Isolation and characterization of two novel antibacterial cyclic hexapeptides from streptomyces alboflavus 313

Article abstract of DOI:10.1002/cbdv.201100364

Two novel cyclic hexapeptides, designated NW-G08 (1) and NW-G09 (2), were isolated from the fermentation broth of Streptomyces alboflavus 313. Their structures were elucidated on the basis of extensive spectroscopic analysis, MS experiments, and chemical

Full text of DOI:10.1002/cbdv.201100364

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Isolation and Characterization of Two Novel Antibacterial Cyclic
Hexapeptides from Streptomyces alboflavus 313
by Zhiqin Ji*a), Gang Qiaoa), Shaopeng Weib), Lixia Fanb), and Wenjun Wub)c)
a) State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling,
Shaanxi 712100, P. R. China (phone: þ86-29-87093987; fax:þ86-29-87093987;
e-mail: jizhiqin@nwsuaf.edu.cn)
b) Shaanxi Province Key Laboratory of Research and Development on Botanical Pesticide,
Northwest A & F University, Yangling, Shaanxi 712100, P. R. China
c) Key Laboratory of Plant Protection Resources and Pest Integrated Management, Ministry of
Education, College of Plant Protection, Northwest A & F University, Yangling, Shaanxi 712100,
P. R. China
Two novel cyclic hexapeptides, designated NW-G08 (1) and NW-G09 (2), were isolated from the
fermentation broth of Streptomyces alboflavus 313. Their structures were elucidated on the basis of
extensive spectroscopic analysis, MS experiments, and chemical analysis. Their antibacterial activities
against several strains of bacteria were evaluated by micro-broth dilution method. NW-G08 (1) and NW-
G09 (2) were highly antibacterial against Gram-positive but not active against Gram-negative bactaria.
The minimum inhibitory concentrations (MICs) of 1 against Bacillus cereus, Bacillus subtilis, and
Staphylococcus aureus were 6.25, 6.25, and 1.56 mg/ml, respectively. It is worth noting that 1 and 2
exhibited much higher activities against methicillin-resistant Staphylococcus aureus (MRSA) than
ampicillin, which implies that they might be potential candidates for the development of novel drugs
against resistant pathogenic bacteria.
Introduction. – Peptides are the largest chemical group of natural antibiotics
produced by microorganisms, in which cyclic peptides have attracted more attention
due to the diversity of their biological activities [1]. Since monamycins were first
isolated from Streptomyces jamaicensis in 1995 [2], a series of natural compounds which
contain the piperazine-3-carboxylic acid moiety have been found among the
metabolites of microorganisms, such as azinothricins [3–6], chloptosin [7], himastatin
[8–10], and piperazimycin [11]. These natural products exhibit impressive antimicro-
bial, antitumor and anti-HIVactivities. Attracted by their intriguing chemical structure
and a medicinally relevant biological activity profile, the piperazic acid-containing
natural products mentioned above have been successfully synthesized in recent years
[12].
In the previous investigation, our group also isolated a piperazic acid-containing
cyclic hexapeptide, NW-G01, from the fermentation broth of Streptomyces alboflavus
313 [13–15]. NW-G01 exhibited strong antimicrobial activity against Gram-positive
bacteria. The structures of himastatin, chloptosin, and NW-G01 consist of valine (Val),
piperazic acid (PA), and a pyrroloindoline derivative (Trp). Although they are
significantly different in the amino acid content, their antimicrobial activities are
similar. All the compounds could selectively inhibit Gram-positive bacteria, including
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
methicillin-resistant Staphylococcus aureus (MRSA), whereas they are not active
against Gram-negative bacteria. In our further investigation, two novel cyclic
hexapeptides, NW-G08 (1) and NW-G09 (2), were isolated from the producer of
NW-G01 by large-scale fermentation (see Fig. 1). We report here the structure
elucidation and antimicrobial activity of these cyclic hexapeptides.
Fig. 1. Structures of NW-G01, NW-G08 (1), and NW-G09 (2)
Results and Discussion. – Chemistry. NW-G08 (1). The IR spectrum of 1 exhibited
typical absorption bands of amide CO groups at 1660 cmꢀ1. Its molecular formula was
established as C36H51ClN10O7 by HR-ESI-MS (m/z 771.3716, ([MþH]þ ,
C36H52ClN10Oþ7 ; calc. 771.3709). The presence of the Cl-atom was suggested by the
isotope abundance peaks in the MS spectrum. The 13C-NMR and DEPT spectra
(Table 1) of 1 showed 36 C-atom signals, which were attributed to four Me, eleven CH2,
and eight sp3CH groups, one O-bearing sp3 quaternary C-atom, three sp2-CH groups,
three sp2 quaternary C-atoms, and six amide CO C-atoms. 1H- and 13C- chemical-shift
assignments were accomplished by standard 1D- and 2D-NMR techniques, such as
1
DEPT, HSQC, and HMBC. The H-NMR spectrum of 1 exhibited signals of two Me
groups at d(H) 3.00 (s) and 1.34 (d, J¼6.5), and of one CH group at d(H) 5.75 (d, J¼
6.5), which were assigned to the MeN H-atoms, and Hb- and Ha-atoms of N-
methylalanine, respectively, based on the HSQC and HMBC spectra of 1, and by
comparison with the corresponding chemical shifts and coupling constants of NW-G01
[14]. The characteristic signals of CH groupat d(H/C) 5.49/85.6, of O-bearing
quaternary C-atom at d(C) 88.4, of three aromatic CH groups at d(H/C) 6.73/110.7,
6.81/119.2, 7.22/123.7, and three sp2 quaternary C-atoms at d(C) 129.5, 135.0, and 149.2
indicated the presence of 6-chloro-1,2,3,3a,8,8a-hexahydro-3a-hydroxy-pyrrolo-[2,3-
b]indole-2-carboxylic acid; the chlorinated pyrroloindoline moiety usually was
considered to be derived from tryptophan (Trp). Three molecules of piperazic acid
were also identified based on the HSQC and HMBC spectra of 1, and by comparison
with the corresponding chemical-shift and coupling-constant values of NW-G01. The
signals of two CH groups at d(H) 5.13–5.15 (m) and 1.55–1.58 (m), of two Me groups
at d(H) 0.96 (d, J¼7.0) and 0.98 (d, J¼7.0), and of one CH2 group at d(H) 1.59–1.61

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1569
and 1.40–1.42 (m) could be assigned to the Ha, Hg, Hd, Hd’, and Hb of leucine based on
1H,1H-COSY and HMBC correlations (see Table 1). Thus, the structure of 1 was
established as consisting of N-methylalanine (MeAla), 6-chloro-1,2,3,3a,8,8a-hexahy-
dro-3a-hydroxy-pyrrolo[2,3-b]indole-2-carboxylic acid (Trp), leucine (Leu), and three
molecules of piperazic acid (PA).
The sequence linkage of the amino acids in 1 could be readily determined by
comparison of the characteristic MS/MS fragmention peaks in the spectra of 1 and NW-
G01 (Table 2, Scheme 1, and Fig. 2). Fragmentation pathway of NW-G01 was
illustrated first, the product ions were derived mostly from the cleavage of peptide
bonds, and the amino acid content of important fragments were also deduced. Based on
the fragmentation pathway, fragmention peaks at m/z 672, 658, 533, 448, 409, 349, and
310 observed in the MS/MS spectrum of NW-G01 could be attributed to PA1-Trp-Val-
PA3-PA2, PA3-PA2-Ala-PA1-Trp, MeAla-PA1-Trp-Val, PA1-Trp-Val, Val-PA3-PA2-Ala,
PA1-Trp, and PA2-MeAla-PA1, respectively. At the same time, fragmention peaks at m/
z 658, 349, and 310 also appeared in the MS/MS spectrum of 1, indicating that the
sequence linkage of the amino acids in 1 was identical with that in NW-G01, except
valine was replaced with leucine. This could be confirmed by further characteristic
fragmention peaks appearing in the spectrum of 1. Fragmention peaks at m/z 686, 547,
462, and 423 were observed in the spectrum of 1, indicating that each fragment was 14
Da more than the corresponding fragments of PA1-Trp-Val-PA3-PA2, MeAla-PA1-Trp-
Val, PA1-Trp-Val, Val-PA3-PA2-Ala revealed in the spectrum of NW-G01, the molecular
weight of leucine is 14 Da higher than that of valine, so these ions could be elucidated as
PA1-Trp-Leu-PA3-PA2, MeAla-PA1-Trp-Leu, PA1-Trp-Leu, and Leu-PA3-PA2-Ala. In
other words, the m/z values of Leu-containing fragments observed in the spectrum of 1
were 14 Da higher than those Val-containing fragments observed in that of NW-G01,
and the m/z value of fragment in which Leu was not presented was equal to that of NW-
G01. The conclusion could also be supported by the results of the HMBC experiment,
which showed correlation from the a-CH group H-atoms, particular of amino acid
residues, to the CO C-atom of the neighboring residues.
The absolute configurations of the amino acids were determined by application of
the Marfeyꢂs method as we described previously [14], as well as the NOESY spectrum.
By comparing the retention times of their 1-fluoro-2,4-dinitrophenyl-5-l-alanineamide
(FDAA) derivatives with the purchased l-leucine and that of NW-G01, the absolute
stereochemistry of amino acid in 1 was determined as l-leucine, N-methyl-d-alanine,
(3R)-piperazic acid (PA1), (3R)-piperazic acid (PA2), (3S)-perazic acid (PA3),
and(2S,3aR,8aS)-6-chloro-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]indole-2-
carboxylic acid (Fig. 1).
NW-G09 (2). The IR spectrum of 2 exhibited typical absorption bands of amide CO
groups at 1636 cmꢀ1. Its molecular formula was established as C35H49ClN10O8 by HR-
ESI-MS (m/z 773.3511 ([MþH]þ , C35H50ClN10O8þ ; calc. 773.3502). The presence of
the Cl-atom was evidenced by the isotope abundance peaks in the MS spectrum. The
13C-NMR and DEPT spectra (Table 3) of 2 showed 35 C-atom signals, which were
recognized as those of four Me, ten CH2, and eight sp3 CH groups, one O-bearing sp3
quaternary C-atom, two sp2 CH groups, and four sp2 quaternary and six amide CO C-
atoms. 1H- and 13C-chemical-shift assignments were achieved by standard 1D- and 2D-
1
NMR techniques, such as DEPT, HSQC, and HMBC. The H-NMR spectrum of 2

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table 1. The 1H- and 13C-NMR Chemical Shifts of NW-G08a) (1; CD3OD, 1H: 500 MHz, 13C: 125 MHz).
d in ppm, J in Hz.
Position
d(C)
d(H)
1H,1H-COSY
HMBC
NOESY
Leu
CO
a
174.7
48.9
42.0
a, PA3-a
b, g
5.13–5.15 (m)
b
b
b
1.59–1.61 (m, Ha)
1.40–1.42 (m, Hb)
1.55–1.58 (m)
a, b2, g
a, b1, g
b, g
a, g
b, d
b, g, d’
b, g, d
g
24.9
19.7
20.5
d
0.98 (d, J¼7.0)
g
d’
0.96 (d, J¼7.0)
g
Ala
CO
a
174.4
49.1
14.4
30.0
PA1-a, a
b, MeN
a
5.75 (q, J¼6.5)
1.34 (d, J¼6.5)
3.00 (s)
b
a, MeN
b2, MeN
b
a
MeN
b
PA1
CO
a
171.4
49.6
23.4
Trp-2, a, b
b, g
5.50 (d, J¼6.5)
b
b
1.97–2.00 (m, Ha)
1.65–1.67 (m, Hb)
1.63–1.65 (m, Ha)
1.46–1.49 (m, Hb)
3.11–3.13 (m, Ha)
2.79–2.82 (m, Hb)
a, b2, g
a, b1, g
b, g2, d
b, g1, d
g, d2
a, g, d
a, b, d
b, g
g
d
21.0
47.1
g, d1
PA2
CO
a
171.1
51.9
Ala-a, MeN, a
b, g
5.30 (br. s)
b
b2, Trp-8a
b
26.3
2.33–2.35 (m, Ha)
1.59–1.61 (m, Hb)
2.12–2.13 (m, Ha)
1.93–1.96 (m, Hb)
3.11–3.13 (m, Ha)
2.80–2.82 (m, Hb)
a, b2, g
a, b1, g
b, g1, d
b, g2, d
g, d2
a, g, d
a, b, d
b, g
g
d
20.5
46.7
g, d1
PA3
CO
a
171.9
47.9
24.4
PA2-a, a
b, g
5.26 (br. s)
b
b2
b
2.33–2.35 (m, Ha)
1.59–1.61 (m, Hb)
2.12–2.13 (m, Ha)
1.93–1.96 (m, Hb)
3.11–3.13 (m, Ha)
2.80–2.82 (m, Hb)
a, b2, g
a, b1, g
b, g1, d
b, g2, d
g, d2
a, g, d
a, b, d
b, g
g
d
19.8
46.4
g, d1
Trp
CO
C(2)
C(3)
173.7
61.5
40.0
2, 3, Leu-a
4.81 (d, J¼8.0)
3
3-Hb
2, 3-Hb
2, 3-Ha
2.48 (d, J¼14.0, Ha)
2.35 (dd, J¼14.0, 8.0, Hb)
2, 3-Ha
2, 3-Hb
2, 8a
4, 2, 8a
5, 7
C(3a)
C(3b)
88.4
129.5

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1571
Table 1 (cont.)
Position
d(C)
d(H)
1H,1H-COSY
HMBC
NOESY
C(4)
C(5)
C(6)
C(7)
C(7a)
C(8a)
123.7
119.2
135.0
110.7
149.2
85.6
7.22 (d, J¼8.0)
5
4
5
7
4, 7
5
4
5
4
6.81 (dd, J¼8.0,2.0)
6.73 (s)
5.49 (s)
3-Ha, MeN
a) Assignments are based on DEPT,1H,1H-COSY, HSQC, and HMBC experiments.
Table 2. Characteristic MS/MS Fragments of NW-G01, and Compounds 1 and 2
Compound [MþH]þ Fragments of amino acidsa)
P1TLP3P2 or P3P2AP1T or AP1TL or LP3P2A or P2AP1 P1TL or P1T or
P1T’VP3P2
P3P2AP1T’
AP1T’V
VP3P2A
P1T’V
P1T’
NW-G01
1
2
757
771
773
672
686
688
658
658
674
533
547
549
409
423
409
310
310
310
448
462
464
349
349
365
a)P1, PA1; P2, PA2; P3, PA3; T, Trp; V,Val; A, Ala; L, Leu; T’, Trp’.
exhibited signals of two sp3 CH groups at d(H) 5.27 (d, J¼8.0) and 2.02–2.04 (m), of
two secondary Me groups at d(H) 0.99 (d, J¼7.0) and 0.97 (d, J¼7.0), which were
assigned to the a, b, l, and l’ H-atoms of valine based on the HSQC and HMBC spectra
of 2, and by comparison with the corresponding chemical-shift and coupling-constant
values of NW-G01. N-Me-alanine (MeAla) and three molecules of piperazic acid could
also be identified on the basis of the HSQC and HMBC spectra of 2, and by comparison
with the corresponding chemical-shift and coupling-constant values of NW-G01 and 1.
The characteristic signals of a CH group at d(H/C) 5.33/85.5, O-bearing quaternary C-
atom at d(C) 88.7, two aromatic CH groups at d(H/C) 6.70/112.4, 6.89/111.0, and four
sp2 quaternary C-atoms at d(C) 121.6, 130.8, 140.9, and 146.8 indicated the presence of a
tryptophan derivative (Trp). Careful comparison of the DEPT spectra of 2 with those
of NW-G01 indicated that there were two aromatic CH groups and four sp2 quaternary
C-atoms in 2, whereas there were three aromatic CH groups and three sp2 quaternary
C-atoms in NW-G01, implying that one H-atom of aromatic CH group in 2 was
replaced with a substituent. The molecular formula of 2 and NW-G01 were
C35H49ClN10O8 and C35H49ClN10O7, respectively, indicating that 2 is the hydroxylated
derivative of NW-G01. The cross peaks between HꢀC(4), and C(3a), C(3b), C(5),
C(6), and C(7a), and between HꢀC(7), and C(3b), C(6), and C(7a) were observed in
the spectrum of HMBC, revealing that the free OH group was at C(5) of Trp. Thus, the
sixth amino acid was deduced as 6-chloro-1,2,3,3a,8,8a-hexahydro-3a,5-dihydroxy-
pyrrolo[2,3-b]indole-2-carboxylic acid. The amino acids of 2 were established as N-
methylalanine (MeAla), valine (Val), 6-chloro-1,2,3,3a,8,8a-hexahydro-3a,5-dihy-
droxy-pyrrolo[2,3-b]indole-2-carboxylic acid (Trp’), and three molecules of piperazic
acid (PA).

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme 1. Fragmentation Pathway of NW-G08 (1) in Positive-Ion-Mode MS/MS
The sequence linkage of the amino acids in 2 could be readily determined by
comparison of the characteristic MS/MS fragmention peaks in the spectra of NW-G01
as described above (Table 2, Scheme 2, and Fig. 2). The common fragmention peaks of
Val-PA3-PA2-Ala (m/z 409) and PA2-MeAla-PA1 (m/z 310) could be observed in the
MS/MS spectra of 2 and NW-G01. Other fragmention peaks at m/z 688, 674, 549, 464,
and 365 were observed in the spectrum of 2; each fragment was 16 Da higher than the
corresponding ions revealed in the spectrum of NW-G01, and they were assigned to
PA1-Trp’-Val-PA3-PA2, PA3-PA2-Ala-PA1-Trp’, MeAla-PA1-Trp’-Val, PA1-Trp’-Val,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1573
Fig. 2. CID Spectra of a) NW-G01, b) NW-G08 (1), and c) NW-G09 (2) in positive-ion-mode MS/MS
PA1-Trp’, respectively. Obviously, the sequence linkage of the amino acids in 2 was
identical with that in NW-G01, except Trp in 2 was hydroxylated.
The absolute configurations of the amino acids were determined by the Marfeyꢂs
method as described above, as well as the NOESY spectrum. The amino acids in 2 were
thus identified as d-valine, N-Me-d-alanine, (3R)-piperazic acid (PA1), (3R)-piperazic

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table 3. The 1H- and 13C-NMR Chemical Shifts of NW-G09a) (2; CD3OD, 1H: 500 and 13C: 125 MHz).
d in ppm, J in Hz.
Position
d(C)
d(H)
1H,1H-COSY
HMBC
NOESY
Val
CO
a
174.7
54.9
29.9
17.4
18.9
a, PA3-a
b, g, g’
a, g, g’
a, b, g’
a, b, g
5.27 (d, J¼8.0)
2.02–2.04 (m)
0.99 (d, J¼7.0)
0.97 (d, J¼7.0)
b, g, g’
a, g, g’
a, b, g’
a, b, g
b, g, g’
b
g
g’
Ala
CO
a
174.2
48.5
13.8
29.7
PA1-a, a
b, MeN
5.75 (q, J¼6.5)
1.34 (d, J¼6.5)
2.96 (s)
b
a, MeN
b2, MeN
b
a
a
MeN
b
PA1
CO
a
171.6
49.9
23.4
Trp-2,a, b
b, g
5.51 (d, J¼6.5)
b
b
2.05–2.08 (m, Ha)
1.76–1.80 (m, Hb)
1.76–1.80 (m, Ha)
1.56–1.60 (m, Hb)
3.14–3.17 (m, Ha)
2.80–2.82 (m, Hb)
a, b2, g
a, b1, g
b, g2, d
b, g1, d
g, d2
a, g, d
a, b, d
b, g
g
d
21.2
47.3
g, d1
PA2
CO
a
171.2
52.3
25.1
Ala-a,MeN, a
b, g
5.38 (d, J¼4.5)
b
b2, Trp-8a
b
2.30–2.33 (m, Ha)
1.54–1.57 (m, Hb)
2.13–2.15 (m, Ha)
1.95–2.00 (m, Hb)
3.14–3.17 (m, Ha)
2.80–2.82 (m, Hb)
a, b2, g
a, b1, g
b, g1, d
b, g2, d
g, d2
a, g, d
a, b, d
b, g
g
d
19.9
46.3
g, d1
PA3
CO
a
172.3
47.3
24.5
PA2-a, a
b, g
5.22 (br. s)
b
b2
b
2.30–2.33 (m)
1.54–1.57 (m)
2.13–2.15 (m)
1.95–2.00 (m)
3.14–3.17 (m)
2.80–2.82 (m)
a, b2, g
a, b1, g
b, g1, d
b, g2, d
g, d2
a, g, d
a, b, d
b, g
g
d
19.8
46.4
g, d1
Trp’
CO
C(2)
C(3)
173.4
61.6
39.6
2, 3, Val-a
3
4.87 (d, J¼8.0)
3
3-Hb
2, 3-Hb
2, 3-Ha
2.54 (d, J¼13.5, Ha)
2.29 (dd, J¼13.5, 8.0, Hb)
2, 3-Ha
2, 3-Hb
2, 8a
4, 2, 8a
4, 7
C(3a)
C(3b)
C(4)
88.7
130.8
111.0
140.9
6.89 (s)
C(5)
4

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1575
Table 3 (cont.)
Position
d(C)
d(H)
1H,1H-COSY
HMBC
4, 7
NOESY
C(6)
C(7)
C(7a)
C(8a)
121.6
112.4
146.8
85.5
6.70 (s)
4, 7
5.33 (s)
3-Ha,MeN
a
1
) Assignments are based on DEPT, H,1H-COSY, HSQC and HMBC experiments.
Scheme 2. Fragmentation Pathway of NW-G09 (2) in Positive-Ion-Mode MS/MS

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
acid (PA2), (3S)-piperazic acid (PA3), and (2S,3aR,8aS)-6-chloro-1,2,3,3a,8,8a-hexa-
hydro-3a,5-dihydroxy-pyrrolo-[2,3-b]indole-2-carboxylic acid (Fig. 1).
Antibacterial Activity. The antibacterial activities of 1 and 2 against several strains
of bacteria were evaluated by micro-broth dilution method as described in [16]. The
results indicated that 1 and 2 could effectively inhibit Gram-positive bacteria, such as
Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, and methicillin-resistant S.
aureus (MRSA), whereas they were inactive to Gram-negative bacteria (Table 4). The
minimum inhibition concentrations (MICs) of 1 against B. cereus, B. subtilis, S. aureus,
and MRSA were 6.25, 6.25, 1.56, and 1.56 mg/ml, respectively, of 2 25.0, 25.0, 12.5, and
12.5 mg/ml, respectively, of NW-G01 12.5, 12.5, 6.25, and 6.25 mg/ml, respectively, and of
ampicillin sodium were 6.25, 1.56, 6.25, and >100.0 mg/ml, respectively. Compound 1
showed stronger antibacterial activity than NW-G01; because the only difference
between their structures was that valine in NW-G01 was replaced by leucine in 1, it
indicated that leucine was more beneficial to the activity than valine. The activity of 2
was slightly lower than that of NW-G01, revealing that the introduction of a free OH
group at C(5) of Trp’ was not contributing to the activity. The structureꢀactivity
relationship of these three antibiotics could be explained by the hydrophobic character
of the molecules. Compared with NW-G01, the incorporation of leucine into 1
increased the hydrophobic property, whereas the hydroxylation of Trp decreased the
hydrophobic nature of 2, evidencing that high hydrophobic nature was benefical for the
activity. We assume that the higher hydrophobicity could promote the penetration of
antibiotics through biomembrane, or increase the binding affinity with receptor. It is
worth noting that these cyclic peptides showed the same sensitivity to S. aureus and
MRSA. MRSA is of particular clinical importance, because it is not only predictably
cross-resistant to all currently licensed b-lactam antibiotics, but it is also typically
resistant to other multiple antibiotics. The results indicated that these cyclic peptides
may have different mechanisms of action, so they could be potential candidates for the
development of novel drugs.
Table 4. MIC Values of Compounds 1 and 2 against Several Strains of Bacteria
Tested bacteria
MIC [mg/ml]
1
2
NW-G01
Ampicillin
Bacillus cereus ( CGMCC 1.1846)
Bacillus subtilis (CGMCC 1.88)
Staphylococcus aureus (CGMCC 1.89)
Escherichia coil (CGMCC 1.1574)
Pseudomonas aeruginosa (CGMCC 1.2031)
MRSA
6.25
6.25
1.56
25
25
12.5
12.5
12.5
6.25
6.25
1.56
6.25
>100
>100
1.56
>100
>100
12.5
>100
>100
6.25
3.13
>100
>100
Experimental Part
General. M.p.: WPR apparatus; uncorrected (Shanghai Jingke Co.).Optical rotations: Horiba SEPA
300 polarimeter. UV Spectra: Shimadzu UV-2401A spectrometer. IR Spectra: Nicolet FT-IR 750
spectrometer. 1H- and13C-NMR, DEPT, 1H,1H-COSY, HSQC, HMBC, and NOESY spectra: Bruker
Avance III-500 NMR spectrometer (Bruker Daltonics Inc.), d in ppm, with TMS as internal standard,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1577
and J in Hz (1H: 500, 13C: 125 MHz). ESI-MS/MS and HR-ESI-MS: Finnigan LCQ Advantage ion-trap
mass spectrometer (Thermo Fisher Co.) and an APEX II FT-ICR mass spectrometer (Bruker Daltonics
Inc.).
Microorganism and Fermentation. The producing strain S. alboflavus 313 was isolated from a soil
sample collected in Qinling Mountain, Shaanxi Province, P. R. China, and identified by its morphology,
physiology, biochemistry, and 16S rRNA gene sequence. The voucher specimen of this streptomycete was
deposited with the Institute of Pesticide Science, Northwest A & F University, P. R. China.
The spores of S. alboflavus 313 grown on Gauseꢂs No. 1 agar were used to inoculate in a 250-ml flask
containing 50 ml of a sterile seed medium consisting of glucose (0.8%), soluble starch (0.8%), beef
extract (0.6%), peptone (1.0%), and NaCl (0.5%); pH 7.2. The flask was shaken on a shaker at 180 rpm.
for 24 h at 288. Ten ml of the seed culture were transferred to 250-ml flasks containing 50 ml of a sterile
producing medium consisting of glucose (3.0%), millet steep liquor (1.0%), peptone (1.5%), NaCl
(0.5%), and CaCO3 (0.5%); pH 7.2. Fermentation was carried out at 180 rpm. for 5 d at 288 on a rotary
shaker.
Extraction and Isolation. The culture of 200 l was filtered through cheese cloth to separate the
medium and culture liquid. The filtrate was absorbed onto HPD400 macroporous resin (Baoen Co., Ltd.,
Hebei, P. R. China), and then eluted with H2O and MeOH in sequence. The MeOH fraction was
evaporated in vacuum. The concentrate was subjected to column chromatography (CC; SiO2 (600 g, 200–
300 mesh, Qingdao Marine Chemical Co., Ltd., Shandong, P. R. China); AcOEt/MeOH 75 :25 (v/v)).
The antimicrobial fraction was concentrated in vacuum and then subjected to flash chromatography (FC;
ODS-AP (50 mm, Daiso Co., Ltd., Osaka, Japan); eluted with the mixture of MeOH/H2O at the ratio of
60 :40 (v/v)). The active fractions were concentrated in vacuum and further purified on a Shimadzu 6AD
HPLC apparatus (Shimadzu Co., Ltd., Tokyo, Japan) equipped with a column of Hypersil ODS-BP (20ꢁ
250 mm, 10 mm, flow rate 8.0 ml/min) to afford two novel cyclic hexapeptides, NW-G08 (1; 8.0 mg) and
NW-G09 (2; 7.6 mg).
Marfeyꢀs Analysis: Approximately 1.0 mg of tested sample was hydrolyzed with 100 ml of 6n HCl at
1108 for 24 h. The acid hydrolysate was evaporated to dryness and dissolved in 100 ml of 0.1n HCl. To
50 ml of the acidic soln., 80 ml of 1n NaHCO3 and 400 mg of 1-fluoro-2,4-dinitrophenyl-5-l-alanineamide
(FDAA; Marfeyꢂs reagent) with 40 ml of acetone was added, the mixture was then heated at 508 for 1 h.
After cooling the mixture to r.t., it was neutralized with 40 ml of 1n HCl and evaporated to dryness. The
residue was dissolved in 40 ml of MeCN, and the FDAA derivative soln. was analyzed by RP-HPLC. The
analysis of the derivatives was performed on a Hypersil ODS-BP (250ꢁ4.6 mm, 5 mm) column
maintained at 308 with UV detector at 340 nm. The mixture MeCN/50 mmEt3N phosphate buffer
(pH 5.0) 40 :60 (v/v) was used as mobile phase, and the flow rate was 1.0 ml per min. The FDAA
derivatives of the acid hydrolysate were identified by comparing the retention times with FDAA-
derivatized standard amino acids.
Antibacterial Assay. The standard bacterial strains Bacillus cereus (1.1846), Bacillus subtilis (1.88),
Staphylococcus aureus (1.89), Escherichia coil (1.1574), and Pseudomonas aeruginosa (1.2031) were
obtained from China General Microbiological Culture Collection Center. A clinical isolate of MRSA was
obtained from Nanjing Medical University. Ampicillin sodium (Sigma, Shanghai, P. R. China) was used
as positive control. The antibacterial activities of compounds against six strains of bacteria were
evaluated by the micro-broth dilution method in 96-well plates [16]. The inoculum was prepared by
suspending several colonies from an overnight culture of tested bacteria from 0.5% sheep blood agar
media in MꢁllerꢀHinton broth (Hangzhou Microbial Reagent Co. Ltd., Zhejiang, P. R. China) and
adjusting to a 0.5 McFarland standard (ca. 1.5 ꢁ108 colony-forming units per ml). A further dilution of
1:200 was performed by placing 0.25 ml of the adjusted suspension into 49.75 ml of MꢁllerꢀHinton broth.
The tested compounds were first dissolved in DMSO at the concentration of 10 mg/ml, and it was diluted
ten-fold with sterile H2O to give the stock soln. Twofold serial dilutions of the tested compounds were
prepared in MꢁllerꢀHinton broth. Then, the dilutions and inoculated suspension of the bacteria were
delivered to wells of a 96-well plate at the ratio of 1:1. The final concentration of inoculum in each well
was 3.7ꢁ105 colony-forming units per ml. After incubation for 24 h at 308, the MICs were examined.
Experiments were run in triplicate, and standard ampicillin sodium was used as the positive control.

1578
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
NW-G08 (¼(4aS,10aR,13S,19aR,21aS,26bR,27aS,30S)-24-Chloro-2,3,4,4a,8,9,10,10a,12,13,17,18,19,
19a,22,26b,27,27a,29,30-icosahydro-26b-hydroxy-12,13-dimethyl-30-(2-methylpropyl)-7H,21aH-tripyrida-
zino[1’’,6’’:10’,11’;1’’’,6’’’:16’,17’;1’’’’,6’’’’:7’,8’][1,4,7,10,13,16]hexaazacyclooctadecino[1’,2’:1,5]pyrrolo[2,3-
b]indole-5,11,14,20,28,31(1H,16H)-hexone; 1). Amorphous white powder. M.p. 192.2–194.08. [a]D ¼
ꢀ44.5 (c¼0.25, MeOH). UV (MeOH): 205, 235. IR (KBr): 3397, 2956, 2933, 1660, 1415, 1336, 1249,
1072, 752.1H- and 13C-NMR: see Table 1. HR-ESI-MS (pos.): 771.3716 ([MþH]þ , C36H52ClN10Oþ7 ; calc.
771.3709).
NW-G09 (¼(4aS,10aR,13S,19aR,21aS,26bR,27aS,30R)-24-Chloro-2,3,4,4a,8,9,10,10a,12,13,17,18,19,
19a,22,26b,27,27a,29,30-icosahydro-25,26b-dihydroxy-12,13-dimethyl-30-(propan-2-yl)-7H,21aH-tripyri-
dazino[1’’,6’’:10’,11’;1’’’,6’’’:16’,17’;1’’’’,6’’’’:7’,8’][1,4,7,10,13,16]hexaazacyclooctadecino[1’,2’:1,5]pyrro-
lo[2,3-b]indole-5,11,14,20,28,31(1H,16H)-hexone; 2). Amorphous white powder. M.p. 225.0–226.08.
[a]D ¼ ꢀ28.0 (c¼0.25, MeOH). UV (MeOH): 205, 240. IR (KBr): 3427, 2960, 2931, 1636, 1443, 1407,
1335, 1248, 1155, 1084, 642.1H- and 13C-NMR: see Table 3. HR-ESI-MS (pos.): 773.3511 ([MþH]þ
C35H50ClN10Oþ8 ; calc. 773.3502).
,
This study was supported part by the grant of the National Key Basic Research Program (973
Program, 2010CB126100) from Science and Technology Ministry of China, the National Natural Science
Foundation of China (No.30971935), Postdoctoral Science Foundation of China (20100471644 and
201104683), and Program for New Century Excellent Talents in University from Education Ministry of
China.
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Received November 5, 2011