New phenethylamine derivatives from Arenibacter nanhaiticus sp. nov. NH36A T and their antimicrobial activity

Article abstract of DOI:10.1038/ja.2013.65

Five new phenethylamine (PEA) derivatives (1-5) were isolated from the strain of Arenibacter nanhaiticus sp. nov. NH36A T derived from the marine sediment of the South China Sea by bioassay-guided fractionation. Their structures were elucidated by spectro

Full text of DOI:10.1038/ja.2013.65

The Journal of Antibiotics (2013), 1–7
2013 Japan Antibiotics Research Association All rights reserved 0021-8820/13
www.nature.com/ja
&
ORIGINAL ARTICLE
New phenethylamine derivatives from Arenibacter
nanhaiticus sp. nov. NH36AT and their antimicrobial
activity
Yanping Chen1, Jinshan Tang1, Xixiang Tang2, Chuanxi Wang1, Yunyang Lian3, Zongze Shao2, Xinsheng Yao1
and Hao Gao1
Five new phenethylamine (PEA) derivatives (1–5) were isolated from the strain of Arenibacter nanhaiticus sp. nov. NH36AT
derived from the marine sediment of the South China Sea by bioassay-guided fractionation. Their structures were elucidated by
spectroscopic methods including UV, IR, HR-MS and NMR. Interestingly, compounds 1–4 existed as enantiomers, which were
resolved by chiral liquid chromatography. The resolved configuration of each enantiomer was assigned by the Marfey’s method.
Of these compounds, 5 showed weak antimicrobial activity against Staphylococcus aureus and Bacillus subtilis with MIC values
of 0.50 and 0.25 mg mlꢀ1, respectively.
The Journal of Antibiotics advance online publication, 17 July 2013; doi:10.1038/ja.2013.65
Keywords: absolute configurations; antimicrobial activity; enantiomers; Marfey’s method; phenethylamine derivatives
INTRODUCTION
Phenethylamine (PEA), the simplest endogenous aromatic amine and 380.1974) in the HR-ESI-TOF MS, indicating 12 degrees of
the quasimolecular ion at m/z 380.1976 [M þ H]þ (calcd
1
potential pharmacophore, provides the basic chemical structure for unsaturation. The H and 13C NMR spectra of 1 (Table 1) showed
several classes of drugs with wide biological activity.1–4 Owing to this resonances for three amide N-H proton signals (dH 7.83 (1H, t,
reason, PEA derivatives have attracted intensive interests recently and 5.2), 7.96 (1H, brs) and 8.12 (1H, brs)) and three ester/amide
a series of new ones has been obtained from natural organisms and carbonyl carbon signals (dC 167.2, 167.3 and 171.1), which further
chemical syntheses, which exhibited diverse bioactivities, including suggested that it was a cyclopeptide. The characteristic methine
bronchiectasia, antidepressant, stimulant, etc.5,6 Nowadays, they have resonances of a-CH at d 3.01/52.8 and 4.12/55.4 indicated that it
emerged as a promising biologically active scaffold in the search of was a cyclodipeptide. The presence of a cluster of 10 aromatic
1
new agents against human diseases.
protons at dH 7.17–7.28 in the H-NMR spectrum suggested that it
In our ongoing work to search for antimicrobial components from contained two monosubstituted benzene rings. Detailed analysis
marine microorganisms, five new PEA derivatives (1–5) were isolated of 1H_1H COSY, HSQC and HMBC data of 1 established
from the strain of Arenibacter nanhaiticus sp. nov. NH36AT derived three residues as phenylalanine (Phe), glutamic acid (Glu) and
from the marine sediment of the South China Sea. Here, we reported N-phenethyl group (Figure 2a). According to the HMBC correla-
the isolation and structural elucidation of new compounds (1–5). tion between H2-100 and C-5 (Glu), the structure of 1 was
Moreover, the chiral resolution of enantiomers and the determination established to be cyclo (Phe-Glu) substituted with N-phenethyl,
of their absolute configurations were also discussed.
which was a new PEA derivative.
The optical rotation value of 1 was almost zero, which suggested it
should be a racemate. Chiral resolution of 1 was performed by Lux
Amylose-2 chiral liquid chromatography (phenomenex, f 5 mm,
4.6 ꢁ 250 mm) using 100% CH3CN at 1.0 mlminꢀ1. Two enantio-
mers were obtained from 1 (Figure 3a), namely 1a and 1b, at tR 17.11
and 24.72 min, respectively, and their relative content was nearly
identical according to their relative peak areas (53.4% for 1a and
46.6% for 1b) in the HPLC chromatogram.
RESULTS
Structure determination
Compounds 1–4 (Figure 1) gave negative reaction with ninhydrin,
but were positive after hydrolysis with concentrated HCl (6.0N),
which suggested that they were cyclo-peptides.7
Compound 1 was obtained as a white amorphous powder.
Its molecular formula was determined to be C22H25N3O3 based on
1Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou, PR China; 2Key Laboratory of Marine Biogenetic Resources,
The Third Institute of Oceanography, State Oceanic Administration, Xiamen, PR China and 3Fujian Provincial Key Laboratory of Screening for Novel Microbial Products, Fujian
Institute of Microbiology, Fuzhou, PR China
Correspondence: Dr J Tang or Dr H Gao, Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China.
E-mail: gztangjinshan@126.com or tghao@jnu.edu.cn
Received 23 December 2012; revised 17 May 2013; accepted 2 June 2013

New PEA derivatives from A. nanhaiticus sp. nov. NH36AT
Y Chen et al
2
Figure 1 Chemical structures of compounds 1–5 from Arenibacter nanhaiticus sp. nov. NH36AT.
Table 1 1H and 13C NMR data assignments of compounds 1–4 (400MHz for 1H and 100 MHz for 13C)
1a
2a
3b
4b
Unit
NO
dC
dH (J in Hz)
dC
dH (J in Hz)
Unit
NO
dC
dH (J in Hz)
dC
dH (J in Hz)
Glu
1
2
167.3
52.8
166.8
53.3
Glu
1
2
170.3
55.0
170.2
54.7
3.01 (1H, t, 5.2)
7.96 (1H, br.s)
1.77 (2H, m)
3.08 (1H, m)
8.02 (1H, br.s)
1.30 (2H, m)
4.08 (1H, t, 5.2)
2.10 (2H, m)
2.27 (2H, m)
3.95 (1H, t, 4.8)
2-NH
3
2-NH
3
28.1
30.2
29.7
30.6
29.5
32.1
32.1
33.0
2.08 (1H, m),
2.03 (1H, m)
4
5
2.07 (1H, m),
1.97 (1H, m)
1.65 (2H, t, 6.6)
4
5
2.32 (2H, t, 7.6)
171.1
171.2
175.0
174.7
Phe
10
20
20-NH
30
167.2
55.4
166.3
55.3
Leu
10
20
20-NH
30
171.4
55.1
171.4
55.7
4.12 (1H, m)
4.17 (1H, m)
8.13 (1H,br.s)
3.94 (1H, t, 6.4)
1.70 (2H, t, 6.8)
1.83 (1H, m)
3.92 (1H, t, 6.4)
8.12 (1H, br.s)
38.4 2.90 (1H, dd, 13.6, 4.4)
3.09 (1H, dd, 13.6, 4.4)
136.0
38.1 2.89 (1H, dd, 12.9, 4.5)
3.13 (1H, dd, 13.6, 4.4)
136.1
43.7
45.5
1.74 (1H, m),
1.66 (1H, m)
1.85 (1H, m)
40
40
50
60
25.4
25.4
50,90
60,80
70
130.0
128.2
126.0
7.17–7.28 (1H, m)
7.17–7.28 (1H, m)
7.17–7.28 (1H, m)
130.2
128.3
126.1
7.13–7.32 (1H, m)
7.13–7.32 (1H, m)
7.13–7.32 (1H, m)
23.5 0.96 (3H, d, 4.8)
22.4 0.98 (3H, d, 4.8)
23.7 0.97 (3H, d, 4.8)
22.2 0.99 (3H, d, 4.8)
N-phenethyl
100
100-NH
200
40.1
3.25 (2H, m)
7.83 (1H, t, 5.2)
2.68 (2H, t, 7.6)
40.1
3.23 (2H, m)
7.61 (1H, t, 5.7)
2.71 (2H, t, 7.6)
N-phenethyl
100
100-NH
200
42.2
3.41 (2H, t, 7.6)
2.80 (2H, t, 7.6)
42.1
3.43 (2H, t, 7.2)
2.81 (2H, t, 7.6)
35.1
35.1
139.6
128.6
128.1
126.6
36.6
36.6
300
139.5
300
140.7
140.7
400,800 128.5
500,700 128.1
600
7.17–7.28 (1H, m)
7.17–7.28 (1H, m)
7.17–7.28 (1H, m)
7.13–7.32 (1H, m)
7.13–7.32 (1H, m)
7.13–7.32 (1H, m)
400,800 130.0 7.17–7.27 (1H, m) 130.0 7.18–7.30 (1H, m)
500,700 129.6 7.17–7.27 (1H, m) 129.6 7.18–7.30 (1H, m)
600
126.7
127.5 7.17–7.27 (1H, m) 127.5 7.18–7.30 (1H, m)
aRecorded in DMSO-d6.
bRecorded in CD3OD.
The Journal of Antibiotics

New PEA derivatives from A. nanhaiticus sp. nov. NH36AT
Y Chen et al
3
Figure 2 Key 1H–1H COSY and HMBC correlations of compounds 1–5. (a) For compound 1/2; (b) for compound 3/4; (c) for compound 5.
Figure 3 Chiral HPLC analytical chromatograms of compounds 1–4. (a) Chiral HPLC analytical chromatogram of compound 1; (b) Chiral HPLC analytical
chromatogram of compound 2; (c) Chiral HPLC analytical chromatogram of compound 3; (d) Chiral HPLC analytical chromatogram of compound 4. A full
color version of this figure is available at The Journal of Antibiotics journal online.
Marfey’s method8,9 was applied to assign the absolute configuration formula of C22H26N3O3 (m/z 380.1977 [M þ H]þ , calcd 380.1974),
1
of enantiomers 1a and 1b. The FDAA derivatives of the acid the same as that of 1. The H and 13C NMR data of 2 were almost
hydrolysate of 1a/1b and the authentic D- and L-amino acids were identical with those of 1 (Table 1), which revealed that they had
subjected to HPLC analysis (Cosmosil C18 column, f 5 mm, closely similar structures. However, they showed different retention
4.6 ꢁ 250 mm, 0.8 ml minꢀ1) at 351C using the following gradient behaviors in normal and reversed-phase chromatography. Interpreta-
program: solvent A, H2O (0.1% HCOOH); solvent B, CH3CN; linear tion of 1D and 2D NMR spectra indicated that 2 had the same planar
gradient, 30–80% of B in A over 30min with UV detection at 340 nm. structure to that of 1.
The absolute configurations of amino acid residues in 1a/1b were
In view of the existence of enantiomers of 1, compound 2 was also
established by comparison of retention times of their derivatives with subjected to Lux Amylose-2 chiral liquid chromatography analysis.
those of corresponding authentic D- and L-amino-acid derivatives. Two enantiomers were obtained at tR 8.19min for 2a and 10.54 min
Therefore, absolute configurations of the Glu and Phe residues of 1a for 2b (Figure 3b), and their relative content ratio was B9:1
were assigned to be D-Glu and L-Phe, respectively (Figure 4a). The according to their relative peak areas in the HPLC chromatogram
absolute configurations of Glu and Phe residues of 1b were assigned (89.3% for 2a and 10.7% for 2b).
to be L-Glu and D-Phe, respectively (Figure 4b).
As the relative contents of enantiomers 2a and 2b were of great
Compound 2 was obtained as a white amorphous powder. disparity, and available amount of 2 was also small, the absolute
[a]D25 ꢀ26.3 (c 0.2, MeOH). HR-ESI-TOF MS gave the molecular configurations of compunds 2a and 2b were determined directly by
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New PEA derivatives from A. nanhaiticus sp. nov. NH36AT
Y Chen et al
4
Figure 4 Determination of the absolute configurations of compounds 1 and 2. (a) HPLC chromatogram of derivatives of the acid hydrolysate of 1a and those
of corresponding authentic amino acids; (b) for 1b and (c) for 2. A full color version of this figure is available at The Journal of Antibiotics journal online.
the Marfey’s method without chiral resolution of 2. Results revealed at tR 8.91 and 15.26 min, respectively (Figure 3c), and their relative
that configurations of Glu and Phe residues of 2a were L-Glu and contents were nearly identical according to their relative peak areas in
L-Phe, respectively, and the opposite to those of 2b (Figure 4c).
Compound 3 was obtained as a faint yellow amorphous powder. Its
the HPLC chromatogram (44.9% for 3a and 55.1% for 3b).
The absolute configurations of the Glu and Leu residues of 3a were
molecular formula was determined to be C19H27N3O3 based on the assigned to be L-Glu and D-Leu, respectively, by Marfey’s method8,9
quasimolecular ion at m/z 346.2130 [Mþ H]þ (calcd 346.2131) in (Figure 5a), and opposite to those of 3b (Figure 5b).
the HR-ESI-TOF MS, indicating 8 degrees of unsaturation. The 13C
Compound 4 was obtained as a faint yellow amorphous powder.
NMR spectrum of 3 (Table 1) showed three ester/amide carbonyl [a]D25 ꢀ17.7 (c 0.5, MeOH). HR-ESI-TOF MS gave the molecular
carbon signals (dC 167.2, 167.3 and 171.1). The characteristic methine formula of C19H28N3O3 (m/z 346.2130 [Mþ H]þ , calcd 346.2131),
resonances of a-CH at d 3.94/55.1 and 4.08/55.0 indicated that it was the same as that of 3. The 1H and 13C NMR data were almost
a dipeptide. The presence of a cluster of five aromatic protons at d identical with those of 3 (Table 1), which revealed that they had
7.17–7.27 suggested the presence of one monosubstituted benzene closely similar structures. However, they showed different retention
ring in the 1H-NMR spectrum of 3. Detailed analyses of 1H_1H behaviors in normal and reverse-phase chromatography. Interpreta-
COSY, HSQC and HMBC data of 3 established the three residues as tion of 1D and 2D NMR spectra indicated that 4 has the same planar
leucine (Leu), glutamic acid (Glu) and N-phenethyl (Figure 2b). structure as that of 3.
According to the HMBC correlation between H2-100 and C-5 (Glu),
the structure of 3 was established to be cyclo (Leu-Glu) substituted subjected to Lux Amylose-2 chiral liquid chromatography analysis.
with N-phenethyl group, which was a new PEA derivative. Two enantiomers were obtained at tR 8.30min for 4a and 10.90 min
In view of the existence of enantiomers of 3, compound 4 was also
The optical rotation value of 3 was almost zero, which suggested for 4b (Figure 3d), and their relative content ratio was B9:1
that it was also a racemate. Further chiral resolution was performed according to their relative peak areas in the HPLC chromatogram
by Lux Amylose-2 chiral liquid chromatography (phenomenex, f (89.2% for 4a and 10.8% for 4b).
5 mm, 4.6 ꢁ 250 mm) using 100% CH3CN at 1.0 mlminꢀ1. Two
As the relative contents of enantiomers 4a and 4b were of great
enantiomers were obtained from compound 3, namely 3a and 3b, disparity, and the available amount of 4 was also small, the absolute
The Journal of Antibiotics

New PEA derivatives from A. nanhaiticus sp. nov. NH36AT
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5
Figure 5 Determination of the absolute configuration of compounds 3 and 4. (a) HPLC chromatogram of derivatives of the acid hydrolysate of 3a and those
of corresponding authentic amino acids; (b) for 3b and (c) for 4. A full color version of this figure is available at The Journal of Antibiotics journal online.
1
Table 2 1H and 13C NMR data assignments of compound 5
(400 MHz for 1H and 100 MHz for 13C)
the HR-ESI-TOF MS, indicating 16 degrees of unsaturation. The H
and 13C NMR spectra of 5 (Table 2) showed two methylenes and
three phenyl rings (including a monosubstituted benzene ring and
two para-disubstituted benzene rings). Detailed analyses of 1H–1H
COSY, HSQC and HMBC correlations further revealed the presence
of an N-phenethyl group and two para-disubstituted benzene
residues. In the HMBC spectra of 5, correlations between H2-100
(dH 3.82, 2H, t, J ¼ 7.2 Hz) and C-1 (dC 172.9) and C-10 (dC 172.9),
H-4/H-8 (dH 6.75, 2H, d, J ¼ 8.8 Hz ) and C-2 (dC 135.5), H-400/H-800
(dH 7.21–7.25, 2H, m) and C-20 (dC 135.5) suggested that the
N-phenethyl group and para-disubstituted benzene residues were
linked with a 1H-pyrrole-2,5-dione ring (Figure 2c), which was
further confirmed by comparison of its NMR data with that
of 1-benzyl-3,4-bis(4-hydroxyphenyl)-1H-pyrrole-2,5-dione.10 Thus,
compound 5 was established to be 1-(2-phenylethyl)-3,4-bis(4-
hydroxyphenyl)-1H-pyrrole-2,5-dione, which was a new compound.
5
5
NO
dC
dH (J in Hz)
NO
dC
dH (J in Hz)
100
200
300
40.6
35.6
3.82 (2H, t, 7.2)
2.96 (2H, t, 7.2)
1, 10
2, 20
3, 30
172.9
135.5
121.6
139.9
400, 800 130.1 7.21–7.25 (2H, m) 4, 8, 40, 80 132.7 6.75 (4H, d, 8.8)
500, 700 129.7 7.21–7.25 (2H, m) 5, 7, 50, 70 116.5 7.29 (4H, d, 8.8)
600
127.7 7.21–7.25 (1H, m)
6, 60
160.5
Recorded in Acetone-d6.
configurations of compunds 4a and 4b were determined directly by
the Marfey’s method without chiral resolution of 4. Results revealed Biological activity
that configurations of Glu and Leu residues of 4a were L-Glu and The antimicrobial activity of compounds 1–5 was evaluated by an
agar dilution method.11 The results showed that compound 5
L-Leu, respectively, and are opposite to those of 4b (Figure 5c).
Compound 5 was obtained as a red amorphous powder. Its displayed antimicrobial activity against S. aureus and B. subtilis,
molecular formula was determined to be C24H19NO4 based on the whereas compounds 1–4 were inactive against the two strains (MIC
quasimolecular ion at m/z 386.1386 [M þ H]þ (calcd 386.1371) in 41.00mgmlꢀ1).
The Journal of Antibiotics

New PEA derivatives from A. nanhaiticus sp. nov. NH36AT
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6
METHODS
General experiments
(38.5g). The extract was subjected to silica gel CC using CHCl3-CH3OH
gradient elution to obtain 10 fractions (F1–F10). The fraction F4 (1.82 g) was
subjected to ODS medium-pressure liquid chromatography eluted with a
gradient of MeOH in H2O (30, 50, 70 and 100%) to afford six subfractions
(F4-1–F4-6). F4-6 (0.27 g) was subjected to a Sephadex LH-20 CC eluted with
1:1 CHCl3-MeOH to yield three subfractions (F4-6-a–F4-6-c). F4-6-c
(35.5mg) was purified by reversed-phase HPLC on an YMC-Pack ODS-A
column (5 mm, 10.0ꢁ 250 mm, 4.0mlminꢀ1), with CH3CN and H2O (50:50,
v/v) as the eluents to yield compound 5 (3.0mg, tR ¼ 16.5min).
Optical rotations were measured on a JASCO P-1020 digital polarimeter at
room temperature (JASCO Inc., Tokyo, Japan). IR spectra were recorded on a
JASCO FT/IR-480 plus Fourier transform infrared spectrometer (JASCO Inc.).
UV spectra were recorded in CH3OH by using a JASCO V-550 UV/VIS
spectrophotometer (JASCO Inc.). HR-ESI-MS was recorded on an Agilent
6200 ESI/TOF mass spectrometer (Agilent Technologies, Santa Clara, CA,
USA). 1D NMR (1H, 13C, DEPT-135) and 2D NMR (1H–1H COSY, HSQC
and HMBC) were recorded on a Bruker AVANCE 400 spectrometer at 300 K
(Bruker Amazon AV400, Zurich, Switzerland). The chemical shifts were given
in d, with the solvent signals (DMSO-d6: dH 2.49/dC 39.5; CD3OD: dH 3.31/dC
49.2; Acetone-d6: dH 2.05/dC 29.9, 206.7) as an internal standard (CIL Inc.,
Berkeley, CA, USA). TLC was carried out on Silica gel GF254 (YanTai Chemical
Inst., Yantai, China) plates. HPLC was performed on a SHIMADZU LC-6AD
LC-6AD Liquid Chromatography with SPD-20A Detector by using an ODS
column (Welch Materials XB-C18 column (f 5 mm, 4.6 ꢁ 250mm), YMC-Pack
ODS-A (f 5 mm, 10.0ꢁ 250 mm)) (Nacalai Tesque Inc., Kyoto, Japan) at 220
and 254 nm. Chiral HPLC analysis was performed using a phenomenex Lux
Amylose-2 column (f 5 mm, 4.6 ꢁ 250 mm) (Phenomen, Torrance, CA, USA).
Column chromatography (CC) was carried out on silica gel (200–300 mesh;
Qingdao Marine Chemical Ltd., Qingdao, China), Sephadex LH-20 (50mm;
Amersham Pharmacia Biotech AB, Uppsala, Sweden) and ODS (60–80 mesh;
YMC Ltd., Kyoto, Japan). All solvents used in CC were of analytical grade
(Tianjin Damao Chemical Plant, Tianjin, China).
The fraction F7 (0.78 g) was subjected to ODS medium-pressure liquid
chromatography eluted with a gradient of MeOH in H2O (30, 50, 70 and
100%) to afford five subfractions (F7-1–F7-5). F7-5 (0.28 g) was purified
by reversed-phase HPLC on an YMC-Pack ODS-A column (5mm,
10.0 ꢁ 250 mm, 4.0mlminꢀ1) with CH3OH and H2O (40: 60, v/v) as the
eluent to yield compounds 1 (5.0mg, tR ¼ 27.2min), 2 (2.4mg, tR ¼ 38.1min),
3 (4.1mg, tR ¼ 24.5 min) and 4 (3.8mg, tR ¼ 32.0min).
Physico-chemical properties
Table 3 summarized the physico-chemical properties of compounds 1–5.
Determination of absolute configuration by the Marfey’s method
Marfey’s method8,9 was applied to assign the absolute configuration of amino-
acid residues of each enantiomer. Compound 1a (0.5mg) in 6.0 N HCl (1.0ml)
was heated at 155 1C for 2 h. Upon removal of excess HCl under vacuum, the
hydrolysates were placed in a 1-ml reaction vial and treated with 1% solution
of 1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide (FDAA; 150 ml) in acetone,
followed by 1.0 N NaHCO3 (40 ml). The reaction mixtures were heated at 451C
for 1.5 h, cooled to room temperature and acidified with 2.0N HCl (20 ml).
Similarly, the standard L- and D-amino acids were derivatized according to the
method mentioned above. The derivatives of the hydrolysates and the standard
amino acids were subjected to HPLC analysis (Kromasil C18 column; f 5 mm,
4.6 ꢁ 250 mm; 1.0 mlminꢀ1) at 351C using the following gradient program:
solvent A, H2O (0.1% HCOOH); solvent B, CH3CN; linear gradient, 30–80%
of B in A over 30 min with UV detection at 340 nm. Absolute configurations of
compounds 1b, 2, 3a, 3b and 4 were also analyzed and determined by the
Marfey’s method, just like that of 1a.
Strain
The strain of A. nanhaiticus sp. nov. NH36AT was isolated from a sandy
sediment sample containing shell fragments collected 0from a water depth of
36m in the South China Sea (191 44.7750 N 1111 6.192 E) in May 2007.12 16S
rRNA gene sequence analysis revealed that strain NH36AT was most closely
related to members of the genus Arenibacter. On the basis of phenotypic,
chemotaxonomic and phylogenetic data, this organism was classified as a
representative of a novel species in the genus Arenibacter. A voucher specimen
was deposited at the China Center for Type Culture Collection (CCTCC AB
208315T) and at the Marine Culture Collection of China (MCCC 1A04137T).
Fermentation of strain
Antimicrobial assays
The producing strain was cultured and fermented as follows. The monoclonal
strain growing on marine 2216E plate13 was inoculated into a 250-ml Erlenmeyer
flask containing 100 ml marine 2216E culture medium and cultured at 30 1C for
24 h on a rotary shaker at 180 r.p.m. Then, 20 ml of the resultant seed culture was
inoculated into 1000-ml Erlenmeyer flasks, each containing 500 ml of the above
culture medium and incubated for 2 days at the same conditions. After
centrifugation, the total 60 l of fermentation broth was collected.
The antimicrobial activities of compounds 1–5 against S. aureus and B. subtilis
were evaluated by an agar dilution method.11 The tested strains were cultivated
on MH agar plates for bacteria at 35 1C. Compounds 1–5 and positive controls
(Tobramycin) were dissolved in EtOH at different concentrations from 1000 to
0.25 mg mlꢀ1 by the continuous two-fold dilution methods. A 10-ml quantity of
test solution was absorbed by a paper disk (5mm diameter) and placed on the
assay plates. After 20h incubation, zones of inhibition (mm in diameter) were
recorded. The MICs were defined as the lowest concentration at which no
microbial growth could be observed. Experimental results showed
Extraction and purification
The fermentation broth was extracted repeatedly with EtOAc, and the organic that compound 5 displayed a weak antimicrobial activity against S. aureus
solvent was evaporated under reduced pressure to afford the crude extract and B. subtilis, with MIC values of 0.50 and 0.25mgmlꢀ1, respectively
Table 3 Physico-chemical properties of compounds 1–5
1
2
3
4
5
Appearance
White powder
White powder
C22H25N3O3
Faint yellow powder
C19H27N3O3
Faint yellow powder
C19H27N3O3
Red powder
C24H19NO4
Molecular fomula
[a]eq \o(\s\up
5(25),\s\do 2(D))
HR FAB MS (m/z)
Found:
C22H25N3O3
Almost zero (c 0.5, MeOH)
ꢀ26.3 (c 0.2, MeOH)
Almost zero (c 0.3, MeOH)
ꢀ17.7 (c 0.5, MeOH)
380.1976 [Mþ H]þ
380.1974
380.1977 [Mþ H]þ
380.1974
346.2130 [Mþ H]þ
346.2131
346.2130 [Mþ H]þ
346.2131
386.1386 [Mþ H]þ
386.1371
Calculated:
IR (KBr) lmax
3189, 3061, 1671, 1546,
1457, 1334, 758, 701
207 (3.2)
3340, 3061, 1671,
1456, 1098, 708, 696
210 (3.7)
3329, 3189, 2958, 1671,
1461, 1090, 697
208 (3.8)
3432, 3322, 3160, 2981,
1679, 1448, 1118, 692
205 (3.0)
3414, 2928, 2361,
1686, 1406, 837, 577
190 (3.2), 231(3.0), 410
(3.5)
UV lmax nm (loge)
(MeOH)
The Journal of Antibiotics

New PEA derivatives from A. nanhaiticus sp. nov. NH36AT
Y Chen et al
7
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ACKNOWLEDGEMENTS
This work was supported by grants from the Ministry of Science and
Technology of China (No. 2009CB522300 and No. 2012ZX09301002-003),
by Guangdong Natural Science Funds for Distinguished Young Scholar
(No. S2013050014287), by the program for New Century Excellent Talents in
University (No. NCET-10-0120) and Fok Ying Tung Education Foundation
(121039) from the Ministry of Education of China.
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