Short Communication
Isolation and identification of chemical constituents from the
bacterium Bacillus sp. and their nematicidal activities
Liming Zeng1, 2, Hui Jin1,4, Dengxue Lu3, Xiaoyan Yang1, Le Pan1,2, Haiyan
Cui1,2, Xiaofeng He1, Hongdeng Qiu1, Bo Qin1*
1
Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key
Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical
Physics, Chinese Academy of Sciences, Lanzhou, China.
2
University of Chinese Academy of Sciences, Beijing, China
3
Institute of Biology, Gansu Academy of Sciences, Lanzhou, China
4
Key Laboratory of Tobacco Diseases and Insect Pests Monitoring Controlling
and Integrated Management, Institute of Tobacco Research, Chinese Academy of
Agricultural Sciences, Qingdao, China
DOI 10.1002/jobm.201400798
Supporting Information

Isolation procedures of compounds from strain SMrs28

Details on the bioassay

Spectroscopic data and elucidation of the compounds

Isolation procedures of compounds from strain SMrs28
The -20℃-stored strain was activated on the basic solid medium, identical to the
formula for preservation, prior to inoculating into a 10-L fermentor with 6 L seed
medium. After being cultured at 28℃ and 180 rpm for 48 h, 5 L of mature seed
medium was fed into 100-L fermentor for large-scale fermentation. The temperature,
agitation rate and fermentation time were set to the same as those above. The pH was
kept at 7.2 with automatic injecting of 1 M NaOH or HCl. 40-60% of dissolved
oxygen was provided during the whole process. After fermentation, the 100 L of
fermentation broth was placed for 24 h, and then filtered twice with 4-layer gauze to
remove the observable impurities. Afterwards, the 1/2 of the filtrate was applied to the
pre-packed chromatography column (150.0 cm ×11.5 cm i.d.) with 3.5 kg non-polar
macroporous resin D101, repeated twice. Initially, the column was eluted with double
distilled water to flush out the high polar substances, followed by washing with 4BV
of 90% (v/v) ethanol aqueous solution. The flow rate was maintained at a relative low
level to ensure enough absorption time. The remaining part of filtrate was conducted
as the same protocols mentioned above, and finally the eluate was concentrated with a
rotary evaporator to ca. 200 g crude extract. After being further dried, the crude
extract was dissolved in methanol, mixing with the silica gel at the ratio of 1:1.5. Up
till the solvent was completely evaporated, the homogeneous mixture was
chromatographed on a silica gel column (130.0 cm×7.5 cm i.d.) using
chloroform/acetone (98:2 to 1:1, v/v) as eluting agent, yielding 21 fractions, P1-21.
Fraction P1 was purified on a silica gel column with a gradient elution of petroleum
ether/acetone (30:1 to 1:1, v/v), obtaining compound 6 (13 mg). Fraction P4 was
subjected to silica gel column with a stepwise eluting fashion of increasing petroleum
ether in acetone (30:1 to 1:1, v/v), affording a subfraction P4-1 (500 mg). Subfraction
P4-1 was repeatedly purified using petroleum ether/acetone (100:1 to 0:1, v/v),
offering compound 5 (350 mg). Fraction 10 was isolated on a silica gel column
eluting with a gradient of petroleum ether/acetone (10:1 to 1:1, v/v) to give compound
3 (25 mg). Fraction P11 was subjected to a silica gel column with petroleum
ether/acetone (10:1 to 1:1, v/v) as eluting solvent, furnishing subfraction P11-4 (70
mg). The subfraction was then fractioned by silica gel column eluting with
chloroform/ethyl acetate (10:1 to 2:1, v/v), and further purified on Sephadex LH-20
column to yield compound 1 (45 mg). Fraction P15 was chromatographed over a
silica gel column with chloroform/acetone (20:1 to 0:1, v/v) to obtain subfraction
P15-5. Further purification of P15-5 was performed with a gradient elution of ethyl
acetate/ methanol (10:1 to 0:1, v/v) on a silica gel, and repeatedly washed over
Sephadex LH-20 column using chloroform/methanol (1:1, v/v), obtaining compound
2 (15 mg). Fraction 13 was loaded on a silica gel column using chloroform/ethyl
acetate (1:1 to 1:10, v/v) and then chloroform/acetone (10:1 to 0:1, v/v) as eluting
solvent,
finally
further
purified
on
Sephadex
LH-20
column
with
chloroform/methanol (10:1, v/v) to obtain compound 4 (20 mg).

Details on the bioassay
The compounds was prepared in dimethyl sulfoxide (DMSO) to overcome
insolubility to 80 mg/ml diluting with double distilled water as stock solutions, stored
at -20℃ until use. The experiment was conducted in 24-well plates (Nunc, Roskilde,
Denmark); a volume of 495μl of the suspension containing about 100 nematodes
obtained by serial dilutions was transferred to each of three wells per treatment. An
aliquot of 5 μl of compound stock solution was also added into the wells, resulting in
the final concentrations of 50, 100, 200, 400, 800 μg/ml. The concentrations of
DMSO in each well was limited at less than 1%, since the paralysis in solvent (1%
DMSO aqueous solution) did not show significant difference with that observed in
double distilled water. The bioassay was carried out twice, with the same
concentration of DMSO served as a control. Multi-well plates were covered,
parafilmed to avoid evaporation, and maintained in a humid chamber at 28℃. To
compare the effect of different compounds against nematodes, the mobile and
immobile individuals was counted at three randomed sites using a light microscope
after different incubation times (24, 48, 72h).

Spectroscopic data and elucidation of the compounds
Compound 1
1
H-NMR (400 MHz, CD3OD): δH=7.02 (d, 2H, J=8.4 Hz, 2H-7, 8), 6.69 (d, 2H, J=8.4
Hz, 2H-6, 9), 3.67 (t, 2H, J=7.2 Hz, 2H-3), 2.70 (t, 2H, J=7.2 Hz, 2H-2); 13C-NMR
(100 MHz, CD3OD): δC =156.70 (C-5), 130.94 (C-1), 130.85 (d, C-7, 8), 116.08 (C-6,
9); 64.54 (C-3), 39.36 (C-2).
Compound 2
1
H-NMR (400 MHz, CD3OD): δH= 6.41 (br.s, NH), 4.00 (dd, J= 6.8, 8.8 Hz, H-C8a),
3.97 (dd, J= 6.8, 6.8 Hz, H-3), 3.30-3.53 (m, 2H-6), 2.27-2.33 (m, Hb-8), 1.91-2.04 (m,
2H-7, Ha-8), 1.37 (d, J= 6.8 Hz, Me). 13C-NMR (100 MHz, CD3OD): δC= 172.58
(C-4), 169.05 (C-1), 60.45 (C-8a), 52.07 (C-3), 46.42 (C-6), 29.18 (C-8), 23.62 (C-7),
15.71 (C-1’).
Compound 3
H-NMR (400 MHz, CDCl3): δH = 7.34 (2H, t, J= 7.6 Hz, 2H-3,5), 7.33 (2H, d, J= 6.4
Hz, 2H-2,6), 7.30 (1H, t, J= 7.2 Hz, H-4); 13C-NMR (100 MHz, CDCl3): δC= 174.24
(C-8), 134.74 (C-1), 128.96 (C-3,5), 128.06 (C-2,6), 126.46 (C-4), 40.59 (C-7).
Compound 4
1
H-NMR (400 MHz, CD3OD): δ= 4.20 (1H, br.t, J= 7.2 Hz; Pro-Hα), 4.06 (1H, br.s;
Val-Hα), 3.64 (1H, dt, J= 12.1, 8.1 Hz; Pro-Hδ), 3.50 (1H, ddd, J= 12.1, 9.5, 2.9 Hz;
Pro-Hδ), 2.48 (1H, d·sept, J= 2.6, 7.0 Hz; Val-Hβ), 2.33 (1H, m; Pro-Hβ), 1.99 (2H, m;
Pro-Hβ2 and Pro-Hγ1), 1.92 (1H, m; Pro-Hγ2), 1.09 (3H, d, J= 7.2 Hz; Val-Hγ1), 0.92
(3H, d, J= 6.8 Hz; Val-Hγ2); 13C-NMR (100 MHz, CD3OD): δC= 172.57 (C-4),
167.54 (C-1), 61.48 (C-8a), 59.99 (C-3), 46.14 (C-6), 29.84 (C-1’), 29.49 (C-8), 23.24
(C-7), 18.85 (C-2’), 16.65 (C-3’).
Compound 5
1
H-NMR (400 MHz, CDCl3): δH= 2.33-2.37 (t, 2H, J= 7.6 Hz, 2H-2a, 2H-2b),
1.60-1.67 (m, 2H), 1.26-1.32 (m, 18H, H2-3, 8×CH2), 0.85 (t, 3H, J= 6.8 Hz，CH3-12);
13
C-NMR (100 MHz, CDCl3): δC= 178.84 (C-1), 33.86 (C-2), 31.95 (C-3), 29.72
(C-4), 29.61 (C-5), 29.46 (C-6), 29.38 (C-7), 29.26 (C-8), 29.09 (C-9), 24.72 (C-10),
22.72 (C-11), 14.13 (C-12).
Compound 6
1
H-NMR (400 MHz, CDCl3): δH= 5.34 (s, 1H), 3.66 (s, 3H), 2.30 (t, J= 7.2Hz, 2H),
2.08-1.95 (m, 2H), 1.49-1.70 (m, 2H), 1.37-1.26 (m, 24H), 0.88 (t, J = 6.2Hz, 3H).
13
C-NMR (100 MHz, CDCl3) δC= 174.10 (C-1), 129.85 (C-2), 129. 61 (C-3), 51.22
(C-4), 34.24 (C-5), 31.87 (C-6), 30.09 (C-7), 29.62 (C-8), 29.54 (C-9), 29.47 (C-10),
29.37 (C-11), 29.27 (C-12), 29.16 (C-13), 29.07(C-14), 28.63 (C-15), 27.10 (C-16),
24.85 (C-17), 22.60 (C-18), 14.12 (C-19).
1
Compound 2 was isolated as a colorless crystal. The molecular formula C8H12O2N2
was determined by HR-ESI-MS (m/z 191.0794 [M+Na]+, calcd. for C8H12O2N2Na,
191.0791). The 1H and
13
C NMR spectrum showed characteristic signals of a
diketopiperazine ring unit, which included two amide carbonyl signals at δC 172.58
and 169.05 and a methine signal at δC 52.07. The 1H NMR spectrum exhibited signal
for a methyl at δH 1.37 (3H, d, J= 6.8Hz). Moreover, the proton resonances at δH
3.30-3.53 (m, 2H-6), 2.27-2.33 (m, Hb-8), and 1.91-2.04 (m, 2H-7, Ha-8), and the
carbon signals at δC 46.42 (C-6), 29.18 (C-8), 23.62 (C-7) indicated the presence of
three methylene groups. The connective details of the cyclic dipeptide structure were
further confirmed by HMBC spectral data. In the HMBC spectrum, the correlations
from Hb-8 (δH 2.27-2.33) to C-6 (δC 46.42), C-7 (δC 23.62) and C-8a (δC 60.45), from
Ha-8 (δH 1.91-2.04) to C-7 (δC 23.62) and C-8a (δC 60.45), from H-C8a (δH 4.00) C-8
(δC 29.18), from H-7 (δH 1.91-2.04) to C-6 (δC 46.62) and C-8 (δC 29.18), from H-6
(δH 3.30-3.53) to C-7 (δC 23.62) and C-8 (δC 29.18), from H-3 (δH 3.97) to C-1’ (δC
15.71), CH3(δH 1.37) to C-3(δC 52.07) were found. Therefore, based on the above
spectroscopic data and comparison with the previously reported data, the structure of
2
was
established
and
named
(3S,
8aS)-hexahydro-3-methylpyrro[1,2-a]pyrazine-1,4-dione (Fig. 1). Compound 3 was
obtained as a white needle crystal. Based on the data reported in the literature, the
compound was identified as phenylacetamide (Fig. 1). Compound 4 was an
amorphous powder solid. The compound exhibited similar spectroscopic data to those
of 2. Comparison of the NMR spectroscopic data of these two compounds, indicated
that they are closely correlated, except that the signal for methyl at δC 15.71 in 2 was
replaced by a isopropyl group for C-1’ (δC 29.84, δH 1.92, m), C-2’ (δC 18.85, δH 1.09,
d, J= 7.2 Hz), and C-3’ (δC 16.65, δH 0.92, d, J= 6.8 Hz). Thus, the structure of 4 was
established as Cyclo ( L-Pro-L-Val) (Fig.1). Compound 5 was obtained as a colorless
crystal. The compound was determined to be lauric acid (Fig. 1). Compound 6 was
isolated as Colorless oil. The compound was identified as methyl elaidate (Fig. 1).