MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2007; 45: 434–438
Published online 19 March 2007 in Wiley InterScience
([Link]) DOI: 10.1002/mrc.1974
Spectral Assignments and Reference Data
1H and 13 C NMR assignments for two peak in the HR-EIMS at m/z 388.1144 (calcd 388.1153) in conjunction
with the NMR data allowed a molecular formula of C20 H20 O8
anthraquinones and two xanthones from the (eleven degrees of unsaturation) to be assigned to compound 1.
mangrove fungus (ZSUH-36) The 13 C NMR and DEPT data showed that compound 1 had one
methylene carbon, one methine carbon, two hydroxyl methylene
carbons together with two methoxyl groups, two carbonyl carbons
Changlun Shao,1 Zhigang She,1 Zhiyong Guo,1 and twelve olefinic carbons. The 1 H NMR spectrum displayed two
Hong Peng,1 Xiaoling Cai,1 Shining Zhou,2 Yucheng Gu3 meta-coupled aromatic proton signals at υH 6.97 and 7.24, one singlet
due to an aromatic proton at υH 7.13 and indicated two methoxyl
and Yongcheng Lin1∗
groups (υH 4.00, 4.00), one hydroxyl methylene group (υH 3.72, 3.78)
1 and one methylene group (υH 1.97). Another hydroxyl methylene
School of Chemistry and Chemical Engineering, Sun Yat-Sen (Zhong-
group and one methine group were covered up by the broad water
shan) University, Guangzhou, 510275, PR China
2
peak. Thus, variations in the 1 H NMR spectrum of 6,8-di-O-methyl
College of Life Science, Sun Yat-Sen (Zhongshan) University, versiconol (1) were observed in a temperature range of 25–75 ° C.
Guangzhou, 510275, PR China With the temperature raised, the water peak signal shifted upfield,
3
Jealott’s Hill International Research Centre, Syngenta, Bracknell, while the chemical shifts of the proton signals of compound 1 (Fig. 1)
Berkshire, RG42 6EY, UK did not change much. All the covered proton signals emerged
when the temperature was raised to 75 ° C (Fig. 6). 1 H NMR and
Received 1 December 2006; revised 8 January 2007; accepted 10 January 2007 DEPT data and the molecular formula indicated that there must
be four exchangeable protons, requiring the presence of three free
We report the unambiguous assignments of the 1 H hydroxyl groups and one chelated hydroxy. The 1 H and 13 C NMR
and 13 C NMR spectra of one new natural product, (Table 1) spectra indicated that compound 1 was similar to versiconol
namely, 6,8-di-O-methyl versiconol (1) together with one except that two methoxyl groups on compound 1 were replaced by
known anthraquinone aversin (2) and two xanthones two hydroxyl groups. Detailed assignments for carbon and proton
5-methoxysterigmatocystin (3) and sterigmatocystin (4). were unambiguously accomplished by analysis of 2D NMR spectral
These compounds were all isolated from the mangrove data (Table 1). The structure of the four-carbon chain moiety was
endophytic fungus ZSUH-36 from the South China confirmed by the sequent correlations of H–H COSY from H-10 to H-
Sea. 1D and 2D NMR experiments including COSY, 20 , H-30 and H-40 . The key HMBC correlation was observed between
HMQC and HMBC were used to elucidate the structures. H-10 and C-2 indicating that the four-carbon chain moiety was linked
Variations in the 1 H NMR spectrum of 6,8-di-O-methyl to the C-2 position. The compound was identified as 6,8-di-O-methyl
versiconol (1) were also observed in the temperature versiconol (1) based on these spectral data.
range 25–75 °C. In addition, the plausible biogenetic Compound 2 (Fig. 2) was isolated as a yellow solid. The
path from 1 to 2 is discussed. Copyright 2007 John molecular formula of 2 was established as C20 H16 O7 on the results
Wiley & Sons, Ltd. of LC-MS. and NMR data. The 13 C NMR and DEPT data showed
that compound 2 had one methylene carbon, one methine carbon,
KEYWORDS: NMR; 1 H NMR; 13 C NMR; 2D NMR; anthraquinone; one ether methylene carbon, one acetyl carbon, two methoxyl
xanthone; secondary metabolites; variable temperature; structure groups, two carbonyl carbons and twelve olefinic carbons. The
elucidation 1 H NMR spectrum (Table 2) displayed two meta-coupled aromatic
proton signals at υH 6.97 and 7.24, one singlet due to an aromatic
proton at υH 7.13, two methoxyl group (υH 4.00, 4.00), one hydroxyl
INTRODUCTION methylene group (υH 3.72, 3.78) and one methylene group (υH 1.97).
Detailed assignments for carbon and proton were unambiguously
In recent years, chemists of natural products have become interested
accomplished by analysis of 2D NMR spectral data (Table 2). This
in marine fungi as sources of bioactive compounds because marine
is the first report on aversin, isolated from marine fungus. From
organisms provide interesting and diverse chemical structures.1 So
the biogenetic point of view, compound 2 should be derived from
far, many of these chemists have studied marine fungi as the vast
compound 1 (Fig. 5).
number of unique and previously unrecognized microorganisms
have not been studied before. In this paper, we report one
new natural product 6,8-di-O-methyl versiconol (1) together with
one known anthraquinone aversin (2) and two xanthones 5-
methoxysterigmatocystin (3) and sterigmatocystin (4) from the
mangrove endophytic fungus ZSUH-36 collected from the South
China Sea. The 1 and 2D NMR experimental results, particularly
those of COSY, HMQC and HMBC, were used to determine the
structures of these compounds. These known compounds have
been reported in previous literature.2 – 4 However, for compound
2, only the 1 H NMR spectrum of its derivative O-methylaversin was
reported and the NMR results for compound 3 are incomplete and
have not been updated; only proton results have been published.
Therefore, it is useful for reference purposes to report the 2D NMR
Figure 1. The structure of compound 1.
data of these compounds. Signal shifting in the 1 H NMR spectrum
of 6,8-di-O-methyl versiconol (1) was also observed in a temperature
range of 25–75° . In addition, the plausible biogenetic path from 1 to
2 is discussed.
RESULTS AND DISCUSSION
6,8-di-O-methyl versiconol (1) was obtained as an optically active red
amorphous solid f[˛]D 22 32.2 (c, 0.05, DMSO)g. A molecular ion
Ł
Correspondence to: Yongcheng Lin, School of chemistry and chemical
engineering, Sun Yat-Sen (Zhongshan) University, Guangzhou, PR China.
E-mail: ceslyc@[Link] Figure 2. The structure of compound 2.
Copyright 2007 John Wiley & Sons, Ltd.
435
Spectral Assignments and Reference Data
Table 1. 1 H and 13 C NMR data of 1 (υ in ppm, J in Hz)
C no. 13 C NMR(DEPT) 1H NMR H–H COSY HMBC
1 163.14 – – –
2 123.27 – – –
3 162.04 – – –
4 106.71CH 7.13(s) – C-2, C-3, C-10, C-13
5 104.43CH 7.24(d, 2.4) H-7 C-6, C-7, C-10, C-11, C-12
6 164.57 – – –
7 104.48CH 6.97(d, 2.4) H-5 C-5, C-8, C-12
8 162.87 – – –
9 185.96 – – –
10 181.85 – – –
11 136.47 – – –
12 114.01 – – –
13 109.40 – – –
14 131.05 – – –
15 55.98 OCH3 4.00(s) – C-8
16 56.53 OCH3 4.00(s) – C-6
10 62.79 CH2 3.78(dd, 10.2, 7.2) H-20 C-2
– – 3.72(dd, 10.2, 6.6) – –
20 35.22 CH 3.50(dt, 7.2, 6.9) H-10 , H-30 –
30 32.63 CH2 1.97(dd, 14.4, 7.2) H-20 , H-40 C-40
40 60.02 CH2 3.38(dd, 10.5, 7.2) H-30 C-20
– – 3.29(dd, 10.5, 6.9) – –
1-OH – 14.00(s) – C-1, C-2, C-13
3-OH – 10.87(s) – –
10 -OH, 40 -OH – 4.32(s), 4.76(s) – –
Figure 4. The structure of compound 4.
Figure 3. The structure of compound 3.
Compound 4 (Fig. 4) was isolated as a pale yellow solid. The
Compound 3 (Fig. 3) was also isolated as a yellow solid. The molecular formula of this compound was C18 H12 O6 , which was
molecular formula of 3 was established as C19 H14 O7 based on confirmed by LC-MS, which showed the [M H] ion peak at m/z
NMR data and LC-MS experiments. The 13 C NMR and DEPT data 323. It was identified by comparison of its 1 H NMR and 13 C NMR
showed that compound 3 had one methine carbon, one acetyl carbon, data with the published values. This compound was the second one
two methoxyl groups, one carbonyl carbon and fourteen olefinic to be isolated from marine fungus.
carbons. The 1 H NMR spectrum (Table 3) displayed two ortho-
coupled aromatic proton signals at υH 6.69 and 7.20, one-singlet due
to an aromatic proton at υH 6.45 and indicated two methoxyl groups CONCLUSIONS
(υH 3.93, 4.00). The 1 H and 13 C NMR (Table 3) spectra indicated that
compound 3 is 5-methoxysterigmatocystin. Detailed assignments for The new natural product 6,8-di-O-methyl versiconol (1) together
carbon and proton were unambiguously accomplished by analysis with aversin, 5-methoxysterigmatocystin (3) and sterigmatocystin
of 2D NMR spectral data (Table 3). This is also the first report on (4) was isolated from the mangrove endophytic fungus ZSUH-36
isolation of 5-methoxysterigmatocystin from marine fungus. from the South China Sea. This is the first time that aversin (2)
Copyright 2007 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2007; 45: 434–438
DOI: 10.1002/mrc
436
Spectral Assignments and Reference Data
Table 2. 1 H and 13 C NMR data of 2 (υ in ppm, J in Hz)
C no. 13 C NMR(DEPT) 1H NMR H–H COSY HMBC
1 160.29 – – –
2 120.13 – – –
3 165.11 – – –
4 101.17CH 7.22(s) – C-2, C-3, C-10, C-13, C-14
5 104.15CH 7.45(d, 2.4) H-7 C-6, C-7, C-10, C-11, C-12
6 165.03 – – –
7 104.87CH 6.78(d, 2.4) H-5 C-5, C-6, C-8, C-12
8 162.91 – – –
9 187.15 – – –
10 182.52 – – –
11 137.53 – – –
12 115.02 – – –
13 112.61 – – –
14 134.92 – – –
15 56.65 OCH3 4.02(s) – C-8
16 56.03 OCH3 3.99(s) – C-6
10 112.94 CH2 6.45(d, 5.6) H-20 C-2, C-3, C-20 , C-30 , C-40
20 44.46 CH 4.15(dd, 7.2, 6.9) H-10 , H-30 C-40 , C-2, C-3
30 30.77 CH2 2.23(dd, 14.4, 7.2) H-20 , H-40 C-2, C-10 , C-20 ,C-40
– 2.35(dd, 12.0, 5.2) – – –
40 67.75 CH2 3.66(dd, 10.5, 7.5) H-30 C-10 , C-20 ,
– – 4.15(dd, 10.5, 6.9) – –
Table 3. 1 H and 13 C NMR data of 3 (υ in ppm, J in Hz)
C no. 13 C NMR(DEPT) 1H NMR H–H COSY HMBC
1 163.29 – – –
2 90.61CH 6.45(s) – C-1, C-3, C-4, C-12
3 164.57 – – –
4 106.77 – – –
5 139.39 – – –
6 120.42CH 7.20(d, 9.0) H-7 C-5, C-8, C-10
7 109.50CH 6.69(d, 9.0) H-6 C-5, C-8, C-11
8 155.27 – – –
9 181.28 – – –
10 153.97 – – –
11 109.50 – – –
12 106.06 – – –
13 144.71 – – –
14 57.71 OCH3 3.93(s) – C-5
15 56.79 OCH3 4.00(s) – C-1
10 113.30 CH 6.84(d, 7.0) H-20 C-3, C-4, C-20 , C-30 , C-40
20 48.13 CH 4.87(ddd, 7.0, 2.5, 2.0) H-10 C-4, C-10 , C-30 , C-40
30 102.57CH 5.51(dd, 2.5, 2.5) H-40 C-10 , C-20 , C-40
40 145.25CH 6.50(dd, 2.0, 2.5) H-30 C-10 , C-20 , C-30
8-OH – 12.61(s) – C-7, C-8, C-11
and 5-methoxysterigmatocystin (3) have been isolated from marine temperatures. As the temperature was raised, the water signal also
fungus. In the 1 H NMR spectrum of 6,8-di-O-methyl versiconol(1), moved to a higher field. As a result of this, the proton signals of H-20
the proton signals of H-20 and H-40 have been overlapped with the and H-40 became visible and so helped to resolve the structures of
big undeuterated water peak. We recorded the spectra at different the compounds.
Copyright 2007 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2007; 45: 434–438
DOI: 10.1002/mrc
437
Spectral Assignments and Reference Data
Figure 5. The plausible biogenetic path from compound 1 to compound 2.
Figure 6. Variable temperature 1 H NMR spectra of compound 1.
Experimental flask containing 250 mL liquid medium (glucose 10, peptone 2,
General experimental procedures yeast extract 1and sea salt 2.5 g/L). The fungus was cultured in
NMR data were recorded in DMSO-d6 or CDCl3 , using TMS as 70 L and incubated at 28 ° C, for 35 days. The culture was filtered
internal reference on a Varian Inova 500NB NMR spectrometer (1 H, through cheesecloth. The mycelium was air-dried and extracted
500 MHz; 13 C, 125 MHz); mass spectra were recorded on a VG ZAB- with methanol. The filtrate was concentrated to 3 L below 60 ° C and
HS Double Focussing Mass Spectrometer, IR spectra were measured partitioned four times by shaking with an equal volume of ethyl
on a Bruker EQUINOX 55 spectrophotometer, UV spectra were acetate. The CH3 OH extract of the fungal mycelium was subjected to
obtained on a Shimadzu UV-2501PC spectrophotometer. Optical silica gel chromatography using a gradient from petroleum to ethyl
rotations were obtained on a Horiba High Sensitivity Polarimeter acetate and followed by that from acetate to methanol. Compounds
SEPA-300, 1(2.5 mg), 2(8.2 mg), 3(15.6 mg) and 4(40.5 mg) were eluted with 100,
30, 10 and 10% ethyl acetate/petroleum ether (V/V), respectively.
6,8-di-O-methyl versiconol(1): mp: 244–245 ° C. EI MS(m/z):,
Fungal strain
388(8), 370(71), 352(22), 342(60), 339(100), 325(96), 314(34), 301(82),
The strain of the fungus (ZSUH 36) was isolated from the Shenzhen
mangrove Acanthus ilicifolius Linn., and the specimen was stored 296(18), 283(13), 268(11), 253(7). [˛]D 22 32.2 (c, 0.05, DMSO).
at the Department of Applied Chemistry, Zhongshan University,
Guangzhou, China. Starter cultures were maintained on cornmeal NMR data
seawater agar. NMR spectra (1H, COSY, HMQC and HMBC) were recorded at
25 ° C on Varian Inova 500NB NMR spectrometer equipped with
Fermentation, extraction and isolation a triple resonance inverse cold probe cooled to 25 K. The 1 H and
Plugs of agar supporting mycelium growth were cut from solid 13 C chemical shifts were referenced to the residual solvent peak of
culture medium and transferred aseptically to a 500 mL Erlenmeyer DMSO-d6 at υ 2.50 and 39.43 ppm and CDCl3 at υ 7.26 and 77.00 ppm,
Copyright 2007 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2007; 45: 434–438
DOI: 10.1002/mrc
438
Spectral Assignments and Reference Data
for proton and carbon respectively. The 1H sweep width was set at Acknowledgements
7520 Hz for all experiments with a 90° pulse for 1 H of 6.5 µs and a We would like to thank the National Natural Science Foundation
13 C 90° pulse of 14.4 µs. The gradient COSY was acquired with 200
of China (20072058, 20572136), the 863 Foundation of China
F1 increments with 32 scans per increment. A sinebell weighting was (2003AA624010), the Natural Science Foundation of Guangdong
applied to each dimension and zero filled to 2 K points. The gradient province, China (021732), Syngenta limited in the United Kingdom
HMQC was acquired with 13 C sweep width of 32 000 Hz and 200
and Star Lake Biotechnology Co., Inc., in Zhaoqing, Guangdong,
t1 increments. Each increment was acquired with 32 transients. A
China, for their financial support.
Gaussian weighting was applied to the F2 dimension before zero
filled to 2 K points and a sinebell was applied to the F1 and zero filled
to 1 K points before Fourier transformation. A one-bond coupling
constant delay was set using 140 Hz and WURST decoupling was REFERENCES
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Copyright 2007 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2007; 45: 434–438
DOI: 10.1002/mrc