [M + H]+ , ESI-MS
(negative): m/z 353 [M − H]− ; HRESIMS (positive) m/z 355.1541 [M + H]+ (calcd. for C21 H23 O5 ,
355.1545); 1 H- and 13 C-NMR data see Table 1.
−5 M, MeOH) λ
(+)-S-Arugosin M (3a): [α]27
max (∆ε) 209 (+14.24),
D +28.4 (c 0.5, CHCl3 ); ECD (c 8.5 × 10
290 (−6.29), 334 (+6.67), 381 (−3.30) nm.
−5 M, MeOH) λ
(−)-R-Arugosin M (3b): [α]27
max (∆ε) 209 (−12.84),
D −27.6 (c 0.5, CHCl3 ); ECD (c 8.5 × 10
292 (+7.26), 334 (−5.93), 381 (+4.27) nm.
Arugosin N (4): 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one (5):
yellow solid; UV (MeOH) λmax (log ε) 206 (3.75), 222 (3.65), 286 (3.29), 356 (3.16) nm; IR (KBr) νmax
3443, 2917, 1628, 1589, 1420, 1353, 1173, 1052 cm−1 ; ESI-MS (positive): m/z 341 [M + H]+ , m/z 363
[M + Na]+ ; ESI-MS (negative): m/z 339 [M − H]− ; HRESIMS (positive) m/z 363.1213 [M + Na]+ (calcd.
for C20 H20 O5 Na, 363.1208); 1 H- and 13 C-NMR data see Table 2.
3.6. ECD Calculation
Before molecular initial 3D structures were generated with CORINA version 3.4 (Molecular
Networks GmbH, Erlangen, Germany), the molecules of (5S)-1, (5R)-1, (5S)-3, and (5R)-3 were
converted into SMILES codes, respectively. Conformer databases were generated in CONFLEX
version 7.0 (CONFLEX Corporation, Tokyo, Japan) using the MMFF94s force-field, with an energy
window for acceptable conformers (ewindow) of 5 kcal·mol−1 above the ground state, a maximum
number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5 Å.
Then each conformer of the acceptable conformers was optimized with HF/6-31G(d) method in
Gaussian09 [19]. Further optimization at the APFD/6-31G(d) level led the dihedral angles to be got.
After that, eight lowest energy conformers of (5S)-1 and (5R)-1 (see Supplementary Materials Table S6
and Figure S3) were found out. Six lowest energy conformers of (5S)-3 and (5R)-3 (see Supplementary
Materials Table S7 and Figure S4) were found out. The optimized conformers were taken for the ECD
calculations, which were performed with Gaussian09 (APFD/6-311++G(2d,p)). The solvent effect was
taken into account by the polarizable-conductor calculation model (IEFPCM, methanol as the solvent).
Comparisons of the experimental and calculated spectra were done with the software SpecDis [20,21].
It was also used to apply a UV shift to the ECD spectra, Gaussian broadening of the excitations, and
Boltzmann weighting of the spectra.
4. Conclusions
Up to now, about nine isopentenyl dibenzo[b,e]oxepinones with intact 6-7-6 tricyclic
systems have been reported from the genera of Aspergillus, Emericella, Pestalotiopsis, Graphiopsis,
and Penicilium. Except the absolute configuration of cephalanone D that had been verified by
X-ray crystallography [6], the absolute configurations of the others had not been not investigated.
In our study, the absolute configurations of the three pairs of enantiomers ((+)-(5S)-1a, (−)-(5R)-1b,
(+)-(5S)-2a, (−)-(5R)-2b, (+)-(5S)-3a, (−)-(5R)-3b), isolated for the first time from the genus Talaromyces,
were determined by quantum chemical ECD calculations, which is the first report for the
absolute configuration of isopentenyl dibenzo[b,e]oxepinones established by ECD experiments.
Furthermore, 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one (5) is
a known compound, but no NMR data was previously reported. The assignments of NMR data of 5
are thus provided for the first time.
Molecules 2016, 21, 1184
11 of 12
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/21/9/1184/
s1, The 1D and 2D NMR data of 1–5, and quantum chemical ECD calculations of 1 and 3.
Acknowledgments: This work was financially supported by grants from the National Natural Science Foundation
of China (81422054 and 81373306), the Guangdong Natural Science Funds for Distinguished Young Scholar
(S2013050014287), Guangdong Special Support Program (2014TQ01R420), Guangdong Province Universities
and Colleges Pearl River Scholar Funded Scheme (Hao Gao, 2014), Pearl River Nova Program of Guangzhou
(201610010021), K.C.Wong Education Foundation (Hao Gao, 2016), and the high-performance computing platform
of Jinan University.
Author Contributions: Hao Gao and Xin-Sheng Yao initiated and coordinated the project. Hao Gao and
Guo-Dong Chen wrote this paper. Tian-Yu Sun and Run-Qiao Kuang performed the extraction, isolation,
and structural identification of the compounds. Sheng-Ying Qin and Chuan-Xi Wang performed the fermentation
of the fungal strain (AHK07-3). Xing-Zhong Liu, Bin Wu, and Dan Hu performed the isolation and identification
of the fungal strain (AHK07-3).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Ballantine, J.; Francis, D.; Hassall, C.; Wright, J. The biosynthesis of phenols. Part XXI. The molecular
structure of arugosin, a metabolite of a wild-type strain of Aspergillus rugulosus. J. Chem. Soc. C 1970, 9,
1175–1182. [CrossRef]
Ballantine, J.; Ferrito, V.; Hassall, C.; Jenkins, M. Biosynthesis of phenols. Part XXIV. Arugosin C, a metabolite
of a mutant strain of Aspergillus rugulosus. J. Chem. Soc. Perkin Trans. 1 1973, 17, 1825–1830. [CrossRef]
[PubMed]
Chexal, K.; Holker, J.; Simpson, T. Biosynthesis of fungal metabolites. Part VI. Structures and biosynthesis of
minor metabolites from variant strains of Aspergillus variecolor. J. Chem. Soc. Perkin Trans. 1 1975, 6, 549–554.
[CrossRef] [PubMed]
Kralj, A.; Kehraus, S.; Krick, A.; Eguereva, E.; Kelter, G.; Maurer, M.; Wortmann, A.; Fiebig, H.; Koenig, G.
Arugosins G and H: Prenylated polyketides from the narine-derived fungus Emericella nidulans var. acristata.
J. Nat. Prod. 2006, 69, 995–1000. [CrossRef] [PubMed]
Li, E.; Jiang, L.; Guo, L.; Zhang, H.; Che, Y. Pestalachlorides A–C, antifungal metabolites from the plant
endophytic fungus Pestalotiopsis adusta. Bioorg. Med. Chem. 2008, 16, 7894–7899. [CrossRef] [PubMed]
Asai, T.; Otsuki, S.; Sakurai, H.; Yamashita, K.; Ozeki, T.; Oshima, Y. Benzophenones from an Endophytic
Fungus, Graphiopsis chlorocephala, from Paeonia lactiflora Cultivated in the Presence of an NAD+ -Dependent
HDAC Inhibitor. Org. Lett. 2013, 15, 2058–2061. [CrossRef] [PubMed]
Mizogami, K.; Kyo, A.; Nakaike, S.; Ikeda, T.; Hanada, K. Preparation of Dihydrodibenzoxepinone Derivative
as Antitumor Agent. JP 06271561A, 27 September 1994.
Proksa, B. Talaromyces flavus and its metabolites. Chem. Pap. 2010, 64, 696–714. [CrossRef]
Li, H.; Huang, H.; Shao, C.; Huang, H.; Jiang, J.; Zhu, X.; Liu, Y.; Liu, L.; Lu, Y.; Li, M.; et al. Cytotoxic
norsesquiterpene peroxides from the endophytic fungus Talaromyces flavus isolated from the mangrove plant
Sonneratia apetala. J. Nat. Prod. 2011, 74, 1230–1235. [CrossRef] [PubMed]
Bao, Y.; Chen, G.; Wu, Y.; Li, X.; Hu, D.; Liu, X.; Li, Y.; Yao, X.; Gao, H. Stachybisbins A and B, the first cases
of seco-bisabosquals from Stachybotrys bisbyi. Fitoterapia 2015, 105, 151–155. [CrossRef] [PubMed]
Bao, Y.; Chen, G.; Gao, H.; He, R.; Wu, Y.; Li, X.; Hu, D.; Wang, C.; Liu, X.; Li, Y.; et al. 4,5-seco-Probotryenols
A–C, a new type of sesquiterpenoids from Stachybotrys bisbyi. RSC Adv. 2015, 5, 46252–46259. [CrossRef]
He, J.; Mu, Z.; Gao, H.; Chen, G.; Zhao, Q.; Hu, D.; Sun, J.; Li, X.; Li, Y.; Liu, X.; et al. New polyesters from
Talaromyces flavus. Tetrahedron 2014, 70, 4425–4430. [CrossRef]
He, J.; Qin, D.; Gao, H.; Kuang, R.; Yu, Y.; Liu, X.; Yao, X. Two new coumarins from Talaromyces flavus.
Molecules 2014, 19, 20880–20887. [CrossRef] [PubMed]
He, J.; Liang, H.; Gao, H.; Kuang, R.; Chen, G.; Hu, D.; Wang, C.; Liu, X.; Li, Y.; Yao, X. Talaflavuterpenoid A,
a new nardosinane-type sesquiterpene from Talaromyces flavus. J. Asian Nat. Prod. Res. 2014, 16, 1029–1034.
[CrossRef] [PubMed]
Sun, T.; Zou, J.; Chen, G.; Hu, D.; Wu, B.; Liu, X.; Yao, X.; Gao, H. A Set of Interesting Sequoiatone
Stereoisomers from a Wetland Soil-Derived Fungus Talaromyces flavus. Acta Pharm. Sin. B 2016. [CrossRef]
Danielsen, K.; Aksnes, D. NMR study of some anthraquinones from rhubarb. Magn. Reson. Chem. 1992, 30,
359–360. [CrossRef]
Molecules 2016, 21, 1184
17.
18.
19.
20.
21.
12 of 12
Pockrandt, D.; Ludwig, L.; Fan, A.; Koenig, G.; Li, S. New Insights into the Biosynthesis of Prenylated
Xanthones: Xptb from Aspergillus nidulans Catalyses an O-Prenylation of Xanthones. ChemBioChem 2012, 13,
2764–2771. [CrossRef] [PubMed]
Simpson, T. Genetic and biosynthetic studies of the fungal prenylated xanthone shamixanthone and related
metabolites in Aspergillus spp. revisited. ChemBioChem 2012, 13, 1680–1688. [CrossRef] [PubMed]
Frisch, M.; Trucks, G.; Schlegel, H.; Scuseria, G.; Robb, M.; Cheeseman, J. Gaussian 09 Revision D.01;
Gaussian Inc.: Wallingford, CT, USA, 2010.
Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. Version 1.61; University of Würzburg: Würzburg,
Germany, 2013.
Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDics: Quantifying the comparison of
calculated and experimental electronic circular dichroism spectra. Chirality 2013, 25, 243–249. [CrossRef]
[PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).