1
456 The Journal of Natural Products, 2007, Vol. 70, No. 9
Macherla et al.
low to neutral pH values, and rapid hydrolysis to 5 in concentrated
acid (see Figure 2). Lipoxazolidinone A (1) was completely
converted to compound 4 at pH values between 4 and 7 within 6
days. This conversion was very slow at higher pH (20% conversion
after 6 days at pH 9). A pure sample of compound 4 was prepared
by hydrolysis of 1 in an aqueous/organic solution at pH 7.4. The
Media and Culturing Conditions for 1, 2, and 3. Frozen stock
cultures of strain NPS008920 were inoculated in 100 mL of seed
medium and allowed to grow for 3–4 days at 28 °C on a rotary shaker
operating at 250 rpm. The seed medium consisted of the following per
liter of sea water: starch, 10 g; yeast extract, 4 g; and peptone, 2 g.
This seed culture was then divided (5 mL each) between nine 500 mL
flasks containing 100 mL of the same seed medium. After 2–3 days of
growth, 5 mL aliquots of this seed culture were inoculated into 500
mL flasks containing 100 mL of production medium having the same
composition of the seed medium. The number of production flasks
inoculated depended on the desired size of fermentation. After 5 days
of growth, the culture broth was extracted with equal volume of EtOAc,
and the extract was concentrated under reduced pressure.
+
LRESIMS data (m/z 340; [M + H] ) clearly indicated the addition
of one molecule of water. The λmax shift from 310 to 245 nm
indicated that the conjugation in the molecule was broken. Analysis
of the NMR spectra indicated the presence of a new isolated
C H
methylene group (δ 50.3; δ 3.67 and 3.56, each 1H, d, J ) 16.4
Hz) between the C-2′ (δ 193.5) carbonyl carbon and C-2 (δ 166.6),
which is in agreement with a double-bond shift to the nitrogen.
The remaining structural elements were confirmed by COSY,
HSQC, and HMBC data. Lipoxazolidinone A (1) was completely
degraded in the presence of strong acid (6 N HCl) at 40 °C. The
major degradation product (5) was characterized by LC-MS, from
Purification of Compounds 1 and 2. The crude extract (0.38 g)
of NPS008920 obtained as described above was dissolved in MeOH
(
19 mL) and injected in 950 µL aliquots (19 mg each) on preparative
reversed-phase HPLC (column: C18-HL, 5 um, 21 mm i.d. × 15 cm;
detection: DAD) using a solvent gradient of 20% CH CN/H O to 80%
CH CN/H O in 12 min; 80–100% CH CN/H O in 1 min, and then 9
min at 100% CH CN with a flow rate of 14.5 mL/min.
3
2
3
2
3
2
+
which the observed molecular ion (LRESIMS m/z 226 [M + H] )
3
suggested that the oxazolidinone ring had been completely hydro-
lyzed; the expected byproduct of this reaction is 6.
Lipoxazolidinones A (1), B (2), and C (3) and hydrolysis product
Compounds 1 and 2 eluted at 21 and 23 min, respectively. These
compounds were further purified by RP semipreparative HPLC using
a Hamilton Polymeric RP (PRP-1) column (10 mm i.d. × 25 cm; 10
um) using the solvent gradient of 40–80% CH
3
CN/H
2
O in 8 min;
4
were screened against a panel of various Gram-positive and Gram-
8
0–100% CH CN/H O in 1 min, and then 15 min at 100% CH CN
3 2 3
negative bacteria (Table 2). Compound 1 showed broad spectrum
activity, with minimum inhibitory concentration (MIC) values
ranging from 0.5 to 5 µg/mL against Gram-positive bacteria and
with a flow rate of 3 mL/min.
2
1
Lipoxazolidinone A (1): colorless oil, [R]
D
-31 (c 0.02, MeOH);
1
UV (MeOH) λmax (log ε) 310 (4.36), 253 (3.96) nm; H NMR (DMSO-
1
2 µg/mL against two strains of Haemophilus influenzae. The
d , 500 MHz) δ 5.98 (1H, br s, H-3′), 5.28 (1H, s, H-1′), 4.88 (1H, dd,
6
antibacterial spectrum and potency of 1 were similar to those of
J ) 4.7, 6.6 Hz, H-5), 2.11 (3H, br s, H-12′), 2.08 (2H, br t, J ) 7.5
Hz, H-5′), 1.84 (1H, m, H-1a′′), 1.71 (1H, m, H-1b′′), 1.43 (2H, m,
H-6′), 1.31 and 1.30 (2H, m, H-2′′ and H-3′′), 1.28 (2H, m, H-10′),
1.25 (6H, m, H-7′ to H-9′), 0.87 (3H, t, J ) 6.6 Hz, H-4′′), 0.86 (3H,
6
the commercially available antibiotic linezolid (Zyvox). Com-
pounds 2 and 3 also showed broad spectrum antibacterial activity,
albeit with lesser overall potency than 1. In contrast, hydrolysis
product 4 showed only weak activity against MSSA, indicating the
importance of an intact oxazolidinone ring system. While the
oxazolidinone heterocycle is a common structural motif shared by
the lipoxazolidinones and linezolid, the compounds are clearly
distinguished as 4- and 2-oxazolidinones, respectively, and each
class is uniquely substituted. Thus, the 4-oxazolidinones offer a
unique scaffold with antibiotic therapeutic potential.
1
3
t, J ) 6.6 Hz, H-11′); C NMR (DMSO-d
6
, 125 MHz) δ 187.9 (C,
C-2′), 173.8 (C, C-4), 165.3 (C, C-2), 155.4 (C, C-4′), 124.6 (CH, C-3′),
8
2
2
3.6 (CH, C-1′), 77.6 (CH, C-5), 40.4 (CH
9.9 (CH , C-1′′), 28.6 (CH , C-8′), 28.4 (CH
5.6 (CH , C-2′′), 22.0 (CH , C-10′), 21.6 (CH
, C-11′), 13.6 (CH , C-4′′); see Table 1 for H and
; HRESIMS m/z 322.2372 [M + H]+ (calcd
C NMR data in CDCl
, 322.2382; ∆ ) 3.3 ppm).
Lipoxazolidinone B (2): colorless oil, UV (CH O) λmax 310,
2
, C-5′), 31.1 (CH2, C-9′),
, C-7′), 27.0 (CH , C-6′),
, C-3′′), 18.4 (CH
2
2
2
2
2
2
2
3
,
1
C-12′), 13.9 (CH
3
3
13
3
for C19H32NO
3
3
CN/H
55 nm; see Table 1 for H and C NMR data; HRESIMS m/z 336.2527
2
Experimental Section
1
13
2
+
General Experimental Procedures. Optical rotations were mea-
sured on a Rudolph Autopol III polarimeter using a 4 × 100 mm sample
cell. UV spectra were obtained with a Beckman Coulter DU 640
spectrophotometer or from analytical HPLC analysis of the purified
compound using an Agilent HP1100 HPLC equipped with an Agilent
[M + H] (calcd for C20
Purification of Compound 3. The crude extract (1.2 g) of
NPS008920 obtained as described above was dissolved in H O (100
3
H34NO , 336.2539; ∆ ) 3.5 ppm).
2
mL) and extracted with hexanes (3 × 100 mL). The combined organic
layer was concentrated to yield about 570 mg of crude material enriched
in 1–3, which was purified by preparative scale RP HPLC (C18-HL, 5
µm, 21 mm i.d. × 15 cm; DAD detection) using a solvent gradient of
PDA detector; the mobile phase was a mixture of CH
NMR spectra were collected using a 500 MHz Bruker Avance NMR
3 2
CN and H O.
13
spectrometer with an inverse probe, except for the C NMR spectrum,
which was acquired with a broad-band observe probe. All NMR data
3 2 3
50% CH CN/H O to 100% CH CN in 12 min and then 13 min at 100%
CH
3
CN with a flow rate of 14.5 mL/min. Compound 3 eluted at about
were acquired at 298 K in CDCl
3
or DMSO-d
6
, referenced to 7.24 and
18 min as a minor compound. This compound was further purified by
2
.50 ppm in the proton spectra and 77.00 and 39.00 ppm in carbon
RP preparative HPLC (C18-HL, 5 µm, 21 mm i.d. × 15 cm; detection:
spectra, respectively. High-resolution mass spectra were acquired using
a Micromass Q-Tof2 mass spectrometer with ES+. HRESI spectra were
referenced using a poly(ethylene glycol) polymer mixture, which was
coinjected during acquisition as an internal accurate mass standard.
Additional ESI-MS and crude extract analysis experiments were
collected using an Agilent HP1100 HPLC equipped with an Agilent
PDA detector and an 1100 series MSD Agilent mass spectrometer.
HPLC was performed on a Gilson HPLC equipped with a Gilson 215
fraction collector and an Agilent PDA detector. For RP HPLC
separations, an Eclipse XDB-C18, 5 µm, 150 × 21 mm i.d. column
was used at a flow rate of 14.5 mL/min. HPLC solvents were obtained
from Fisher Scientific and VWR, and H O used for HPLC was filtered
2
through a NANOpure Infinity ultrafilter. Deuterated solvents were
obtained from Cambridge Isotope Laboratories, Inc.
Microorganism. The producing culture, NPS008920, was collected
from Cocos Lagoon, Guam, in January 2002. Strain NPS008920 was
deposited with the American Type Culture Collection and assigned the
accession number PTA-6527. The 16S rRNA sequence was submitted
to GenBank and assigned the accession number EF470589. The 16S
rRNA sequence of strain NPS008920 matched closely to those of the
2
DAD) using a solvent gradient of 20–80% MeOH/H O in 12 min; 80%
2
MeOH/H O to 100% MeOH in 1 min, and then 9 min at 100% MeOH
with a flow rate of 14.5 mL/min. Lipoxazolidine C eluted as a pure
compound at about 18.5 min.
Lipoxazolidinone C (3): colorless oil; UV (CH
3
CN/H
2
O) λmax 310,
1
13
255 nm; for H and C NMR data, see Table 1; HRESIMS m/z
+
3
308.2234 [M + H] (calcd for C18H30NO , 308.2226; ∆ ) 2.8 ppm).
Preparation of Compound 4 from 1. Compound 4 was obtained
by adding pH 7.4 buffer (2 mL; 75 mM Tris buffer) into a solution of
1 (4.2 mg) in CH
for about 4 days. The CH
reduced pressure, and the remaining aqueous layer was extracted with
CH Cl
3
CN (2 mL) and letting it stand at room temperature
3
CN was removed by evaporation under
2
2
(3 × 20 mL). The combined organic layer was concentrated
to yield 4 (3.2 mg).
1
3 2
Compound 4: colorless oil; UV (CH CN/H O) λmax 245 nm; H
NMR (CDCl , 500 MHz) δ 6.02 (1H, br s, H-3′), 5.24 (1H, dd, J )
3
3.8, 8.5 Hz, H-5), 3.67 (1H, d, J ) 16.4 Hz, H-1′a), 3.56 (1H, d, J )
16.4 Hz, H-1′b), 2.15 (2H, br t, J ) 7.6 Hz, H-5′), 2.15 (3H, br s,
H-12′), 1.96 (1H, m, H-1′′a), 1.82 (1H, m, H-1′′b), 1.46 (2H, m, H-6′),
1.34–1.31 (4H, m, H-2′′ and H-3′′), 1.26–1.23 (8H, m, H-7′ to H-10′),
4
,5
13
newly proposed genus Marinispora.
0.88 (3H, t, J ) 7 Hz, H-4′′), 0.87(3H, t, J ) 7 Hz, H-11′); C NMR