Lahorenoic acids A-C, ortho-dialkyl-substituted aromatic acids from the biocontrol strain pseudomonas aurantiaca PB-St2

Article abstract of DOI:10.1021/np3005166

Three new aromatic acids, named lahorenoic acids A (1), B (2), and C (3), have been isolated along with the known compounds phenazine-1-carboxylic acid (4), 2- hydroxyphenazine-1-carboxylic acid (5), 2-hydroxyphenazine (6), 2,8-dihydroxyphenazine (7), cyclo-Pro-Tyr (8), cyclo-Pro- Val (9), cyclo-Pro-Met (10), and WLIP (11) and characterized from the biocontrol strain Pseudomonas aurantiaca PBSt2. The structures of these compounds were deduced by 1D and 2D NMR spectroscopic and mass spectral data interpretation. Compounds 2, 4, and 7 showed moderate antibacterial activity against mycobacteria and other Grampositive bacteria, while 4 was also found to exhibit cytotoxic and antifungal properties.

Full text of DOI:10.1021/np3005166

Article
pubs.acs.org/jnp
Lahorenoic Acids A−C, ortho-Dialkyl-Substituted Aromatic Acids
from the Biocontrol Strain Pseudomonas aurantiaca PB-St2
Samina Mehnaz,*,† Rahman Shah Zaib Saleem,‡ Basit Yameen,‡ Isabelle Pianet,§ Gregor Schnakenburg,⊥
Halina Pietraszkiewicz,∥ Fred Valeriote,∥ Michaele Josten,▽ Hans-Georg Sahl,▽ Scott G. Franzblau,○
and Harald Gross*,#
†Department of Biological Sciences, Forman Christian College University, Lahore 54600, Pakistan
‡Department of Chemistry, School of Sciences and Engineering, Lahore University of Management Sciences, Lahore 54792, Pakistan
§CESAMO-ISM, UMR 5255, CNRS, Universite
́ ́
Bordeaux I, 351 Cours de la Liberation, F-33405 Talence, France
⊥Institute of Inorganic Chemistry, University of Bonn, Gerhard-Domagk-Straße 1, 53121 Bonn, Germany
∥Henry Ford Health System, Detroit, Michigan 48202, United States
▽Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), Pharmaceutical Microbiology Unit, University of
Bonn, 53115 Bonn, Germany
○Institute for Tuberculosis Research, University of Illinois at Chicago, Chicago, Illinois 60612, United States
#Institute for Pharmaceutical Biology, University of Tubingen, Auf der Morgenstelle 8, 72076 Tubingen, Germany
̈
̈
S
* Supporting Information
ABSTRACT: Three new aromatic acids, named lahorenoic
acids A (1), B (2), and C (3), have been isolated along with
the known compounds phenazine-1-carboxylic acid (4), 2-
hydroxyphenazine-1-carboxylic acid (5), 2-hydroxyphenazine
(6), 2,8-dihydroxyphenazine (7), cyclo-Pro-Tyr (8), cyclo-Pro-
Val (9), cyclo-Pro-Met (10), and WLIP (11) and charac-
terized from the biocontrol strain Pseudomonas aurantiaca PB-
St2. The structures of these compounds were deduced by 1D
and 2D NMR spectroscopic and mass spectral data
interpretation. Compounds 2, 4, and 7 showed moderate
antibacterial activity against mycobacteria and other Gram-
positive bacteria, while 4 was also found to exhibit cytotoxic and antifungal properties.
ungal phytopathogens such as Fusarium, Rhizoctonia, or
P. chlororaphis MA342 (Cedomon, Cerall),14 P. fluorescens
FColletotrichum species cause worldwide damage to crop A506 (BlightBan A506),15 and P. sp. DSMZ 13134
(Proradix).16,17
production. In the last decades, disease management has relied
In our continuing effort to explore the chemistry of
pseudomonads,10,12,13 we describe here the chemical inves-
tigation of the biocontrol strain Pseudomonas aurantiaca PB-
St2,18,19 which was found to inhibit the growth of
Colletotrichum falcatum, the causative agent of the red rot
disease of sugar cane (Saccharum sp. hybrids).20,21 Strain PB-
St2 was known to produce at least five secondary metabolites:
phenazine-1-carboxylic acid (4), 2-hydroxyphenazine (6), N-
hexanoyl homoserine lactone, hydrogen cyanide, and a
siderophore of the hydroxamate type.18,19 The antifungal
properties of the strain have been attributed to the production
of phenazine-1-carboxylic acid (4) and 2-hydroxyphenazine
(6).18 The high capacity for secondary metabolite production
in this group of bacteria, as shown by genomic sequence data
for several Pseudomonas spp.,22 prompted us to continue the
mainly on the use of synthetic fungicides. However, this
strategy is challenged due to the accumulation of these
compounds in the ecosystem and due to resistant fungal
strains. One way of reducing the use of synthetic fungicides in
agriculture is by using bacterial strains,1 which aggressively
colonize the plant rhizosphere and are able to protect their
hosts from infection by soil-borne fungal pathogens.2 The
biological mechanisms underlying this phenomenon, known as
biocontrol, include the secretion of both enzymes and small
molecules.2−5 One bacterial genus used for this purpose is
Pseudomonas.6 This group of bacteria is frequently described as
being rhizosphere colonizers and are known to produce a
diverse array of potent antifungal compounds, which include
2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid,7−9 pyr-
rolnitrin, pyoluteorin, hydrogen cyanide, lipopeptides,10,11 and
rhizoxin congeners.12,13 On the basis of their capacity to control
soil-borne phytopathogens, some Pseudomonas biocontrol
strains have been developed into commercial products, e.g.,
Received: July 25, 2012
Published: February 12, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Compounds isolated from Pseudomonas aurantiaca PB-St2.
Table 1. NMR Spectroscopic Data (300 MHz, d4-MeOH) for Lahorenoic Acids A (1), B (2), and C (3)
lahorenoic acid A (1)
lahorenoic acid B (2)
lahorenoic acid C (3)
δC, type δH (J in Hz)
position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
a
a
1
177.8, C
175.9, C
178.1, C
a
2
34.5, CH2
26.0, CH2
29.9, CH2
32.7, CH2
34.0, CH2
144.0, C
2.33, t (7.0)
1.68, m
34.7, CH2
25.8, CH2
29.8, CH2
32.6, CH2
34.0, CH2
144.0, C
2.36, t (7.4)
1.68, m
34.6, CH2
25.9, CH2
33.7, CH2
131.9, CH
130.1, CH
137.4, C
2.37, t (7.4)
1.82, p (7.4)
2.29, q (7.1)
3
4
1.44, m
1.43, m
5
1.62, m
1.62, m
6.17, dt (7.1, 15.8)
6.60, d (15.8)
6
2.85, t (7.7)
2.85, t (7.6)
7
8
131.4, CH
131.5, CH
127.7, CH
127.7, CH
134.0, C
7.29, d (7.5)
131.4, CH
131.5, CH
127.7, CH
127.7, CH
134.0, C
7.29, d (7.5)
126.4, CH
128.0, CH
127.5, CH
130.5, CH
136.7, C
7.51, brd (6.4)
7.20, m
9
7.37, dd (7.2, 7.5)
7.28, dd (7.2, 7.9)
7.70, d (7.9)
7.38, dd (7.2, 7.5)
7.28, dd (7.2, 7.9)
7.71, d (7.9)
10
11
12
13
14
15
16
17
7.17, m
7.13, m
142.7, CH
128.8, CH
201.2, C
8.00, d (16.0)
6.78, d (16.0)
142.7, CH
128.8, CH
201.1, C
8.00, d (16.0)
6.78, d (16.0)
128.5, CH
135.9, CH
22.8, CH2
14.5, CH3
6.49, d (11.7)
5.78, dt (11.7, 7.5)
2.11, m
27.7, CH3
2.44, s
27.8, CH3
52.0, CH3
2.44, s
3.68, s
1.02, t (7.5)
a
Indicated carbons showed only weak resonances in the 13C NMR spectrum, but their presence and connectivity were clearly evidenced by all
conducted 2D NMR experiments.
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1
chemical investigation of this Pseudomonas strain. Using H
NMR guided fractionation, we isolated three new ortho-dialkyl-
substituted aromatic acids (1−3), along with a suite of
previously reported metabolites belonging to the class of
phenazines (4−7), diketopiperazines (8−10), and cyclic
lipopetides (11). To date, ortho-dialkyl-substituted aromatic
acids are rare and so far exclusively produced by Actinomycetes
(e.g., serpentenes,23,24 and demetric acid25) or by the marine
bacterium Pseudoalteromonas rubra (rubrenoic acids A−C26,27),
respectively. Indeed, lahorenoic acids A (1) and B (2) represent
the first members of this family that bear a keto functionality,
and this is the first report of the occurrence of this compound
class in the genus Pseudomonas.
Spectroscopic analysis of compound 2 revealed that it was
very closely related to lahorenoic acid A (1) but differed from 1
by 14 mass units. Comparison of the 1H and 13C chemical shifts
for 1 and 2 showed a slight upfield shift for C-1 by 1.9 ppm in
the 13C NMR spectrum and the presence of an oxygenated
1
methyl group (δH 3.68, s; δC 52.0) in the H NMR and 13C
spectrum of 2 (Table 1). These differences could be explained
by methylation of the free carboxylic acid in 2,28 as confirmed
by an HMBC correlation from the 3H singlet at δH 3.68 (H3-
17) to the C-1 resonance (δC 175.9). Therefore, the structure
of 2 was assigned as methyl 6-(2-((E)-3-oxobut-1-enyl)-
phenyl)hexanoate and given the trivial name lahorenoic acid
B. Since during the purification process methanol was applied
as eluent, compound 2 could possibly represent an isolation
artifact of 1. However, LC-MS investigations using MeCN as
mobile phase proved that compound 2 is already present in the
crude ethyl acetate extract. Thus, we currently consider
lahorenoic acid B (2) to be a real product of a biosynthesis
pathway, and lahorenoic acid A (1) appears to be an
intermediate in the biosynthesis of lahorenoic acid B (2).
High-resolution mass measurement of the sodiated molecular
ion for 3 yielded a molecular formula of C16H20O2 with 7
degrees of unsaturation. Analysis of the NMR data (Table 1)
indicated that compound 3 shared many structural features
with lahorenoic acid A (1). Differences between them, evident
in their 1D spectra, resulted from the absence of one carbonyl
group in 3 that was formerly observed at δC 201.2 in the
spectrum for 1, from the upfield shift of H3-16, and from the
appearance of signals representing a second double bond (δH
6.17 and 6.60) in the spectra for compound 3. Analysis of the
COSY and HMBC spectra revealed that compound 3
possessed a 1-butene side chain that is attached to a benzene
ring and a 5-hexenoic acid, bonded to the benzene ring in ortho
position. The configurations of the double bonds were found to
RESULTS AND DISCUSSION
■
All compounds (1−11) were isolated by the use of solvent
partitioning, reversed-phase SPE (solid phase extraction), and
HPLC, and their structures assigned by detailed spectroscopic
analysis as detailed below.
The molecular formula of lahorenoic acid A (1) was
determined to be C16H20O3 by HRESIMS. The 13C NMR
spectrum of 1 showed 16 13C resonances (Table 1) for one
methyl carbon (δC 27.7), five methylenes, a benzene ring, a
carbon−carbon double bond (δC 128.8 and 142.7), a
ketocarbonyl carbon (δC 201.2), and a carboxylic acid group
(δC 177.8 and IR 1708 cm−1). The benzene ring was suggested
to be ortho-substituted on the basis of the coupling pattern of
1
1
the aromatic protons in the H NMR and in the H−13C-
HSQC spectrum, which showed four aromatic proton
resonances at δ 7.28 (1H, dd, J = 7.2, 7.9; H-10), 7.29 (1H,
d, J = 7.5; H-8), 7.37 (1H, dd, J = 7.2, 7.5; H-9), and 7.70 (1H,
d, J = 7.9; H-11). H−1H COSY correlations between H-8/H-
9, H-9/H-10, and H-10/H-11 confirmed the connectivity
1
deduced from coupling constant analysis. Further analysis of
1
3
the H−1H-COSY NMR spectrum revealed two additional
be 5E, 13Z, as deduced from the coupling constants JH5−H6 of
isolated 1H spin systems (H2-2 through H2-6 and H-13/H-14),
representing partial structures of the phenyl substituents in 1
(Figure 2). HMBC correlations observed from the olefinic H-
3
15.8 Hz and JH13−H14 of 11.7 Hz, respectively. The deduction
of the 13Z configuration was also supported by the typical
upfield shift of δH 7.7 to 7.1 for the conjugated ring proton H-
11, which is usually observed in this case for this compound
class.23 Therefore, compound 3 was identified as (5E)-6-(2-
((Z)-but-1-enyl)phenyl)hex-5-enoic acid and given the name
lahorenoic acid C (3).
The structures of phenazines 4−7,29−31 diketopiperazines 8−
10,29,32−35 and the cyclic lipodepsipeptide “white line inducing
principle” (WLIP) 1136 were established either by direct
comparison of their spectral data with literature data or by an
independent spectral analysis (see Supporting Information).
Regarding the analysis of WLIP (11), it is noteworthy to
mention that the strain PB-St2 provided false negative results in
the white line in agar (WLA) test37 (see Supporting
Information). We therefore suggest that the WLA test should
no longer be considered a means of unequivocal proof of WLIP
production by a Pseudomonas strain. The application of X-ray
crystallography in combination with chemical degradation
studies and subsequent Marfey’s analysis38 allowed the
unambiguous confirmation that compound 11 is indeed
WLIP and not its epimer viscosin.39
Figure 2. 1H−1H-COSY and 1H−13C long-range key correlations of 1.
13 signal to three aromatic carbon resonances for methine C-
11, quaternary C-12, and quaternary C-7 (δC 144.0), along with
correlations from olefinic H-14 to the ketone resonance at δC
201.2 (C-15), which was in turn correlated with H3-16,
established a 3-buten-2-one side chain located at C-12 of the
benzene ring. In a similar fashion, on the basis of HMBC
correlations observed between H2-2/C-1, H2-6/C-7, and H2-6/
CH-8, the remaining second spin system could be extended and
identified as a hexanoic acid, bonded to the benzene ring via
quaternary C-7. The double-bond geometry of Δ13,14 was E-
configured, as determined from the coupling constant 3JH13−H14
of 16.0 Hz. Therefore, compound 1 was identified as 6-(2-((E)-
3-oxobut-1-enyl)phenyl)hexanoic acid and was given, according
to its origin (Lahore, Pakistan) and chemical structure, the
name lahorenoic acid A (1).
Bioassay. The antifungal, antimicrobial, and cytotoxic
effects of compounds 2, 4, 6, 7, and 11 were investigated.
Lack of material precluded the bioassays with compounds 1, 3,
and 5. Diketopiperazines 8−10 were not included in the
bioassay investigations since they were considered isolation
artifacts.40,41 While compounds 6 and 11 were inactive in all
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2192T and ATCC 33618T) and “P. reactans” LMG 5329 were obtained
from the BCCM/LMG Bacteria Collection, Ghent, Belgium. P.
fluorescens SWB 25 was kindly provided by J. M. Raaijmakers
(Wageningen University, Wageningen, The Netherlands). The fungal
phytopathogens Colletotrichum falcatum (BF166, C01148, CP77400,
SPF234) and Rhizoctonia solani AG 2-2 and AG 442 were kindly
provided by the Shukur Gunj Research Institute, Jhang, Pakistan, and
assays conducted, compounds 2, 4, and 7 were found to be
active. Lahorenoic acid B (2) showed a moderate inhibition of
Mycobacterium tuberculosis (MIC = 49.5 μg/mL) and was
noncytotoxic and not antifungal. For phenazine-1-carboxylic
acid (4) a 50% growth inhibition (IC50) value of 15.6 μM was
determined toward HCT-116 cells. In disc diffusion assays, 4
showed selectivity toward the solid tumor cell lines H-125,
MCF-7, and LNCaP (see Supporting Information). In
antimicrobial assays, compound 4 showed a moderate activity
toward M. tuberculosis (MIC = 32.9 μg/mL) and demonstrated
a 0.2 cm growth inhibition zone (reduced hyphal density)
toward the fungus Colletotrichum falcatum at 100 μg per filter
disc. 2,8-Dihydroxyphenazine (7) was inactive in antifungal and
cytotoxicity assays but demonstrated in antibacterial tests a
moderate activity toward Arthrobacter crystallopoietes DSM
20117 (MIC = 25 μg/mL) and Bacillus cereus (MIC = 25 μg/
mL).
This study demonstrated, by proof of production of the new
ortho-dialkyl-substituted aromatic acids 1−3 and the cyclic
lipopeptide WLIP (11), that P. aurantiaca PB-St2 posssess a
higher biosynthetic capacity than previously anticipated.
Evaluation of the toxic and antimicrobial potential of the
major metabolites confirmed at the 100 μg level phenazine-1-
carboxylic acid (4) to be the antifungal component of the
strain. However, since in parallel compound 4 exhibited also
cytotoxic properties toward eukaryotic cells, the antifungal
activity has to be classified as nonspecific. 2-Hydroxyphenazine
(6), which was previously identified as an antifungal
component,18 was active only above the 200 μg level and
therefore reassessed in our strict assays as not active.
Furthermore, this study revealed that the secrected metabolites
of PB-St2 possess moderate antibacterial properties that might
additionally contribute to the biocontrol effect of the strain.
M. Hofte, University of Ghent, Belgium, respectively. M. tuberculosis
̈
H37Rv was obtained from the American Type Culture Collection
(ATCC 27294).
Fermentation and Isolation. P. aurantiaca PB-St-2 was grown
with shaking (140 rpm) at 26 °C in four 5 L Erlenmeyer flasks, each
containing 1 L of LB medium. After 72 h of incubation, cell and
supernatant fractions of the cultures were separated by centrifugation
at 3000 rpm for 20 min. Supernatant was acidified to pH 3 with HCl
and extracted four times with chloroform (1:1, v/v), to yield 1.6 g of
crude extract. The extract was fractionated by vacuum liquid
chromatography (VLC) over reversed-phase silica gel using a stepwise
gradient of methanol−water and finally dichloromethane to give six
fractions. 1H NMR profiling of these fractions indicated the 20% H2O
in MeOH fraction and the 100% MeOH fraction to be of further
1
interest due to a large number of aromatic resonances in its H NMR
spectrum. Separation of the (80:20) VLC fraction by RP-HPLC by
using a linear gradient of 55:45−100:0 MeOH−H2O (0.05% TFA)
over a period of 40 min, followed by isocratic elution at 100% MeOH
for an additional 20 min (Phenomenex Luna C18(2)-100A, 10 × 250, 5
μm column in combination with a Phenomenex SecurityGuard Luna-
C18(2) 10 × 10 mm precolumn; 1.9 mL/min flow rate; UV monitoring
1
at 215 nm), produced 10 subfractions. H NMR profiling of these
HPLC subfractions showed 3, 4, and 8 to be of further interest. HPLC
subfraction 3 represented pure 2,8-dihydroxyphenazine (7) (10.6 mg).
HPLC subfractions 4 and 8 appeared to be mixtures of aromatic
compounds. While the chloroform-soluble parts of HPLC subfraction
4 yielded readily pure phenazine-1-carboxylic acid (4) (2.0 mg),
rechromatography of its methanol-soluble parts by RP-HPLC applying
a linear gradient of 65:35−80:20 MeOH−H2O (0.05% TFA) over a
period of 30 min (Phenomenex Luna C18(2)-100A, 10 × 250 mm, 5
μm column in combination with a Phenomenex SecurityGuard Luna-
C18(2) 10 × 10 mm precolumn; 2.0 mL/min flow rate; UV monitoring
at 215 nm) provided pure 2-hydroxyphenazine (6) (3.5 mg). HPLC
subfraction 8 of the (80:20) VLC fraction was also purified by C18
HPLC, this time using a linear gradient of 50:50−75:25 MeOH−H2O
(0.05% TFA) over a period of 30 min, followed by isocratic elution at
75:25 MeOH−H2O (0.05% TFA) for an additional 30 min (column:
Waters XTerra, 4.6 × 250 mm; 0.8 mL/min flow rate; UV monitoring
at 210 nm), to yield (in order of elution) compounds 1 and 2 in a
semipure form. Rechromatography of these fractions by RP-HPLC
employing two different RP18-HPLC columns in series (Macherey-
Nagel Nucleosil 120-5, 4.6 × 250 mm column, followed by a
Macherey-Nagel Nucleodur 100-5 HILIC, 4.6 × 250 mm) with a
linear gradient of 50−80% ACN in H2O (0.05% TFA) over 30 min at
0.5 mL/min, followed by isocratic elution at 80:20 ACN−H2O (0.05%
TFA) for an additional 30 min, provided compounds 1 (0.9 mg) and 2
(4.3 mg) in a pure form. RP-HPLC separation of the 100% MeOH
VLC fraction (Phenomenex Luna C18(2)-100A, 10 × 250 mm, 5 μm
column in combination with a Phenomenex SecurityGuard Luna-
C18(2) 10 × 10 mm precolumn; 2.0 mL/min flow rate; UV monitoring
at 210 nm) using gradient elution from 80% to 100% MeOH in H2O
(0.05% TFA) over 45 min, followed by isocratic elution at 100%
MeOH for an additional 15 min, gave compounds 5 (1.2 mg) and
impure WLIP (11) and provided additional quantities of compounds 4
and 7. The WLIP (11)-containing fraction was purified twice by RP-
HPLC, first by employing a Macherey-Nagel Nucleosil 120-5, 4 × 250
mm column (1.0 mL/min, 40 min gradient elution from 60% to 100%
MeOH−H2O (0.05% TFA) followed by a 20 min hold) and
subsequently a Waters SymmetryShield RP18, 4.6 × 250 mm column
(1.0 mL/min, 40 min gradient elution from 50% to 100% ACN−H2O
(0.05% TFA) followed by a 20 min hold), to yield 6 mg of pure WLIP
(11). Higher yields of 11 were achieved by variation of the extraction
solvent (e.g., EtOAc instead of CHCl3) or the fermentation medium
EXPERIMENTAL SECTION
■
General Experimental Procedures. High-performance liquid
chromatography (HPLC) was performed using either a Merck-Hitachi
system consisting of an L-6200 pump, an L-7420 UV−vis detector, a
Rheodyne 7125 injector, and a Knauer interface box, controlled by
Knauer EuroChrom software, or a Waters system consisting of a 600
pump, a 996 photodiode array detector, a Rheodyne 7161 injector, and
a Perkin-Elmer Series 200 vacuum degasser. 1D and 2D NMR
experiments were carried out on a Bruker Avance 300 DPX
spectrometer equipped with a 5 mm broadband observe (BBO)
probe head or a 2.5 mm selective inverse (SEI) microprobe head and
on a Bruker AV-III 600 MHz spectrometer using an inverse broadband
probe head, respectively. For samples, obtained in small quantities,
advanced 5 mm microtubes, matched to CDCl3, MeOH, or DMSO
(Shigemi, Allison Park, PA, USA), were applied for 13C NMR and
DEPT135 NMR experiments. Spectra were referenced to residual
solvent signals with resonances at δH/C 7.26/77.0 (CDCl3), δH/C 3.35/
49.0 (CD3OD), and 2.50/39.5 (d6-DMSO). UV spectra were taken on
Perkin-Elmer Lambda 40 and Thermo Scientific Evolution 300
instruments, respectively. Infrared spectra were obtained employing
Perkin-Elmer Spectrum BX and Bruker Vector 22 FT-IR instruments,
respectively. EIMS and ESIMS spectra were recorded on a Kratos MS
50 and Bruker Daltonic’s micrOTOF-Q instrument, respectively.
Macherey-Nagel Polygoprep 50−60 reversed-phase silica gel was used
for vacuum liquid chromatography. All solvents were purchased as
HPLC grade.
Biological Material and Collection. P. aurantiaca PB-St2 was
isolated by one of the authors (S.M.) from surface-sterilized stems of
sugar cane grown in Pakistan.18,19 The identity of the strain was
proven by 16S rRNA gene sequence analysis. The 16S sequence for
PB-St2 was deposited in GenBank (accession number EU761590).
Reference strains P. tolaasii LMG 2342T (also known as NCPPB
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(Davis Minimal Broth without dextrose, supplemented with 20 mM
glycerol (DMBgly) instead of LB medium). Adaption of the extraction
procedure, using CH2Cl2 without prior pH adjustment by acidification
of the supernatant, led to the isolation of diketopiperazines 8−10.
Under these conditions, 490 mg of crude extract was obtained from a
4.5 L LB-medium-based culture. The ACN-soluble parts were directly
subjected to C8 HPLC (Knauer Eurospher II C8 100-5, 8 × 250 mm
column 2.0 mL/min, 40 min gradient elution from 35% to 70% ACN−
H2O followed by a 20 min hold) to give compound 8 (2.0 mg) and a
mixture of 9 and 10 and provided additional quantities of compounds
4 and 6. Rechromatography of the diketopiperazine mixture using a
C18 Macherey-Nagel Nucleosil 120-5, 4.0 × 250 mm column (1.0 mL/
min, gradient elution from 35% to 80% ACN in 40 min, followed by a
20 min hold) gave pure 9 (2.8 mg) and 10 (0.8 mg).
KappaApexII diffractometer for 11 using graphite-monochromated Mo
Kα radiation (λ = 0.71073 Å). Both diffractometers were equipped
with a low-temperature device (Cryostream, Oxford Cryosystems,
123(2) K, Bruker Kryoflex, 100(2) K). Intensities were measured by
fine-slicing psi and omega scans and corrected for background,
polarization, and Lorentz effects. A semiempirical absorption
correction from equivalent reflections was applied for the data sets
following Blessing’s method.45 The structures were solved by direct
methods and refined anisotropically by the least-squares procedure
implemented in the SHELX program system.46 The hydrogen atoms
were included isotropically using the riding model on the bound
carbon atoms. The illustrations of the molecular structures were
prepared with Diamond 2.1c.47 Crystals of 2-hydroxyphenazine (6)
were obtained by slow evaporation from an ethyl acetate solution
stored at 4 °C. Monoclinic space group C2/c with a = 18.9536(13) Å,
b = 4.3422(2) Å, c = 22.3807(17) Å, α = 90.00°, β = 96.752(3)°, γ =
90.00°, V = 1829.2(2) Å3 at 123(2) K with Z = 8; 5879 reflections
measured, 2154 independent reflections (Rint = 0.0734). The final R1
values were 0.0485 (I > 2σ(I)). The final wR(F2) values were 0.1089 (I
> 2σ(I)). The final R1 values were 0.0890 (all data). The final wR(F2)
values were 0.1234 (all data). Crystals of WLIP (11) were generated in
a two-staged process: 6.3 mg of WLIP (11) was dissolved in a vial in
0.5 mL of DMSO, followed by addition of 1.5 mL of methanol. Seed
crystals were obtained by slow evaporation of the solvent in a 100 mL
round-bottom flask at room temperature after 24 h. Upon freeze-
drying of the sample, suitable seed crystals were selected and inserted
into a DMSO−methanol solution of 3.1 mg of compound 11. Crystals
suitable for X-ray analysis were obtained by slow evaporation at room
temperature after several weeks. Orthorhombic space group P212121
with a = 14.1536(5) Å, b = 18.7258(6) Å, c = 24.3604(8) Å, V =
6456.4(4) Å3 at 100(2) K with Z = 4; 25 141 reflections measured,
8473 independent reflections (Rint = 0.0207). The final R1 values were
0.0370 (I > 2σ(I)). The final wR(F2) values were 0.0933 (I > 2σ(I)).
The final R1 values were 0.0443 (all data). The final wR(F2) values
were 0.0994 (all data). CCDC 874817 (6) and CCDC 919229 (11)
contain the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/
cif. The absolute configuration fixed by the side-chain stereochemistry
of threonine and glutamic acid was supported by a Marfey analysis.38
Absolute Configuration of the Glu2 and Thr3 Residues of
WLIP (11). WLIP (1.0 mg) was dissolved in 400 μL of 6 N HCl and
was hydrolyzed in a sealed vial at 110 °C for 15 h, using an oil bath.
The solvent was removed by using a stream of dry N2. A 1 M
NaHCO3 solution (100 μL) was added to the dried hydrolysate of 11,
as well as to standards of L-Glu, D-Glu, L-Thr, L-allo-Thr, D-Thr, and D-
allo-Thr. A solution of 1% L-FDAA in acetone (50 μL) was added to
each reaction batch. Each vial was sealed and incubated at 80 °C for 30
min. To quench the reactions, 50 μL of 2 N HCl was added to each
sample and evaporated to dryness. The Marfey’s derivatives of the
hydrolysate and standards were resuspended in a mixture of DMSO
(150 μL) and ACN (150 μL), and analyzed by C18 HPLC (1.0 mL/
min, 40 min gradient elution from 25% to 100% ACN−H2O acidified
with 0.05% TFA, UV detection at 340 nm). Retention times for the
amino acid standards were L-Thr 10.5 min, L-allo-Thr 10.6 min, D-Thr
11.5 min, D-allo-Thr 10.9 min, L-Glu 12.8 min, and D-Glu 12.1 min,
while the hydrolysate gave peaks at 10.9 and 12.1 min. Co-injection of
the hydrolysate with the corresponding standards unequivocally
established the analyzed residues as D-Glu and D-allo-Thr.
Further adaption of the cultivation and extraction procedure,
employing DMBgly medium on a 4.5 L scale, with only 25 h of growth
and extraction with CH2Cl2 without prior pH adjustment yielded 3.
The ACN-soluble parts of the crude extract (110 mg) were directly
subjected to HPLC (Knauer Eurospher II C8 100-5, 8 × 250 mm
column 2.0 mL/min, 30 min gradient elution from 50% to 75% ACN−
H2O followed by a 30 min hold) to afford 1.2 mg of compound 3.
Lahorenoic acid A (1): pale yellow, amorphous solid; UV (MeOH)
λmax 210 nm (ε 4.23), 225sh nm (ε 4.12), 285 nm (ε 4.19); FT-IR
(ATR) νmax 2930, 2857, 1708, 1668, 1596, 1416, 1359, 1257, 1180,
1
974, 754 cm−1; H and 13C NMR data, see Table 1; ESIMS m/z
(positive) 283.1 [M + Na]+; HRESIMS m/z 283.1302 (calcd for
C16H20NaO3, 283.1310).
Lahorenoic acid B (2): pale yellow, amorphous solid; UV (MeOH)
λmax 211 nm (ε 4.23), 226sh nm (ε 4.14), 288 nm (ε 4.32); FT-IR
(ATR) νmax 2927, 2854, 1735, 1670, 1597, 1435, 1358, 1254, 1174,
1
975, 753 cm−1; H and 13C NMR data, see Table 1; ESIMS m/z
(positive) 297.2 [M + Na]+; HRESIMS m/z 297.1461 (calcd for
C17H22NaO3, 297.1467).
Lahorenoic acid C (3): pale yellow, amorphous solid; UV (MeOH)
λmax 244 nm (ε 5.31); FT-IR νmax 2930, 2871, 1711, 1452, 1404, 1249,
1
1159, 967 cm−1; H and 13C NMR data, see Table 1; ESIMS m/z
(positive) 267.1 [M + Na]+; HRESIMS m/z 267.1341 (calcd for
C16H20NaO2, 267.1356).
Phenazine-1-carboxylic acid (tubermycin B) (4): yellow needles;
1H and 13C NMR data, see Table S1 of the Supporting Information;
HRESIMS [M + Na]+ m/z 247.0475 (calcd for C13H8N2NaO2,
247.0483).
2-Hydroxyphenazine-1-carboxylic acid (5): orange powder; 1H
and 13C NMR data, see Table S2 of the Supporting Information;
HREIMS [M]+ m/z 240.0537 (calcd for C13H8N2O3, 240.0535).
1
2-Hydroxyphenazine (6): orange powder; H and 13C NMR data,
see Table S3 of the Supporting Information; HRESIMS [M + H]+ m/z
197.0709 (calcd for C12H9N2O, 197.0715).
2,8-Dihydroxyphenazine (7): brownish-orange, amorphous solid;
1H and 13C NMR data, see Table S4 of the Supporting Information;
HRESIMS [M + H]+ m/z 213.0663 (calcd for C12H9N2O2, 213.0664).
cyclo-(L-Pro-L-Tyr) (maculosin-1) (8): colorless, amorphous solid;
[α]2D2 −131.3 (c 0.14, EtOH) (lit.,29 −126.1 in EtOH, lit.,32 −144.5 in
MeOH); HRESIMS [M + H]+ m/z 261.1235 (calcd for C14H17N2O3,
1
261.1239); H and 13C NMR data, see Supporting Information.
cyclo-(L-Pro-L-Val) (9): colorless, amorphous solid; [α]2D2 −142.8 (c
0.20, EtOH) (lit.,29 −139.4 in EtOH, lit.,43 ranging from −134 to
−180 in EtOH, lit.,44 −128 in MeOH); HRESIMS [M + Na]+ m/z
219.110 (calcd for C10H16N2NaO2, 219.1109); 1H and 13C NMR data,
see Supporting Information.
White-Line-in-Agar Test. The white line test was performed
according to the method of Wong and Preece.37 P. aurantiaca was
streaked on King’s B medium at 1 cm distance next to Pseudomonas
tolaasii LMG 2342T, and the formation of a white precipitate in the
agar was evaluated after 24 to 72 h growth at 26 °C. Pseudomonas
reactans LMG 5329 (WLIP producer) and Pseudomonas fluorescens
SWB 25 (viscosin producer)48 were included as positive and negative
controls, respectively. Assays were carried out in duplicates and
repeated twice.
cyclo-(L-Pro-L-Met) (10): colorless, amorphous solid; [α]2D2 −85.7
(c 0.057, EtOH) (lit.,29 −82.2 in EtOH); HRESIMS [M + Na]+ m/z
1
251.0839 (calcd for C10H16N2NaO2S, 251.0830); H and 13C NMR
data, see Supporting Information.
WLIP (11): yellowish glass; 1H and 13C NMR data, see Table S5 in
the Supporting Information); HRESIMS [M + Na]+ m/z 1148.6798
(calcd for C54H95N9O16Na, 1148.6794).
X-ray Crystallographic Analysis of 2-Hydroxyphenazine (6)
and WLIP (11). The data collection was performed on a NONIUS
KappaCCD diffractometer (area detector) for 6 and a Bruker X8-
Antibacterial Assay. The antibacterial activity was determined as
previously published49 by agar diffusion assays. In brief, culture plates
139
dx.doi.org/10.1021/np3005166 | J. Nat. Prod. 2013, 76, 135−141

Journal of Natural Products
Article
(5% sheep blood Columbia agar, BD) were overlaid with 3 mL of
tryptic soy soft agar, inoculated with tryptic soy broth (TSB, Oxoid)
growth suspension of the bacteria to be tested. Compounds were
diluted with DMSO to a concentration of 1 mg/mL, and 3 μL of this
dilution was spotted on the surface of the agar; the diameter of the
inhibition zone was measured after 24 h incubation at 37 °C. The
indicator strains represent clinical isolates from distinct patients.49
These strains were maintained on Mueller−Hinton agar or on blood
agar. MIC determinations were carried out in microtiter plates.
Arthrobacter crystallopoietes DSM 20117 was grown in tryptic soy broth
(Oxoid); all other strains were grown in half-concentrated Mueller−
Hinton broth (Oxoid). MICs with 2-fold serial dilution steps were
performed (1:2). Bacteria were added to give a final concentration of
105 CFU in a volume of 0.2 mL. After incubation for 24 h at 37 °C, the
MIC was read as the lowest compound concentration causing
inhibition of visible growth. Results are mean values of three
independent determinations. Pure compounds demonstrating an
MIC higher than 50 μg/mL were regarded as inactive samples.
Antituberculosis Bioassay. The virulent H37Rv strain of M.
tuberculosis (ATCC27294, American Type Culture Collection, Rock-
ville, MD, USA) was used for the antituberculosis bioassay.50,51 The
test materials were dissolved in DMSO at 10 mg/mL and tested in a
series of 2-fold dilutions with the highest concentration of 100 μg/mL
(and 1% v/v DMSO). Samples were incubated at 37 °C for 7 days
with M. tuberculosis in a 96-well plate, and then cell growth was
determined using the Alamar Blue dye with fluorometric detection.
The MIC was defined as the lowest concentration resulting in 90% or
greater inhibition of fluorescence compared to controls (M. tuberculosis
without sample). Rifampicin and INH were used as positive controls,
exhibiting MICs of 0.02−0.07 and 0.02−0.24 μg/mL, respectively,
while DMSO at a final concentration of 1% v/v served as a negative
control. Pure compounds demonstrating an MIC higher than 50 μg/
mL were regarded as inactive samples.
bovine serum containing 1% penicillin−streptomycin and 1%
glutamine)54 at 37 °C and 5% CO2 from a starting cell density of 5
× 104 cells/T25 flask. On day 3, the cells were exposed to different
concentrations of the corresponding pure compound. The flasks were
incubated for 120 h (5 days) at 5% CO2 and 37 °C, and the cells were
harvested with trypsin, washed once with Hanks’ balanced salt
solution, resuspended in Hanks’ balanced salt solution, and counted
using a hemocytometer. The results were normalized to an untreated
control. The 50% inhibitory concentration (IC50) was determined by
nonlinear regression analysis using Prism 4.0 software (GraphPad, San
Diego, CA, USA). Pure compounds demonstrating an IC50 value
higher than 20 μg/mL were regarded as inactive samples.
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR and MS spectral data of compounds 1−11, X-ray
structures of 6 and 11, and detailed results of biological assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*(S.M.) Tel: +92 42 99231581, ext. 544. Fax: 92 42 99230703.
E-mail: saminamehnaz@fccollege.edu.pk. (H.G.) Tel: +49 7071
2976970. E-mail: harald.gross@uni-tuebingen.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge M. Hofte, Ghent University,
■
̈
Antifungal Bioassay. The antifungal activity of PB-St2 was
examined using four strains of Colletotrichum falcatum, isolated from
local sugar cane varieties (BF166, C01148, CP77400, and SPF234).
Potato dextrose agar (PDA; Difco, MI, USA) was used for these
assays. A small plug cut from a growing edge of the mycelium of the
target fungus was seeded at the center of the medium in a Petri plate.
Aliquots (25, 50, 75, 100, 150, and 200 μg) of purified compounds
dissolved in suitable solvents (methanol, chloroform, or DMSO) were
applied on sterilized filter paper discs, placed 2 cm away from the
fungal plug, and air-dried in a laminar flow cabinet before incubation.
Plates were incubated for 10−15 days at 25 °C. Antifungal activity was
observed by formation of a zone of inhibition of mycelial growth
around the disc. Discs with methanol, chloroform, and DMSO were
used as negative controls. These assays were repeated three times in
duplicate. Pure substances demonstrating no effect up to 100 μg per
disc were regarded as inactive.
Belgium, and J. M. Raaijmakers, Wageningen University, The
Netherlands, for providing the strains Rhizoctonia solani AG2-
2/AG4 and Pseudomonas fluorescens SWB 25, respectively. We
thank S. Kehraus, University of Bonn, for invaluable help
regarding structure elucidation. S.M. thanks Q. Zaheer-ul-Haq,
University of Karachi, for technical support. H.G. and S.M.
would like to gratefully acknowledge the generous contribution
of the Alexander von Humboldt Foundation, which provided
financial support for conducting this research (Georg Forster
Fellowship awarded to S.M.). G.S. thanks A. C. Filippou,
University of Bonn, for support. This research was also
supported by the Deutsche Forschungsgemeinschaft (DFG)
grants GR 2673/2-1 (H.G.) and Sa292/13-1 (H.G.S.) within
the Research Unit FOR854.
Disc Diffusion Soft-Agar Colony Formation Assay. An in vitro
cell-based assay was employed to identify the cytotoxicity and the solid
tumor selectivity for pure compounds. Cell lines used in this study
included cell lines of murine (L1210 leukemia, C38 colon carcinoma,
CFU-GM normal) and human origin (H116 colon carcinoma, H125
lung adenocarcinoma, MCF-7 and MDA-MB-231 breast adenocarci-
noma, LNCaP prostate adenocarcinoma, OVCAR-3 ovarian adeno-
carcinoma, U251N glioma, Panc-1 pancreatic carcinoma, CCRF-CEM
leukemia, CFU-GM normal). Samples were dissolved in 250 μL of
DMSO, and a 15 μL aliquot was applied to a cellulose disc in an agar
plate containing cells. After a period of incubation, a zone of cell
colony inhibition (z) was measured from the edge of the disc to the
edge of colony growth and expressed as zone units (zu), where 200 zu
was equal to 6 mm. General cytotoxic activity for a given sample was
defined as an antiproliferation zone of 300 zu or greater. The
differential cytotoxicity52 of a pure compound was expressed by
observing a zone differential of 250 units or greater between any solid-
tumor cell (murine or human) and their corresponding leukemia cells
or normal cells.
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