A new polyketide from the mangrove endophytic fungus penicillium sp. sk14JW2P

Article abstract of DOI:10.1002/hlca.201300248

Two polyketides were isolated from the mangrove endophytic fungus, Penicillium sp. sk14JW2P, and one derivative with a modified structure. The structures were elucidated by spectroscopic analyses, the structure of the compound 1 was further confirmed by single-crystal X-ray diffraction, and its absolute configuration was determined. Compound 1 and 2 showed acetylcholinesterase (AchE) inhibitory activities with IC₅₀ values of 12±0.3 and 79±2 nM, respectively. Copyright

Full text of DOI:10.1002/hlca.201300248

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Helvetica Chimica Acta – Vol. 97 (2014)
A New Polyketide from the Mangrove Endophytic Fungus Penicillium sp.
sk14JW2P
by Xi-Shan Huang, Bin Yang, Xue-Feng Sun, Guo-Ping Xia, Ya-Yue Liu, Lin Ma, and Zhi-Gang She*
School of Chemistry and Chemical Engineering, Sun Yat-sen University, #135, Xingang West Road,
Haizhu District, Guangzhou 510275, P. R. China
(phone: þ 86-20-84113356; cesshzhg@mail.sysu.edu.cn)
Two polyketides were isolated from the mangrove endophytic fungus, Penicillium sp. sk14JW2P, and
one derivative with a modified structure. The structures were elucidated by spectroscopic analyses, the
structure of the compound 1 was further confirmed by single-crystal X-ray diffraction, and its absolute
configuration was determined. Compound 1 and 2 showed acetylcholinesterase (AchE) inhibitory
activities with IC50 values of 12 ꢀ 0.3 and 79 ꢀ 2 nm, respectively.
Introduction. – Palitantin is a polyketide and cyclitol compound, which was isolated
by Birkinshaw and Raistrick from fungus in 1936 [1]. Palitantin, with the ability to
mimic the parent saccharides, rendered it a potential candidate as inhibitor of
glycosidase [2], and so it has received a high degree of attention regarding its synthesis
[3 – 5]. In the past decade, we have been dedicated to investigating novel bioactive
compounds from mangrove endophytic fungi collected from the South China Sea
[6 – 9], and an endophytic fungus, strain sk14JW2P, identified as Penicillium sp., was
isolated from the mangrove. Study of the extract of this fungus led to a new
(þ)-palitantin analog, named 13-hydroxypalitantin (1), (þ)-palitantin (2), and 1,4-
diacetoxypalitantin (2a), a new derivative by structure modification of 2. Herein, we
report the isolation, structure elucidation, and acetylcholinesterase (AchE) inhibitory
activities of these compounds.
Results and Discussion. – Compound 1 was isolated as colorless block crystals
(MeOH). The molecular formula, C14H22O5, was determined by NMR (Table 1) and
HR-EI-MS (m/z 270.1459 (Mþ)). The formula indicated four degrees of unsaturation.
The IR spectrum of 1 displayed absorption bands at 3383, 3018, 2970, 2921, 2882, and
1735 cmꢁ1, which indicating the presence of OH, C¼O, and CH¼CH groups. The 1D-
NMR and HSQC spectra of 1 showed signals corresponding to five CH (d(C) 40.4, 56.1,
68.5, 73.8, and 78.8), two CH¼CH (d(C) 131.1, 133.0, 133.3, and 134.5), three CH2
ꢀ 2014 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 97 (2014)
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Table 1. 1H- and 13C-NMR Data (400 and 100 MHz, resp., CD3OD) of Compound 1. d in ppm, J in Hz.
Arbitrary atom numbering as indicated in the Formulae.
Position
d(H)
d(C)
HSQC
HMBC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
3.69 – 3.76 (m, 2 H)
2.44 (dddd, J ¼ 11.8, 6.1, 2.9, 1.0, 1 H)
59.3
56.1
210.8
78.8
73.8
37.9
CH2
CH
C¼O
CH
CH
CH2
CH
CH
CH
CH
CH
CH2
CH
Me
3, 2, 7
3, 1, 7
4.32 (dd, J ¼ 3.4, 1.0, 1 H)
4.29 (dd, J ¼ 6.2, 3.4, 1 H)
1.95 – 2.03 (m, 2 H)
5, 3, 2
4, 3, 6
5, 7
2, 6, 8
7, 9
8, 10
9, 11
10, 12
11, 13
12, 14
13, 12
2.76 (dtd, J ¼ 11.6, 9.3, 6.8, 2.9, 1 H)
40.4
5.50 – 5.59 (m, 1 H)
134.5
133.3
133.0
131.1
43.4
6.13 (dd, J ¼ 10.2, 18.4, 1 H)
6.10 (dd, J ¼ 9.5, 18.4, 1 H)
5.69 (dd, J ¼ 9.5, 6.8, 1 H)
2.16 – 2.30 (m, 2 H)
3.79 (dd, J ¼ 12.4, 6.2, 1 H)
68.5
23.0
1.16 (d, J ¼ 6.2, 3 H)
(d(C) 37.9, 43.4, and 59.3) groups, and one Me (d(C) 23.0) and one C¼O group (d(C)
210.8); thus, the structure must contain one ring. The 1H,1H-COSY spectrum displayed
the cross-peaks HꢁC(4)/HꢁC(5)/CH2(6) and CH2(6)/HꢁC(7)/HꢁC(2), indicating a
1H,1H spin system of CH(4)ꢁCH(5)ꢁCH2(6)ꢁCH(7)ꢁCH(2). Combined with the
HMBC cross-peaks (Fig. 1) HꢁC(2)/C(3) and C(7), and cross-peaks HꢁC(4)/C(3)
and C(5), the structure of the six-membered ring was established. The 2D-NMR
spectrum revealed that two CH¼CH groups were conjugated and attached to
1
CH(7), based on CH(8) showing H,1H-COSY and HMBC correlations with CH(7).
The HꢁC(12) showed HMBCs with C(11) and C(13), and, combined with the 1D-
NMR and 1H,1H-COSY, C(11) was connected to CH2CH(OH)Me (C(12)/C(13)/
C(14)). Ultimately, the structure of 1 was further confirmed by single-crystal X-ray
diffraction, and the absolute configuration was determined using copper radiation. The
new compound 1 named 13-hydroxylpalitantin (Fig. 2) with absolute configuration
(2R,4R,5R,7S,13S).
Compound 2 was obtained as colorless block crystals. The NMR spectrum was
almost identical to that of 1 and the data were consistent with those (þ)-palitantin
([a]2D3 ¼ þ4.49 (c ¼ 0.32, CHCl3); [a]D25 (2) ¼ þ6.80 (c ¼ 0.32, CHCl3)) [4]. The NOE
experiment revealed the same relative configuration as that of 1, and, together with the
biogenenic pathway analysis, we deduced the absolute configuration of 2 as that of 1.
Thus, 2 was determined as (þ)-palitantin.
Fig. 1. Main HMBCs (H ! C) of 1 and 2a

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Helvetica Chimica Acta – Vol. 97 (2014)
Fig. 2. X-Ray crystal structure of 1
Compound 2a was obtained as colorless powder. It was obtained by structure
modification of 2 via the reaction with the AcOH in pyridine. Compared to the NMR
data of 2 [4], the spectra of 2a exhibited two more Me singlet signals at d(H) 2.21 (4-
AcO) and 1.99 (1-AcO), plus four more C-atom signals at d(C) 171.0, 169.6, 21.0, and
20.7, attributed to two AcO groups. In the HMBC (Fig. 1), HꢁC(1) signal showed
correlation to that at d(C) 171.0, and HꢁC(4) signal correlated with that at d(C) 169.6,
suggesting that the OH groups at C(1) and C(4) were acetylated, different from the
reported compound 4,5-diacetoxypalitantin [10].
Finally, compound 2a was identified as a new compound and named 1,4-
diacetoxypalitantin.
All compounds were evaluated for their inhibitory activities against AChE in vitro
according to the modified Ellman method [11]. Compound 2 showed significant
inhibitory activity against AchE with an IC50 value of 79 nm (Table 3); however, the
Table 2. 1H- and 13C-NMR Data (400 and 100 MHz, resp., CDCl3) of Compound 2a. d in ppm, J in Hz.
Arbitrary atom numbering as indicated in the Formulae.
Position
d(H)
d(C)
HSQC
HMBC
1
2
3
4
5
6
4.22 – 4.27 (m, 2 H)
2.55 – 2.60 (m, 1 H)
60.4
52.7
200.3
78.8
71.0
CH2
CH
3, 2, 7, 1-MeCO
3, 1, 7, 8
C¼O
5.25 (dd, J ¼ 3.0, 1 H)
4.42 (dd, J ¼ 7.1, 3.0, 1 H)
2.17 (dt, J ¼ 14.5, 4.1, 1 H),
1.87 – 1.94 (m, 1 H)
CH
5, 3, 4-MeCO
4, 3, 6
5, 4, 2, 7
CH
CH2
36.9
3, 2, 1, 6, 8
7
2.78 – 2.87 (m, 1 H)
39.5
CH
8
9
10
11
12
13
14
4-AcO
1-AcO
5.41 (dd, J ¼ 14.7, 9.1, 1 H)
6.08 (dd, J ¼ 9.1, 18.4, 1 H)
6.00 (dd, J ¼ 14.7, 18.4, 1 H)
5.65 (dd, J ¼ 14.7, 6.9, 1 H)
2.02 – 2.07 (m, 2 H)
130.7
132.9
129.5
135.5
34.8
22.5
13.9
20.7, 169.6
21.0, 171.0
CH
CH
CH
CH
CH2
CH2
Me
Me, C
Me, C
2, 6, 7, 9
7, 10
9, 12
10, 12, 13
10, 11, 13, 14
11, 12, 14
12, 13
1.36 – 1.45 (m, 2 H)
0.90 (t, J ¼ 7.4, 3 H)
2.21 (s, 3 H)
1.99 (s, 3 H)

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Table 3. Acetylcholinesterase (AChE) Inhibitory Activity of Compounds 1, 2, and 2aa)
Compound
1
2
2a
HUPb)
0.060
IC50 [mm]
12
0.079
> 200
a) Expressed in IC50 values. b) Huperzine A used as positive control.
corresponding value for 1 showed only 12 mm. Since 1 possesses an additional OH group
at C(13) as the only difference to 2, the results of the biological assay indicated that the
Pr group was essential for inhibitory activity. Similarly, 2a showed no activity, its IC50
was > 200 mm, indicating that the OH groups are indispensable for the AchE inhibitory
activity.
Financial support from the National Natural Science Foundation of China (Nos. 20972197, 41276146,
and 21102020), the 863 Foundation of China (No. 2011AA09070201), the Science & Technology Plan
Project of Guangdong Province of China (Nos. 2010B030600004 and 2011A080403006), and the
Fundamental Research Funds for the Central Universities of China (No. 11lgjc01) are gratefully
acknowledged. We also thank Prof. L. Jiang for his helpful instructions in crystallography.
Experimental Part
General. Column chromatography (CC): silica gel (200 – 300 mesh; Qingdao Marine Chemicals, P. R.
China). M.p.: FisherꢁJohns hot-stage apparatus. Optical rotation: WZ-1 polarimeter at 258. IR Spectra:
Nicolet 5DX-FT-IR, in KBr discs; ˜n in cmꢁ1. NMR: Varian Inova-400 NB spectrometer, d in ppm rel. to
Me4Si as internal standard, J in Hz. HR-EI-MS: VG-ZAB mass spectrometer; in m/z.
Fungal Material. The fungal strain Penicillium sp. sk14JW2P (GenBank accession No. KC545799)
was isolated as endophytic fungus from a mangrove, root of the Kandelia candel (L.) Druce, collected
from Shankou, Guangxi Province, P. R. China, in September 2008. The fungus was deposited with the
School of Chemistry and Chemical Engineering, Sun Yat-sen University.
Fermentation and Extraction. Spores of Penicillium sp. sk14JW2P were directly spread into 4 ꢂ 500-
ml Erlenmeyer flasks, each containing 200 ml of potato-liquid media composed of glucose (20 g/l) and
NaCl (30 g/l), and the final pH was adjusted to 6.9. Four Erlenmeyer flasks of the inoculated media were
incubated at 258 on a rotary shaker at 180 rpm for 72 h to yield the seed liquid. The rice solid-substrate
medium was carried out in 50 Erlenmeyer flasks (1000 ml) each containing 100 g of rice and 20 ml of 3%
sea salt liquid, all were prepared by autoclaving at 1208 ca. 30 min and cooling to r.t. The seed liquid was
transferred into the rice solid-substrate medium under aseptic conditions and incubated at 258 for 28 d.
The mycelia and rice medium were extracted with MeOH. The MeOH layer was concentrated under
reduced pressure to give a dark brown gum (26.6 g).
Purification. The extract was separated by CC (silica gel; CHCl3/MeOH from 1:0 to 1:3) to give four
fractions, Frs. 1 – 4. Fr. 4 (320 mg) with CHCl3/MeOH from 1:0 to 1:3 afforded compound 2 (50 mg).
The Fr. 3 was subjected to CC (Sephadex LH-20; petroleum ether/CHCl3/MeOH 2 :1:1) to furnish
compound 1 (4 mg).
(þ)-13-Hydroxypalitantin (¼(2R,3R,5S,6R)-2,3-Dihydroxy-5-[(1E,3E,6R)-6-hydroxyhepta-1,3-di-
en-1-yl]-6-(hydroxymethyl)cyclohexanone; 1). Colorless crystals. M.p. 144 – 1468. [a]2D0 ¼ þ46.0 (c ¼
0.10, MeOH). IR (KBr): 3383, 3018, 2970, 2921, 2909, 2882, 1735, 1404, 988. 1H- and 13C-, and 2D-
NMR: Table 1. HR-EI-MS: 270.1459 (Mþ, C14H22O5þ ; calc. 270.1467).
(þ)-1,4-Diacetoxypalitantin (¼{(1R,3R,4R,6S)-3-(Acetyloxy)-6-[(1E,3E)-hepta-1,3-dien-1-yl]-4-hy-
droxy-2-oxocyclohexyl}methyl Acetate; 2a). Colorless powder. [a]2D5 ¼ þ40.0 (c ¼ 0.10, MeOH). 1H- and
13C-NMR: Table 2. HR-EI-MS: 338.1729 (Mþ, C18H26O6þ ; calc. 338.1729).
X-Ray Crystallography of 1. Crystal data: C14H22O5, Mr 270.32, crystal size 0.32 ꢂ 0.26 ꢂ 0.25 mm,
monoclinic, space group: P21, unit cell dimensions a ¼ 8.6284(2) ꢂ, b ¼ 5.0503(2) ꢂ, c ¼ 16.0474(4) ꢂ,

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Helvetica Chimica Acta – Vol. 97 (2014)
a ¼ 908, b ¼ 103.753(3)8, g ¼ 908, V¼ 679.23(4) ꢂ3, Z ¼ 2, Dx ¼ 1.322 g/cm3, F(000) ¼ 292. The reflection
data were collected on an Oxford Diffraction XcaliburNova single-crystal diffractometer using CuKa
radiation at 150(2) K, l ¼ 1.54178 ꢂ. A total of 12262 reflections were collected in the range 2.838 ꢃ q ꢃ
67.098, of which 2,397 unique reflections with I > 2s(I) were collected for the analysis. The structure was
solved by direct methods and refined by full-matrix least-squares based on F2 using the SHELXL-97. The
final R and wR2 factors were 0.0294 and 0.0764, resp. The absolute-structure Flack parameter was ꢁ
0.05(16). Crystallographic data for the structure reported in this article has been deposited with the
Cambridge Crystallographic Data Centre (accession No. CCDC 944995).
Biological Assay. Acetylcholinesterase (AChE) inhibitory activity was evaluated in vitro according
to the modified Ellman method [11]. The IC50 value represents the concentration of a compound
required to inhibit the activity by 50%.
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Received June 17, 2013