Structural determination of two new sesquiterpenes biotransformed from germacrone by Mucor alternata

Article abstract of DOI:10.1002/mrc.2148

Eight transformed sesquiterpenes of germacrone by Mucor alternata were obtained. Their structures were characterized on the basis of spectral methods including 2D NMR. Among them, (1S, 4S, 55, 10R)-isozedoarondiol (2) and (1R, 4S, 55, 10R)-diepoxy-12-hydroxygermacrone (3) are new compounds. Copyright

Full text of DOI:10.1002/mrc.2148

Spectral Assignments and Reference Data
Received: 19 April 2007
Revised: 5 October 2007
Accepted: 18 October 2007
Published online in Wiley Interscience: 20 December 2007
(www.interscience.com) DOI 10.1002/mrc.2148
Structural determination of two new
sesquiterpenes biotransformed from
germacrone by Mucor alternata
Niu Jia,a MA Xiao Chia,b∗ Xin Xiu Lan,c Jin Hong Weia and Guo Deana∗
Eight transformed sesquiterpenes of germacrone by Mucor alternata were obtained. Their structures were characterized
on the basis of spectral methods including 2D NMR. Among them, (1S, 4S, 5S, 10R)-isozedoarondiol (2) and (1R, 4S, 5S,
c
10R)-diepoxy-12-hydroxygermacrone (3) are new compounds. Copyright ꢀ 2007 John Wiley & Sons, Ltd.
Keywords: NMR; biotransformation; germacrone; sesquiterpene
Introduction
correlated with the carbon signals of C-1 (δ 51.2), C-9 (δ 50.5) and
C-10 (δ 71.8). These data indicated that compound (2) had the
structure of guaiane, rather than eudesmane skeleton. In addition,
the carbon of C-10 correlated with the protons of δ 1.04 (Me-
15), δ 2.65 (H-1), δ 3.22 (Ha-9) and δ 4.27 (HO-10), suggesting
that compound (2) possessed 10-hydroxyl group. Similarly, the
HMBC correlation of C-4 (δ 80.4) with H-5 (δ 1.85), Ha-3 (δ 1.66),
Me-14 (δ 1.25), Ha-6 (δ 2.30) and HO-4 (δ 4.16) indicated that an
additional hydroxyl group was at C-4. In nuclear overhauser effect
spectroscopy (NOESY) spectrum, the signal at δ 2.65 (H-1) had NOE
enhancements with the signals at δ 1.04 (Me-15) and δ 1.85 (H-5).
Meanwhile, NOE contact between HO-4 (δ 4.16) and H-5(δ 1.85)
was observed. And NOE effects of Me-14 (δ 1.25) with H-1 (δ 2.65)
and H-5 (δ 1.85) were not observed. In addition, the CD spectrum
of (2) showed a positive R-band cotton effect (325 nm, methanol),
and a positive K-band cotton effect at 250 nm base on the n-π∗
transition of α, β unsaturated ketone. To our knowledge, the
B3LYP method of GAUSSIAN-03 package program with 6–31G∗ (d,
p)basissetfunction(BSF)ofthedensityfunctionaltheory(DFT)can
be a useful tool in providing sufficiently accurate conformational
analysis.[11,12] Sothegeometryof(2)wasoptimizedbyusingB3LYP
method and 6–31G∗ BSF.[13,14] The calculated harmonic frequency
indicated that its molecule was located at the minimal and stable
points. And then according to the fully optimized geometry, CD
spectrum and NOESY experiment, the preferred conformation of
(2) was as shown in Fig. 2. On the basis of the above analysis,
compound (2) was identified as (1S, 4S, 5S, 10R)-isozedoarondiol.
All 1H and 13C NMR spectral data were unambiguously assigned
by 2D NMR spectra (Table 1).
Curcuma aromatica (Family Zingiberaceae) is an important medic-
inal plant and its rhizome has been used as a traditional Chinese
medicine with the properties of anticancer and bile secretion
promotion. Germacrone (1), is one of the major constituents
in this plant, and also the most important and basic sesquiter-
pene for biosynthesis. Recently, germacrone was reported to
exhibit hepatoprotective activity.[1] In our pervious work,[2–6] the
biotransformation of some sesquiterpenes such as curdione, de-
hydrocostuslactone, and costunolide were investigated to obtain
some new chemical entities. In an endeavor to improve activities
of germacrone, the microbial transformation of (1) was carried
out. Incubation of (1) with M. alternata for 6 days yielded two new
compounds, (1S, 4S, 5S, 10R)-isozedoarondiol (2) and (1R, 4S, 5S,
10R)-diepoxy-12-hydroxygermacrone (3), together with 6 known
compounds.
Results and Discussion
Incubation of germacrone (1) with M. alternata for 6 days
yielded eight products (Fig. 1). Their structures were identi-
fied as (1S, 4S, 5S, 10R)-isozedoarondiol (2) and (1R, 4S, 5S,
10R)-diepoxy-12-hydroxygermacrone (3), curcumenone (4), ze-
doaronediol (5), 1β, 10α, 4α, 5β-diepoxygerma-crone (6), 1α,
4β-dihydroxyeudesman-8-one (7), 1α, 4α-dihydroxyeudesman-8-
one (8), and 3-hydroxygermacrone (9), respectively. Among them,
(2) and (3) are new compounds.
All 1H and 13C NMR spectral data of known compounds
(4–9) agreed with spectral data reported in the literature.[7–10]
The spectral data of (2) and (3) were compiled in the
Table 1.
∗
Correspondence to: MA Xiao Chi or Guo Dean, School of Pharmaceutical
Compound (2) afforded as colorless oil, [α]2D2 +86.3◦ (c 0.3,
MeOH). Its HR-FABMS showed a quasi-molecular ion [M + H]+ at
m/z 253.1801 (calcd 253.1798), suggesting the molecular formula
as C15H24O3. Compared to that of compound (1), the 1H NMR
spectrum of (2) showed the disappearance of two olefin protons.
The 13C NMR and HMQC spectrum of (2) exhibited two oxygen-
bearing quaternary carbons (δ 71.8 and 80.4), disappearing four
olefin carbons. In HMBC, the proton signals of δ 4.27 (HO-10)
Sciences, Peking University, Beijing 100083, P.R. China.
E-mail: maxc1978@sohu.com; gda@bjmu.edu.cn
a
The State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing 100083, P. R. China
b
c
College of Pharmacy; Dalian Medical University; Dalian 116027; P. R. China
Biotechnology Application Center, Beijing Vocational College of Electronic
Science and Technology, Beijing, P. R. China
c
Magn. Reson. Chem. 2008; 46: 178–181
Copyright ꢀ 2007 John Wiley & Sons, Ltd.

Structural determination of two new sesquiterpenes
15
9
HO
14
O
15
H
1
3
O
10
10
4
2
6
10
1
2
4
1
5
2
3
8
7
8
3
O
9
15
O
5
5
8
7
7
6
4
H
OH
HO
12
O
13
14
11
12
11
12
13
14
13
3
1
2
15
HO
O
O
2
H
10
6
4
9
1
3
14
8
O
O
O
5
7
H
O
H
HO
12
11
13
4
5
6
OH
OH
15
O
O
O
1
9
8
7
10
2
3
5
4
HO
6
12
11
13
H
H
HO
HO
14
7
8
9
Figure 1. Chemical structures of compounds 1–9.
Figure 2. Selected NOE enhancements of compounds 2 and 3.
Compound (3) afforded as colorless oil, [α]2D2 −36.5◦ (c 0.4,
MeOH). Its HR-FABMS showed a quasi-molecular ion [M + H]+ at
m/z 267.1591 (calcd 267.1590), suggesting the molecular formula
asC15H22O4.Comparedwiththatof(1),the1HNMRspectrumof(3)
exhibited three methyl signals at δ 1.73 (s), δ 1.32 (s), and δ 1.01 (s),
and two olefin protons were disappeared. The 13C NMR spectrum
of (3) also showed the disappearance of four olefin carbons and
presence of four oxygen-bearing carbons (δ 57.6, 59.5, 60.5 and
62.6). The carbon signal of C-12 being at δ = 22.0 ppm in (1)
appeared at δ = 61.2 ppm in (3). In heteronuclear single quantum
coherence HSQC spectrum, two protons at δ 4.04 ppm correlated
with an additional oxygen-bearing carbon (δ 61.2), suggesting a
CH2OH group was introduced in the molecule. In HMBC spectrum,
the proton signals (δ 4.04) correlated with carbon signals at δ 140.4
(C-11) and δ 18.2 (C-13), suggesting that a hydroxyl group should
be at C-12. The proton signals of δ 1.01 (Me-14) correlated with
the carbon signals of C-4 (δ 59.5), C-3 (δ 35.0) and C-5 (δ 62.6). The
HMBC correlation of Me-15 with C-1, C-9, and C-10 were observed.
These data indicated that compound (3) was the C-1, 10, and C-4, 5
diepoxide of compound (1). In the NOESY spectrum, NOE contacts
of Me-14 (δ 1.01) with H-1 (δ 2.93) and H-5 (δ 2.75) were observed.
And NOE effects between Me-15 (δ 1.32) and H-1 (δ 2.93) were
c
Magn. Reson. Chem. 2008; 46: 178–181
Copyright ꢀ 2007 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc

N. Jia et al.
Table 1. 1H (500 MHz) and 13C (125 MHz) NMR data of (2) and (3) in DMSO-d6 (δ in ppm, J in Hz)
2
3
No.
1H
13C
HMBC(H → C)
1H
13C
HMBC(H → C)
1
2
2.65 m
1.60 m
1.60 m
1.66 m
1.48 m
4.16 s (OH)
1.85 m
2.30 m
1.74 m
–
51.2
24.7
C-5, C-10
2.93 m
1.88 m
1.39 m
2.03 m
1.20 m
–
60.5
22.3
C-2, C-3, C-9
C-1, C-3, C-4
C-1, C-3, C-10
3
36.5
C-2, C-4, C-5,
35.0
C-2, C-4, C-5, C-14
4
5
6
80.4
52.4
26.8
C-3, C-4, C-5
C-1, C-3, C-4, C-6, C-14
C-1, C-4, C-7, C-8
59.5
62.6
28.0
–
2.75 dd (11.0, 2.5)
2.83 m
2.02 m
–
C-4, C-6
C-4, C-5, C-7, C-8, C-11
7
8
9
134.4
202.7
50.5
–
134.0
207.1
53.8
–
–
–
–
–
3.22 d (16.0)
2.09 d (16.0)
4.27 s (OH)
–
C-1, C-7, C-8, C-10, C-15
3.02 d (11.0)
2.47 d (11.0)
–
C-1, C-8, C-10, C-15
10
11
12
71.8
139.8
22.4
C-1, C-9, C-10, C-15
57.6
140.4
61.2
–
–
–
–
–
1.87 s
C-7, C-11, C-13
4.04 m
4.91 t (6.0, OH)
1.73 s
C-7, C-11, C-13
13
14
1.77 s
1.25 s
21.6
24.8
C-7, C-8, C-11, C-12
C-3, C-4, C-5
18.2
15.2
C-7, C-8, C-11, C-12
C-3, C-4, C-5
1.01 s
4.16 s (OH)
1.04 s
15
32.4
C-1, C-9, C-10
1.32 s
17.1
C-1, C-9, C-10
not observed. The CD spectrum of (3) showed a positive cotton
effect (345 nm) and negative cotton effect (245 nm), base on the
n-π∗ transitionof α, β unsaturatedketone. Similarly, thegeometric
optimizationwasthencarriedoutusingthe6–31G∗ BSFandB3LYP
method included in the Gaussian 03 package program. Harmonic
frequency calculation of (3) verified the nature of minimal points.
This proves, in combination with the CD and NOE results, that
the preferred conformation of (3) was as shown in Fig. 2. On the
basis of the above analysis, compound (3) was identified as (1R, 4S,
5S, 10R)-diepoxy-12-hydroxygermacrone (3). All 1H and 13C NMR
spectral data were unambiguously assigned by 2D NMR spectra
(Table 1).
[θ]345 = +593, [θ]245 = −783; IR (KBr) νmax (cm−1) = 2972, 1055,
1012; HR FAB MS m/z = 267.1591 [M + H]+, calcd for C15H22O4:
267.1590. 1H NMR and 13C NMR data see Table 1.
Culture medium
All culture and biotransformation experiments using filamentous
fungi were performed in the potato medium which was made of
the following composition (L): 200 g potato and 20 g glucose.
NMR spectra
All NMR experiments were performed on a Varian INOVA-
500 NMR spectrometer. 1H and 13C NMR spectra (at 500 and
125 MHz, respectively) were measured at room temperature
(22 ◦C). Chemical shifts are given on the δ scale and are referenced
to tetramethylsilane (TMS) at δ = 0 ppm for 1H and 13C. Coupling
constant(J)aregiveninHz. Thepulseconditionswereasfollowing:
for1H,theobservationfrequencywas499.89 MHz,acquisitiontime
(AQ) 1.882 s, relaxation delay (RD) 1.0 s, pulse 90◦, spectral width
(SW) 4108.0 Hz, and the FT size 16 386 data points; for 13C, the
observation frequency was 125.70 MHz, AQ = 0.700 s, RD = 1.0 s,
pulse 56◦, SW = 30 166.9 Hz, line broadening 3.0 Hz, and FT size
32 716 data points. COSY: RD = 1.0 s, SW = 3701.0 Hz, number
of points (NP) 2048, number of increments (NI) 200. NOESY:
RD = 1.0 s, SW = 3263.7 Hz, NP = 2048, NI = 220, mixing
time 0.5 s. HSQC: AQ = 0.277 s, RD = 1.0 s, SW = 3701.1 (1H) and
20 110.6 Hz(13C), NP = 2048, FTsize1024×2048datapoints. One-
bond 13C, 1H chemical shift correlations were obtained in HMQC
experiment for 1J (C, H) = 140.0 Hz; for the HMBC spectrum,
the parameters were very similar to those used in the HSQC
Experimental
General
Melting points were determined on an XT4A apparatus and are
uncorrected. UV spectra were measured on a YV-1091 UV-Vis
spectrophotometer. IR spectra were obtained on an Avatar 360
FT-TR spectrophotometer. Optical rotations were obtained with
a Perkin-Elmer 243B polarimeter. EI MS data were measured
by MS-QP5050A mass spectrometer. Silica gel (200–300 mesh)
was purchased from Qingdao Marine Chemical Group, China.
Germacrone (1) was isolated from C. aromatica. The purity was
above 98% determined by HPLC. Its structure was characterized
by 1H NMR, 13C NMR, and EI-MS.
(1S, 4S, 5S, 10R)-isozedoarondiol (2): colorless oil (Et2O);
[α]2D2 +86.3◦ (c 0.3, MeOH); CD (c = 0.005, MeOH) [θ]325 = +501,
[θ]250 = +1510; IR (KBr) νmax (cm−1) = 3360, 2922, 1666, 1370,
1161; HR FAB MS m/z = 253.1801 [M + H]+, calcd for C15H24O3:
253.1798. 1H NMR and 13C NMR data see Table 1.
1
(1R, 4S, 5S, 10R)-diepoxy-12-hydroxygermacrone (3): colorless
experiments, and long-range 13C, H chemical shift correlations
oil (Et2O); [α]22 −36.5◦ (c 0.4, MeOH); CD (c = 0.006, MeOH):
were obtained for 2J (C, H) = 8.0 Hz.
D
c
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Copyright ꢀ 2007 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2008; 46: 178–181

Structural determination of two new sesquiterpenes
Computational method
References
The geometry optimization and the frequency calculations were
performed by the B3LYP method included in the GAUSSIAN-03
package program together with 6–31G∗ (d, p) BSF of the DFT. The
minimum natures of all structures were confirmed by frequency
calculations.
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Acknowledgments
We thank the Youth Foundation by National Natural Science
Foundation of China (30701088) and PHR (IHLB) for financial
support.
c
Magn. Reson. Chem. 2008; 46: 178–181
Copyright ꢀ 2007 John Wiley & Sons, Ltd.
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