Two piperazic acid-containing cyclic hexapeptides from Streptomyces alboflavus 313

Article abstract of DOI:10.1007/s00726-012-1303-1

Two novel cyclic hexapeptides, designated NW-G10 (1) and NW-G11 (2), were isolated from the fermentation broth of Streptomyces alboflavus 313. Their relative structures were elucidated on the basis of extensive spectroscopic analysis, and the absolute con

Full text of DOI:10.1007/s00726-012-1303-1

Amino Acids
DOI 10.1007/s00726-012-1303-1
ORIGINAL ARTICLE
Two piperazic acid-containing cyclic hexapeptides
from Streptomyces alboflavus 313
Shaopeng Wei
Zhiqin Ji
•
Lixia Fan
•
Wenjun Wu
•
Received: 31 January 2012 / Accepted: 17 April 2012
Ó Springer-Verlag 2012
Abstract Two novel cyclic hexapeptides, designated
NW-G10 (1) and NW-G11 (2), were isolated from the
fermentation broth of Streptomyces alboflavus 313. Their
relative structures were elucidated on the basis of extensive
spectroscopic analysis, and the absolute configurations of
several constituent amino acids were determined by Mar-
fey’s method. NW-G10 (1) and NW-G11 (2) exhibited
significant activity against Gram-positive bacteria, such as
Bacillus cereus, Bacillus subtilis and Staphylococcus aur-
eus, including methicillin-resistant Staphylococcus aureus
Introduction
Since monamycins were first isolated from Streptomyces
jamaicensis in 1959 (Hassall and Magnus 1959), a series of
natural peptides which contain the moiety of piperazine-3-
carboxylic acid have been found among the metabolites
of microorganisms, such as antrimycin (Shimada et al.
1981), azinothricin (Maehr et al. 1986; Smitka et al. 1988;
Sakai et al. 1997; Maskey et al. 2006), himastatin (Lam
et al. 1990; Leet et al. 1990, 1996), chloptosin (Umezawa
et al. 2000), luzopeptins (Konishi et al. 1981), lydiamycin
(
MRSA), but they are not active against gram negatives.
(Huang et al. 2006), and piperazimycin (Miller et al. 2007).
Keywords Structure elucidation ꢀ Cyclic hexapeptide ꢀ
These natural products exhibit impressive antimicrobial,
antitumour, and anti-HIV activities. Attracted by their
intriguing chemical structures and a medicinally relevant
biological activity profile, many piperazic acid-contain-
ing natural products have been successfully synthesized
in recent years (Kamenecka and Danishefsky 1998a, b;
Yu et al. 2010; Oelke et al. 2010, 2011; Setsuya et al.
Antibacterial activity
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-012-1303-1) contains supplementary
material, which is available to authorized users.
2
011).
In our previous investigation of natural products from
S. Wei ꢀ Z. Ji
College of Plant Protection, Northwest A&F University,
Yangling 712100, Shaanxi, People’s Republic of China
microorganisms for drug discovery, two piperazic acid-
containing cyclic hexapeptides, NW-G01 and NW-G03,
were discovered in the fermentation broth of Streptomyces
alboflavus 313 (Guo et al. 2009, 2010, 2011). These natural
cyclic hexapeptides could selectively inhibit Gram-positive
bacteria, such as Bacillus cereus, Bacillus subtilis and
Staphylococcus aureus, including methicillin-resistant
Staphylococcus aureus (MRSA), whereas they were not
active against Gram-negative bacteria. In our continuous
investigation, two novel analogs of NW-G01, NW-G10 (1)
and NW-G11 (2) were isolated as trace constituents from
the same microorganism by large-scale fermentation.
Herein, we report the structure elucidation and antibacterial
activity of these two novel cyclic hexapeptides.
S. Wei ꢀ L. Fan ꢀ W. Wu
Shaanxi Province Key Laboratory of Research and Development
on Botanical Pesticide, Northwest A&F University,
Yangling 712100, Shaanxi, People’s Republic of China
W. Wu ꢀ Z. Ji
State Key Laboratory of Crop Stress Biology in Arid Areas,
Northwest A&F University, Yangling 712100, Shaanxi,
People’s Republic of China
Z. Ji (&)
Institute of Pesticide Science, Northwest A&F University,
Xinong 61#, Yangling 712100, Shaanxi,
People’s Republic of China
e-mail: jizhiqin@nwsuaf.edu.cn
123

S. Wei et al.
Experimental procedures
and then subjected to an ODS-AP flash chromatography
(50 lm, Daiso Co. Ltd., Osaka, Japan), eluted with the mix-
Materials
ture of MeOH–water 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
(20 9 250 mm, 10 lm, flow rate 8.0 mL/min) to afford two
novel cyclic hexapeptides, NW-G10 (1, 5.8 mg) and NW-
G11 (2, 5.2 mg).
Melting points were measured on a WPR apparatus and
are uncorrected (Shanghai Jingke Co.). IR spectra were
recorded on a Nicolet FT-IR 750 spectrometer. UV spectra
were obtained using a Shimadzu UV-2401A spectrometer.
Optical rotations were measured with a Horiba SEPA300
polarimeter. ESI–MS/MS and HR-ESI–MS spectra were
obtained on a Finnigan LCQ Advantage ion-trap mass spec-
trometer (Thermo Fisher Co.) and an APEX II FT-ICR mass
Acid hydrolysis and Marfey’s analysis
1
13
spectrometer (Bruker Daltonics Inc.). H, C NMR, DEPT,
HSQC, HMBC and NOESY spectra were obtained on Bruker
Avance III-500 NMR spectrometer (Bruker Daltonics Inc.), with
Approximately 1.0 mg of 1 or 2 was hydrolyzed with
100 lL of 6 N HCl at 110 °C for 24 h. The acid hydro-
lysate was evaporated to dryness and dissolved in 100 lL
of distilled water. To 50 lL of the solution, 80 lL of 1 N
1
13
TMS as internal standard ( H at 500 MHz, C at 125 MHz,
respectively). The LC–MS experiments were performed on the
Finnigan LCQ Advantage Max ion-trap mass spectrometer
NaHCO and 100 lL of 1 % acetone solution of L-FDAA
3
(J&K chemical Co. Beijing, China) were added, the mix-
ture was heated at 50 °C for 1 h. After cooling to room
temperature, the reaction mixture was neutralized with
40 lL 1 N HCl, and evaporated to dryness (Fujii et al.
(Thermo Fisher Co.) coupled with a surveyor HPLC.
Methods
1997). The residue was then dissolved in 0.2 mL acetoni-
Microorganism and fermentation
trile and injected for HPLC.
TheproducingstrainS. alboflavus313 wasisolatedfromasoil
sample collected in Qinling Mountain, Shaanxi Province,
China, and identified by its morphology, physiology, bio-
chemistry and 16S rRNA gene sequence. The voucher spec-
imen of this streptomycete was deposited at the Institute of
Pesticide Science, Northwest A&F University, China.
Chromatography
The HPLC instrument used for the analysis was a Finnigan
Surveyor equipped with a photodiode array detector.
Separations were carried out on a Hypersil Gold column
(150 9 2.1 mm) (Thermo, San Jose, USA). Acetonitrile–
0.1 % acetic acid was used as the mobile phase under a
linear gradient elution mode (acetonitrile, 15–70 %,
40 min). The flow rate was 0.2 mL/min with UV detection
at 340 and 200–600 nm by photodiode array detection.
The spores of S. alboflavus 313 grown on Gause’s No. 1
agar were used to inoculate into a 250-mL flask containing
5
0
1
0 mL of a sterile seed medium consisting of glucose
.8 %, soluble starch 0.8 %, beef extract 0.6 %, peptone
.0 %, and NaCl 0.5 %, pH 7.2. The flask was shaken on a
shaker at 180 rpm for 24 h at 28 °C. Ten milliliters of the
seed culture was transferred to 250 mL flasks containing
LC–MS
5
3
0
0 mL of a sterile producing medium consisting of glucose
.0 %, millet steep liquor 1.0 %, peptone 1.5 %, NaCl
The LC–MS experiments were performed on the Finnigan
LCQ Advantage Max ion-trap mass spectrometer (Thermo
Fisher Co.) coupled with a surveyor HPLC. The sample
solution from the outlet of the DAD detector was injected
to the ESI interface directly. The ESI voltage was 5.4 kV
with the sheath and auxiliary gas set at 60 and 5 arbitrary
units, respectively, and the capillary was heated to 250 °C.
A mass range of m/z 200–850 was covered, and data were
collected in positive mode.
.5 %, and CaCO 0.5 %, pH 7.2. Fermentation was car-
3
ried out at 180 rpm for 5 days at 28 °C on a rotary shaker.
Extraction and isolation
The culture of 200 L was filtered with cheesecloth to separate
the medium and culture liquid. The filtrate was absorbed onto
HPD400 macroporous resin (Baoen Co. Ltd., Hebei, China),
and then eluted with water and methanol in sequence. The
methanolfractionwasevaporatedinvacuum. Theconcentrate
was subjected to a silica gel column (600 g, 200–300 mesh,
Qingdao Marine Chemical Co. Ltd., Shandong, China) and
eluted with the mixture of EtOAc–MeOH at the ratio of 75:25
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 the China General
(
v/v). The antimicrobial fraction was concentrated in vacuum,
1
23

Two piperazic acid-containing cyclic hexapeptides
Microbiological Culture Collection Center. A clinical
isolate of MRSA was obtained from Nanjing Medical
University, China. Ampicillin sodium (Sigma, Shanghai,
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. The inoculum was prepared by suspending several
colonies from an overnight culture of tested bacteria from
resin (5 kg), and then eluted with water and methanol in
sequence. The concentrated methanol fraction was subjected
to a column of silica gel and eluted with the mixture of
EtOAc–MeOH, the active fraction was subsequently purified
by reverse phase (RP) flash chromatography and RP-HPLC
to afford two novel cyclic hexapeptides, NW-G10 (1) and
NW-G11 (2).
0
.5 % sheep blood agar media in M u¨ ller-Hinton broth
Hangzhou Microbial Reagent Co. Ltd., Zhejiang, China)
and adjusting to a 0.5 McFarland standard (approximately
Structure elucidation
(
NW-G10 (1) was obtained as a white powder. Its molecular
formula was established as C H ClN O by HR-ESI–
8
1
.5 9 10 colony-forming units per mL). A further dilution
3
?
5
45
10 8
of 1:200 was made by placing 0.25 mL of the adjusted
suspension into 49.75 mL of M u¨ ller-Hinton broth. The
tested compounds were firstly dissolved in DMSO at the
concentration of 10 mg/mL, and it was diluted ten folds
with sterile water to give the stock solution. Twofold serial
dilutions of the tested compounds were prepared in M u¨ ller-
Hinton 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
MS (m/z 769.3182, [M ? H] ; cacld for C H ClN O ,
35 46 10 8
769.3189). The presence of chlorine was suggested by the
isotope abundance peaks in the MS spectrum. Like the IR
spectrum with typical absorption bands of amides carbon-
-
1
yls at 1,658 cm , the NMR data showed typical charac-
teristics of peptides, for example resonances for amide
1
3
carbonyls in the C NMR spectrum and for a proton in the
H NMR spectrum (Table 1). The C NMR and DEPT
1
13
spectra (Table 1) of 1 showed the presence of 35 carbon
signals, which were recognized as three methyls, one
5
well was 3.7 9 10 colony-forming units per mL. After
3
oxygenated methyl, seven methylenes, eight sp methines,
incubation for 24 h at 30 °C, the MICs were examined.
Experiments were repeated triplicate and standard ampi-
cillin sodium was used as the positive control.
3
2
one oxygenated sp quaternary C-atom, five sp methines,
2
four sp quaternary C-atoms, and six amide carbonyl car-
1
13
bons. H and C chemical shift assignments were made by
standard 1D and 2D NMR techniques, such as DEPT,
HSQC, and HMBC.
Chemistry
1
NW-G10 (1)
The H NMR spectrum of 1 showed the presence of
3
two sp methines at d 5.34(d, J = 5.5) and d 2.25(m), two
Amorphous white powder; m.p. 206 °C(dec.); [a] -95.8
c 0.25, MeOH); UV (MeOH): kmax 210, 240, 300 nm; IR
secondary methyl groups at d 0.99(d, J = 7.0) and d
1.02(d, J = 7.0), they were assigned to the a, b, k and k
D
0
(
(
KBr) mmax 3,426, 2,931, 1,750, 1,657, 1,414, 1,339, 1,234,
protons of valine based on the HSQC and HMBC spectra of
1, and by comparison with the corresponding chemical
shifts and coupling constants of NW-G01 and NW-G03. At
the same time, the cross peak between H-a at d 5.34 and
the carbonyl carbon at d 173.4 was observed on the HMBC
spectrum. Thus, the valine (Val) moiety was elucidated.
Alanine (Ala), and two molecules of piperazic acid (PA)
could also be determined based on the 1D, 2D NMR
spectra, and by comparison to the data of NW-G01.
-
1
1
13
,162 and 648 cm ; for H and C NMR data, see
1
?
Table 1; positive HR-ESI–MS m/z 769.3182 [M ? H]
cacld for C H ClN O , 769.3189).
(
3
5
46
10 8
NW-G11 (2)
Amorphous white powder; m.p. 195 °C(dec.); [a] -49.8
(
D
c 0.25, MeOH); UV (MeOH): kmax 220, 230, 305 nm; IR
(
KBr) mmax 3,425, 3,298, 2,935, 1,639, 1,441, 1,409, 1,255,
The signals observed at d 4.83(t, J = 8.0), d 2.36(m) and
d 2.51(m), d 7.23(d, J = 8.0), d 6.83(dd, J = 8.0, 2.0), d
6.73(d, J = 2.0), d 5.68(s) were assigned to the H-2, H-3,
H-4, H-5, H-7, and H-8a protons of the chlorinated
pyrroloindoline moiety (Trp derivative) based on the HSQC
and HMBC spectra of 1, and by comparison with the
corresponding chemical shifts and coupling constants of
NW-G01 and NW-G03. The long-range coupling between
H-a at d 4.83 and the carbonyl carbon at d 172.8 was
observed on the HMBC spectrum. Thus, the 6-chloro-3a-
hydroxy-1,2,3,3a,8,8a-hexhydropyrrolo-[2,3-b]indole-2-
carboxylic acid moiety, a chlorinated pyrroloindoline
-
,102 and 924 cm ; for H and C NMR data, see
1
1
13
1
?
Table 1; positive HR-ESI–MS m/z 781.3193 [M ? H]
cacld for C H ClN O , 781.3189).
(
3
6
46
10 8
Results and discussion
Extraction and isolation
Two hundred liters of filtered fermentation broth of
S. alboflavus 313was passedthrough a column ofmacroporous
123

S. Wei et al.
1
13
3
Table 1 NMR data of NW-G10 (1) and NW-G11(2) ( H measured at 500 MHz, C measured at 125 MHz, in CD OD)
Position
NW-G10 (1)
dC (ppm)
NW-G11 (2)
dC (ppm)
dH (ppm, J = Hz)
dH (ppm, J = Hz)
Valine (Val)
CO
173.4
55.6
29.7
16.3
18.4
172.6
54.6
29.4
17.5
18.5
C
C
C
C
a
b
c
5.34(d, J = 5.5)
2.25(m)
5.22(d, J = 5.5)
2.07, 1.38(m)
0.99(d, J = 7.0)
1.02(d, J = 7.0)
0.92(d, J = 7.0)
1.02(d, J = 7.0)
0
c
Alanine or N-methylalanine (Ala)
CO
175.4
44.8
16.5
–
171.8
48.1
13.1
29.1
C
C
a
5.39(q, J = 7.0)
1.35(d, J = 7.0)
–
5.72(q, J = 7.0)
1.40(d, J = 7.0)
3.10(s)
b
N-CH
3
Piperazic acid (PA
CO
1
)
173.1
52.4
24.3
20.1
46.4
171.9
54.9
24.9
19.0
46.2
C
C
C
C
a
b
c
5.00(m)
5.50(brs)
2.25(m), 2.06(m)
1.93(m), 1.62(m)
3.28(m), 2.96(m)
2.32(m), 1.75(m)
2.34(m), 2.24(m)
d
3.17(dd, J = 12.5, 3.0), 2.91(m)
5
2
-Methoxy-2,3-dihydropyridazine-3-carboxylic acid (PA )
CO
171.8
52.7
170.0
53.1
C
C
C
C
a
b
c
6.30(d, J = 6.0)
6.36(d, J = 6.0)
92.9
5.30(dd, J = 6.0, 2.5)
92.7
5.40(dd, J = 6.0, 2.5)
146.4
136.0
54.2
146.4
135.6
54.1
d
6.86(d, J = 2.5)
6.79(d, J = 2.5)
OCH
3
3.67(s)
3.63(s)
3
Piperazic acid or 2,3,4,5-tetrahydropyridazine-3-carboxylic acid (PA )
CO
171.0
51.5
24.2
19.3
45.9
172.1
50.6
C
C
C
C
a
b
c
4.71(m)
5.18(m)
2.25(m), 2.06(m)
1.93(m), 1.62(m)
3.16(m), 3.00(m)
20.1
2.01(m), 1.60(m)
2.11(m)
17.0
d
144.4
7.13(brs)
6
-Chloro-3a-hydroxy-1,2,3,3a,8,8a-hexhydropyrrolo-[2,3-b]indole-2-carboxylic acid (Trp)
CO
172.8
61.2
171.0
61.7
C
C
C
C
C
C
C
C
C
C
2
4.83(t, J = 8.0)
4.31(t, J = 8.0)
3
40.7
2.51(m), 2.36(m)
38.0
2.72(m), 2.58(m)
3a
3b
4
88.7
86.3
130.0
123.6
119.4
135.0
111.0
149.4
85.9
128.6
124.3
119.2
135.5
111.4
149.9
83.0
7.23(d, J = 8.0)
7.25(d, J = 8.0)
5
6.83(dd, J = 8.0, 2.0)
6.81(dd, J = 8.0, 2.0)
6
7
6.73(d, J = 2.0)
6.75(d, J = 2.0)
7a
8a
5.68(s)
5.30(s)
derivative considered to be derived from tryptophan (Trp),
was elucidated.
were assigned to methine, methine, and quaternary carbon,
respectively, based on the DEPT spectrum. It indicated the
presence of C=C and C=N in the amino acid residue, and in
which one hydrogen atom of the unsaturated carbon was
1
3
In the C NMR spectrum of 1, three unsaturated carbon
signals were observed at d 92.9, d 136.0, and d 146.4, they
1
23

Two piperazic acid-containing cyclic hexapeptides
1
replaced. In H- H-COSY spectrum, there was strong
1
G01 were prepared and analyzed using LC/DAD/MS, the
absolute structure of this peptide has been established
determinately by single-crystal X-ray diffraction and
3
2
coupling signals between sp methine (d 6.30) and sp
methine (d 5.30), the long-range coupling from the sp
3
?
methine proton (d 6.30) to carbonyl (d 171.8) and unsat-
urated carbons (d 92.9, d 146.4) were also observed in the
HMBC spectrum, it diagnosed the presence of a 2,3-
dihydropyridazine-3-carboxylic acid moiety. The signal
observed at dH/C 3.67(s)/54.2 was assigned to the oxy-
genated methyl group based on the spectra of DEPT and
HSQC, and a cross peak between the protons of methyl and
the quaternary carbon at d 146.4 was observed in the
HMBC spectrum. Thus, the last amino acid was deter-
mined as 5-methoxy-2,3-dihydropyridazine-3-carboxylic
acid.
Marfey’s method. Two PA-L-FDAA peaks ([M ? H] , m/z
383) were observed in the selected ion chromatogram (SIC),
and the ratio of their areas was 1:2, it was in accord with the
structure of NW-G01, into which two molecules of R-PA
and one S-PA were incorporated. Because only one peak
was observed in the mass chromatogram of NW-G10 (1),
piperazic acids in this compound could be determined as
R configuration by comparison of its retention time with
that of R-PA-L-FDAA. Unfortunately, FDAA derivatives of
the unsaturated piperazic acid (PA ) and the chlorinated Trp
2
were not observed in Marfey’s experiment, it may due
to they were unstable in strong acidic conditions. The ste-
reochemical configuration of these two rare amino acids
will be assigned in further investigation by synthesis route
(Fig. 1).
The sequence linkage of the amino acids in 1 could be
readily determined by MS/MS experiment (Figs. 2, 3). The
product ions observed in the MS/MS spectrum were
derived mostly from the cleavage of peptide bonds, and the
amino acid content of characteristic fragments could be
deduced based on the mass values of amino acid residues.
Fragment ions observed at m/z 251, 349, 448, 519, 631
were deduced as the moieties of PA -PA (P P ), PA -Trp
NW-G11 (2) was obtained as a white powder. Its
molecular formula was established as C H ClN O by
3
6
45
10 8
?
HR-ESI–MS (m/z 781.3197, [M ? H] ; cacld for
C H ClN O8, 781.3189). The presence of chlorine was
36 46 10
2
3
2
3
1
(
P T), PA -Trp-Val (P TV), Ala-PA -Trp-Val (AP TV)
1
suggested by the isotope abundance peaks in the MS
1
1
1
1
and Ala-PA -Trp-Val-PA (AP TVP ), it revealed most
1
spectrum. Like the IR spectrum with typical absorption
3
1
3
-
bands of amides carbonyls at 1,639 cm , the NMR data
1
information of the sequence linkage of the amino acids in
, except for the position of the unsaturated piperazic acid.
1
The ion observed at m/z 560 was determined as PA -Trp-
showed typical characteristics of peptides, for example
1
resonances for amide carbonyls in the C NMR spectrum
3
1
1
Val-PA (P TVP ), it indicated that 5-methoxy-2,3-dihy-
3
and for a proton in the H NMR spectrum (Table 1). The
C NMR and DEPT spectra (Table 1) of 2 showed the
3
1
1
3
dropyridazine-3-carboxylic acid (PA ) was connected to
2
Ala. The conclusion could also be testified by the results of
HMBC experiment, which showed correlation from the
a-methine protons of amino acid residue to carbonyl
carbon of the neighboring residues.
presence of 36 carbon signals, which were recognized as
four methyls, one oxygenated methyl, six methylenes,
3
eight sp methines, one oxygenated sp quaternary
3
2
C-atom, six sp methines, four sp quaternary C-atoms,
2
1
13
For the determination of the absolute configurations, an
acidic hydrolysate was generated according to the Marfey’s
protocol (Fujii et al. 1997). Subsequent HPLC analysis
indicated the presence of L-alanine and D-valine. Because
of the standard R- and S-piperazic acids were not obtained
commercially, the 1-fluoro-2,4-dinitrophenyl-5-L-alaninea-
mide (FDAA) derivatives of acidic hydrolysate of NW-
and six amide carbonyl carbons. H and C chemical
shift assignments were made by standard 1D and 2D
NMR techniques, such as DEPT, HSQC, and HMBC. The
moieties of valine, N-methyl-alanine, piperazic acid, and
chlorinated pyrroloindoline derivative were elucidated
based on the HSQC and HMBC spectra of 2, and by
comparison with the corresponding chemical shifts and
Fig. 1 Structures of NW-G10
(
1) and NW-G11 (2)
123

S. Wei et al.
coupling constants of NW-G01. 5-Methoxy-2,3-dihy-
dropyridazine-3-carboxylic acid could also be determined
based on the NMR data, and by comparison with the
corresponding data of 1.
The sequence linkage of the amino acids in 2 could be
readily determined by the characteristic MS/MS fragments
as described above (Figs. 2, 3). The conclusion could also
be testified by the results of HMBC experiment, which
showed correlation from the a-methine protons of partic-
ulars amino acid residues to the carbonyl carbon of the
neighboring residues (Fig. 1).
Careful comparison of the DEPT spectra of 2 with that
of 1, an aromatic methine was new appeared at dH/C 7.13/
1
44.4, and this proton was correlated to the b and c carbons
(
d 17.0 and d 20.1) in HMBC spectrum. As only one
N-Methyl-L-alanine, D-valine and R-piperazic acids
could be recognized by application of the Marfey’s method
as described above, whereas the absolute configurations of
other amino acids were not assigned in this investigation,
because their FDAA derivatives were not be obtained by
acid hydrolysis and Marfey’s analysis.
remaining methenyl carbon was present, a 2,3,4,5-tetra-
hydropyridazine-3-carboxylic acid was diagnosed easily.
This could also be testified by the correlation between H-a
and carbonyl carbon (d 172.1), as well as the results of MS/
MS experiment.
HN
N
HN
HN
HN
O
N
N
O
O
O
H
N
O
N
O
N
N
O
O
O
H
N
H
N
O
O
N
N
O
N
H
Cl
OH
N
Cl
N
Cl
H
OH
OH
NH
m/z 448 (P TV)
m/z 251(P P )
1
2
3
m/z 349 (P T)
1
m/z 519 (AP TV)
1
HN
HN
HN
O
H
N
HN
N
N
N
O
N
N
O
O
O
O
O
O
O
H
N
H
N
O
O
O
N
O
O
O
N
H
N
N
N
O
N
NH
N
NH
N
N
H
Cl
H
Cl
OH
OH
NH
N
H
Cl
OH
m/z 631 (AP TVP
1
)
m/z 769
m/z 560 (P TVP )
3
1
3
A
HN
N
N
HN
O
O
N
O
N
O
O
H
N
O
N
O
N
N
N
O
N
N
N
O
O
H
N
O
O
N
O
O
N
H
Cl
OH
N
H
Cl
N
N
OH
m/z 448 (P TV)
m/z 249(P P )
1
2
3
m/z 334 (P P A)
3
2
m/z 533 (AP TV)
1
HN
N
HN
N
O
HN
N
N
N
O
O
N
N
N
O
O
O
O
O
H
N
H
O
O
O
N
N
O
O
O
O
N
H
N
N
O
N
N
N
H
N
N
N
N
Cl
H
Cl
OH
OH
N
H
Cl
OH
m/z 643 (AP TVP
)
m/z 781
m/z 558 (P TVP )
1 3
1
3
B
Fig. 2 Fragment pathway of NW-G10 (a) and NW-G11 (b)
1
23

Two piperazic acid-containing cyclic hexapeptides
Fig. 3 MS/MS spectra of NW-G10 (a) and NW-G11 (b)
Antibacterial activity
B. cereus were 6.25, 25.0 and 6.25 lg/ml, their MICs against
B. subtilis were 3.13, 25.0 and 1.56 lg/ml, their MICs against
S. aureus were 6.25, 25.0 and 6.25 lg/ml, respectively, the
antibacterial activity of 1 was higher than 2. Because two
amino acids are different in their structures, the results could
not give any conclusion on the relationship of structure–
activity. Compared to ampicillin, the MICs of 1 and 2 against
MRSA were equal to their MICs against S. aureus, it revealed
that this type of cyclic hexapeptides has a different mecha-
nism of action from commercial b-lactam antibiotics.
The antibacterial activities of 1 and 2 against several strains
of bacteria have been evaluated by micro-broth dilution
method as we described previously (Ji et al. 2007). The
results indicated that 1 and 2 could effectively inhibit Gram-
positive bacteria, such as Bacillus cereus, B. subtilis Staph-
ylococcus aureus, and MRSA, whereas they were insensitive
to Gram-negative bacteria (Table 2). The minimum inhibi-
tion concentrations (MICs) of 1, 2 and Ampicillin against
123

S. Wei et al.
Ji ZQ, Wang MA, Zhang JW et al (2007) Two new members of
streptothricin class antibiotics from Streptomyces qinlingensis
sp. nov. J Antibiot 60:739–744
Table 2 MICs of NW-G10 (1) and NW-G11(2) against several
strains of bacteria
Tested bacteria
MIC (lg/ml)
Kamenecka TM, Danishefsky SJ (1998a) Studies in the total synthesis
of himastatin: a revision of the stereochemical assignment.
Angew Chem Int Ed 37:2993–2995
Kamenecka TM, Danishefsky SJ (1998b) Total synthesis of himast-
atin: confirmation of the revised stereostructure. Angew Chem
Int Ed 37:2995–2998
Konishi M, Ohkuma H, Sakai F et al (1981) Structures of BBM-928
A, B, and C novel antitumor antibiotics from Actinomadura
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ity. J Antibiot 43:956–960
1
2
Ampicillin
sodium
Bacillus cereus (CGMCC 1.1846)
Bacillus subtilis (CGMCC 1.88)
6.25 25.0 6.25
3.13 25.0 1.56
Staphylococcus aureus (CGMCC 1.89) 6.25 25.0 6.25
Escherichia coil (CGMCC 1.1574)
Pseudomonas aeruginosa (CGMCC
[100 [100 3.13
[100 [100 [100
1
.2031)
MRSA
6.25 25.0 [100
Leet JE, Schroeder DR, Krishnan BS et al (1990) Himastatin, a new
antitumor antibiotic from Streptomyces hygroscopicus II. Isola-
tion and characterization. J Antibiot 43:961–966
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Azinothricin, a novel hexadepsipeptide antibiotic. J Antibiot
Acknowledgments This study was supported part by the grant of
The National Key Basic Research Program (973 Program,
2
010CB126100) 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.
3
9:17–25
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structure elucidation and activity of a new hexadepsipeptide
antibiotic from a terrestrial Streptomyces sp. J Antibiot 59:309–
3
14
Miller ED, Kauffman CA, Jensen PR et al (2007) Piperazimycins:
cytotoxic hexadepsipeptides from a marine-derived bacterium of
the genus Streptomyces. J Org Chem 72:323–330
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