Bioactive Metabolites from Pseudomonas sp.
Journal of Natural Products, 2005, Vol. 68, No. 9 1383
Scheme 1. Lactone Formation of 1 and 2 under Acidic Conditions
for determination of 1H and 13C frequencies and connectivities.
For complete structure elucidation, 1D 1H NMR, COSY,
TOCSY, NOESY, DEPT-HSQC, and HMBC were applied.
Chemical shifts were determined relative to internal CHCl3
(δC 77.23; δH 7.27), DMSO-d5 (δH 2.50), or CH3OH-d3 (δC 49.15;
δH 3.31). Positive and negative mode ESIMS were obtained
on an ion-trap instrument with CH3OH as solvent. LC-MS was
done with the same mass spectrometer coupled to an analytical
HPLC system. HRFABMS was performed on a four-sector
tandem mass spectrometer with glycerol as matrix and PEG
as internal standard. SPE was done with 1 or 10 g prepacked
columns or columns packed in-house with bulk C18 material.
Preparative HPLC was run at a flow of 10 mL/min with UV
monitoring at 210 or 254 nm. Fractions were collected in
polypropylene 2-mL square well plates. For the mobile phase
CH3CN of HPLC gradient grade and deionized filtered H2O
were used.
Isolate Origin and Identity. The psychrotrophic Pseudomo-
nas sp. strain MF 381-IODS was isolated, as previously
described,19 from the roots of a field-grown oilseed rape
specimen [Brassica napus ssp. oleifera (DC.) Metzger (Cru-
ciferae)] collected in Lipperswil, Switzerland, in March 1997.
The strain was identified as belonging to the genus Pseudomo-
nas, RNA group I, by the German Collection of Microorganisms
and Cell Cultures (DSMZ) by means of physiological tests,
cellular fatty acid analysis, and partial sequencing of the 16S
rDNA.
Production of Bacterial Cultures. Cultures of the isolate
MF381-IODS were produced on Mineral Medium (MM) for
Pseudomonas28 modified by adding 125 mg of citric acid, 12.5
mg of Na2MoO4 × 2 H2O, and 250 mg of trichloroacetic acid
per 1000 mL of the medium, with 0.1% glycerol as the sole
carbon source. The MM cultures were started by transferring
10 + 10 mL of 24 h old inocula, which were grown in half-
strength vegetable peptone broth [15 g of VPB in 1000 mL of
deionized H2O] and in nutrient broth [8 g of NB in 1000 mL
of deionized H2O], respectively. The cultures were incubated
on a rotary shaker (120 rpm) for 68-72 h at 20-25 °C. Cells
were removed by centrifugation (11 000 rpm, 15 min, 4 °C),
and cell-free supernatants were immediately fractionated by
SPE or stored at -70 °C before being processed.
Sample Workup and Isolation Procedures. Five liters
of cell-free supernatant was fractionated on a 150-g SPE
column. The column was packed and activated with 500 mL
of CH3CN and equilibrated with 500 mL of H2O before sample
loading. Hydrophilic components were washed out with 500
mL of aqueous 5% CH3CN followed by 500 mL of 20% CH3CN
in H2O before the lipophilic fraction was eluted with 700 mL
of aqueous 95% CH3CN. Fractionation was done in three
HPLC steps with in vitro bioassay verification of antimicrobial
activity in between. The first run was a gradient from 20% to
100% CH3CN in H2O in 10 min with an 8 min hold at 100%.
The second run was isocratic at aqueous 63% CH3CN, yielding
3 (2.0 mg) and 4 (1.5 mg). The final isocratic step was aqueous
53% CH3CN, which yielded 1 (1.7 mg) and 2 (14 mg). The
column used in steps 1 and 3 was a Reprosil-pur C18 (100 ×
20 mm with guard column 30 × 20 mm, 5 µm) and in step 2
a Kromasil C18 (150 × 21.2 mm, 5 µm). CH3OH was used for
sample transfer prior to spectroscopic analysis.
culmorum, Drechslera sorokiniana, and Staphylococcus aureus
were used as test organisms. Aliquots from the HPLC fractions
were transferred into 96-well microtiter plates, and the solvent
was evaporated. Spore suspensions (F. culmorum and D.
sorokiniana) or cell suspensions (S. aureus), 100 µL at a
concentration of 104 spores or cells/mL, were added to the
wells, resulting in sample concentrations 200-400 times
higher than in the culture supernatant. Spore/cell suspensions
were used as positive controls, and sterile medium was used
as negative control. The MIC values of 1 and 2 were deter-
mined using a microtiter plate bioassay with the organisms
Aspergillus fumigatus, Candida albicans, D. sorokiniana, F.
culmorum, F. oxysporum, Heterobasidion annosum, Mi-
crosporum canis, Pseudomonas sevastanoi, P. syringae pv.
syringae, and S. aureus. All MIC tests were performed in
triplicate and repeated once.
(2E,4E,6E)-9-[((2S,3R)-3-Hydroxy-4-{[(3E,5E,7RS)-7-
hydroxy-4-methylhexadeca-3,5-dienoyl]amino}-2-meth-
ylbutanoyl)amino]nona-2,4,6-trienoic acid (pseudotri-
1
enic acid A, 1): colorless oil; H NMR (CD3OD, 600 MHz),
see Table 1; 13C NMR (CD3OD, 150 MHz), see Table 1; ESIMS
m/z 569.2 [M + Na]+; ESIMS m/z 545.2 [M - H]-; HRFABMS
569.3541 (calcd for C31H50O6N2Na, 569.3567).
(2E,4E,6E)-9-[((2S,3R)-3-Hydroxy-4-{[(3E,5E,7RS)-7-
hydroxy-4-methyltetradeca-3,5-dienoyl]amino}-2-meth-
ylbutanoyl)amino]nona-2,4,6-trienoic acid (pseudotri-
1
enic acid B, 2): colorless oil; [R]20 +1.9° (c 0.4, MeOH); H
D
NMR (CD3OD, 600 MHz), see Table 1; 13C NMR (CD3OD, 150
MHz), see Table 1; ESIMS m/z 541.4 [M + Na]+; HRFABMS
541.3208 (calcd for C29H46O6N2Na, 541.3254).
Pseudotrienic acid A lactone (1a): 1H NMR (CD3OD,
600 MHz) δ 7.26 (1H, dd, H-3), 6.59 (1H, dd, H-5), 6.31 (1H,
dd, H-4), 6.26 (1H, dd, H-6), 6.26 (1H, dd, H-19), 6.06 (1H, d,
H-18), 5.94 (1H, m, H-7), 5.85 (1H, d, H-2), 5.66 (1H, dt, H-20),
4.42 (1H, dd, H-16), 3.70 (1H, dt, H-12), 3.41 (1H, ddd, H-13b),
3.28 (2H, dt, H-9), 3.16 (1H, ddd, H-13a), 2.43 (2H, dd, H-15),
2.36 (2H, m, H-8), 2.34 (1H, m, H-11), 2.10 (2H, m, H-21), 1.75
(3H, s, CH3-17), 1.38 (1H, m, H-22b), 1.32 (1H, m, H-22a), 1.31
(10H, m, H-23 to H-26 and H-28), 1.28 (2H, m, H-27), 1.14
(3H, d, CH3-11), 0.90 (3H, t, H-29); 13C NMR (CD3OD, 150
MHz) δ 177.3 (C, C-10), 174.5 (C, C-14), 170.3 (C, C-1), 145.7
(CH, C-3), 141.6 (CH, C-5), 137.3 (C, C-17), 137.1 (CH, C-7),
136.2 (CH, C-20), 132.9 (CH, C-6), 129.6 (CH, C-4), 127.1 (CH,
C-19), 126.7 (CH, C-18), 121.9 (CH, C-2), 75.2 (CH, C-16), 73.1
(CH, C-12), 45.4 (CH, C-11), 44.4 (CH2, C-13), 43.4 (CH2,
C-15), 39.3 (CH2, C-9), 34.1 (CH2, C-21), 33.7 (CH2, C-8), 33.0
(CH2, C-27), 30.5 (4CH2, C-23 to C-26), 26.3 (CH2, C-22),
23.1 (CH2, C-28), 14.7 (CH3, CH3-11), 14.3 (CH3, C-29), 12.4
(CH3, CH3-17); diagnostic HMBC connectivities, H-15fC-14;
CH3-17fC-16, 17, 18; ESIMS m/z 527.5 [M - H]-.
Pseudotrienic acid B lactone (2a): 1H NMR (CD3OD, 600
MHz) δ 7.26 (1H, dd, H-3), 6.59 (1H, dd, H-5), 6.31 (1H, dd,
H-4), 6.26 (1H, dd, H-6), 6.26 (1H, dd, H-19), 6.06 (1H, d, H-18),
5.94 (1H, m, H-7), 5.85 (1H, d, H-2), 5.66 (1H, dt, H-20), 4.42
(1H, dd, H-16), 3.70 (1H, dt, H-12), 3.41 (1H, ddd, H-13b), 3.28
(2H, dt, H-9), 3.16 (1H, ddd, H-13a), 2.43 (2H, dd, H-15), 2.36
(2H, m, H-8), 2.34 (1H, m, H-11), 2.10 (2H, m, H-21), 1.75
(3H, s, CH3-17), 1.38 (1H, m, H-22b), 1.32 (1H, m, H-22a), 1.31
(6H, m, H-23, H-24 and H-26), 1.28 (2H, m, H-25), 1.14 (3H,
d, CH3-11), 0.90 (3H, t, H-27); 13C NMR (CD3OD, 150 MHz) δ
177.3 (C, C-10), 174.5 (C, C-14), 170.3 (C, C-1), 145.7 (CH, C-3),
141.6 (CH, C-5), 137.3 (C, C-17), 137.1 (CH, C-7), 136.2 (CH,
C-20), 132.9 (CH, C-6), 129.6 (CH, C-4), 127.1 (CH, C-19), 126.7
In Vitro Bioassay and MIC Determination. Antimicro-
bial fractions were identified during isolation using a previ-
ously developed protocol, based on inhibition of spore germi-
nation or cell growth in microtiter plates.29,30 Fusarium