1590 Journal of Natural Products, 2008, Vol. 71, No. 9
McAlpine et al.
glycerol, bacto-peptone, and oceanic salt were purchased from Sigma-
Aldrich. Yeast extract and beef extract were from BD Biosciences.
Genome Scanning. The genome of Micromonospora sp. 046Eco-
11, a strain in the Thallion collection, was analyzed by the genome
scanning technique as described.13 The DNA that comprises the ECO-
046D gene cluster is available from the deposited E. coli DH10B
vectors, each harboring a cosmid clone (designated as 046KM and
046KQ, respectively) of a partial biosynthetic locus for 1 and together
spanning the full biosynthetic locus. The strains containing cosmid
clones designated 046KM and 046KQ were assigned deposit accession
numbers IDAC 250203-06, and IDAC 250203-07, respectively, at the
International Depositary Authority of Canada, Bureau of Microbiology,
Health Canada, 1015 Arlington St., Winnipeg, Manitoba, Canada R3E
3R2, on February 25, 2003.
Feeding Experiments. Micromonospora sp. 046Eco-11 was main-
tained as frozen glycerol stock at -80 °C. Inoculum for fermentation
was obtained by streaking freshly thawed frozen stock onto glucose
yeast peptone agar (Sigma G-9663) and incubating at 30 °C until
sporulation was apparent (15 to 20 days). Four loopfuls of vegetative
mycelium and spores were homogenized in 25 mL of KH medium [10
g/L glucose, 20.0 g/L potato dextrin type IV, 5.0 g/L yeast extract, 5.0
g/L NZ Amine, and 1.0 g/L CaCO3 in tap water, with the pH adjusted
to 7.0 before the addition of CaCO3]. The seed (0.5 mL) was used to
inoculate either modified Bennett’s medium [1.0 g/L yeast extract, 1.0
g/L beef extract, 2.0 g/L NZ Amine A, 15.0 g/L glucose, 34.4 g/L
oceanic salt in tap water] (25 mL × 4), in 125 mL flasks supplemented
with 5% activated HP20, or in HI medium [30.0 g/L glycerol, 20.0
g/L potato dextrin, 2.5 g/L bacto-peptone, 8.3 g/L yeast extract, 1.0
g/L CaCO3 in tap water (pH 7.0 adjusted before the addition of CaCO3)]
(25 mL × 4) in 125 mL flasks. Each medium was supplemented with
4,6-D2-3-hydroxyanthranilic acid at 0.5 mg/mL before inoculating with
the vegetative culture at 2%. Control cultures, with or without
3-hydroxyanthranilic acid (0.5 mg/mL), were also prepared in order to
determine any feedback inhibition of 1 production by 3-hydroxyan-
thranilic acid. The culture was incubated either 7 days for HI medium
or 9 days for Bennett’s medium at 28 °C on a rotary shaker (250 rpm).
The labeled precursor feeding experiments conducted at Wyeth were
performed with Micromonospora sp. DPJ15, a related strain to DPJ12,
also isolated from Didemnum proliferum Kott. For fermentation seed
inoculum, cultures of DPJ15 were streaked onto YPSS agar (4.0 g/L yeast
extract, 0.5 g/L K2HPO4, 10.0 g/L Difco soluble starch, 15.0 g/L Bacto
agar, 500 mL of artificial seawater, 500 mL of distilled water, pH 7.2)
from frozen stock. After confluent growth appeared on the agar plates
(about 7 days), spores and mycelia of DPJ15 were scraped into several 25
× 150 mm seed tubes each containing 7 mL of YPSS broth. After 3 days
of incubation at 28 °C with agitation (200 rpm on a rotary shaker), the
seed stages were combined into one vessel and used to inoculate the
production fermentation at 2.5% volume-to-volume.
hydroxyanthranilic acid (0.75 g, 4.8 mmol). The 1H NMR spectrum [δH
7.37, 6.80, 6.47; integration ratio 1:1:8] of the product suggested that it
was a mixture of 4,6-D2-3-hydroxyanthranilic acid and partially deuterated
and nondeuterated 3-hydroxyanthranilic acid, which was used for the
feeding experiment described above.
Extraction. At harvest, cultures of each experiment (four flasks) were
pooled and stored overnight at -20 °C after adjusting to pH 3 with 20%
H2SO4. The culture broth was centrifuged at 3000 rpm (15 min) in order
to separate the mycelial cake, which was then extracted with methanol (3
× 35 mL). The combined methanolic extracts were dried under reduced
pressure and resuspended in MeOH (10 mL). The suspension was filtered
through a 0.45 µm × 13 mm Acrodisc GHP syringe, and the filtrate was
subjected to isolation and liquid chromatography mass spectrometry (LC-
MS) analysis.
LC-MS Analyses. The LC-MS analyses of the MeOH extracts (50
µL) described above were performed on a Waters Alliance 2695 instrument
with electrospray source using a Symmetry C18 5 µm, 60 Å, 4.6 × 150
mm column (Waters). A linear gradient of water/acetonitrile containing
0.2% formic acid (0.2% formic acid in acetonitrile/0.2% formic acid in
water, 50:50-100:0, 0-9 min; acetonitrile alone up to 14 min; 100:0-50:
50, 14-16 min, 50:50 run up to 20 min) was used as eluent, in which 1
eluted at 6.24 min. A standard calibration curve (concentration 0.2-5.0
µg vs area under UV at 290 nm) was plotted in order to calculate the
concentration of 1 in each culture described above.
Isolation of 1. The MeOH extracts from the labeled experiments in
both HI and Bennett’s medium described above were subjected to
semipreparative HPLC purification using a Waters Delta 600 HPLC
coupled with a diode array UV detector. Multiple injections of no more
than 500 µL samples on a Waters RCM Nova-Pak HR C18 6 µm, 60 Å,
25 × 200 mm column (water/acetonitrile 80:20-30:70, 0-8 min; 30:
70-0:100, 8-18 min at 20 mL/min) afforded 1 (tR 13.5 min), 1.5 mg/L
and 0.5 mg/L in HI and Bennett’s medium, respectively (Figure S2,
Supporting Information).
1: UV (MeOH) λmax (log ε) 220 (4.6), 240 (4.4), 290 (3.8) nm; MS
(ESI in positive mode) m/z 463.36 (M + H)+, 271.15, 243.31; MS
(ESI in negative mode) m/z 461.21 (M - H)-, 255.99. The 1H and 13
NMR data are in Table S1, Supporting Information.
C
1
Supporting Information Available: H and 13C NMR assignments
for 1. HPLC (UV scan) of the MeOH extract from the label incorporation
experiment with 4,6-D2-3-hydroxyanthranilic acid in HI medium. Down-
1
field portion of the H NMR spectra of labeled and unlabeled 1. This
References and Notes
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5946–5959.
All precursor feedings at Wyeth were performed in 100 mL of Bennett’s
medium (10.0 g/L dextrose, 0.77 g/L beef extract, 1.0 g/L yeast extract,
2.0 g/L NZ Amine A, in distilled water, pH 7.3) per 500 mL Erlenmeyer
shake flask. The following labeled compounds were added at 500 µg/mL
preautoclaving: L-tryptophan-15N2, L-tryptophan-2′,4′,5′,6′,7′-D5 (indole-
D5), anthranilic acid-15N (Cambridge Isotope Laboratories, Inc.), and 4,6-
D2-3-hydroxyanthranilic acid. Gentle heating in a 50 °C water bath was
used to completely dissolve the precursors, and the pH was readjusted to
7.3. Activated sterile HP20 resin in water was added to each flask at 5%
volume-to-volume post autoclaving. The fermentations were incubated at
28 °C with agitation.
To follow the incorporation of the labeled precursors, 1 mL was sampled
from each fermentation on the third, fourth, and seventh days of incubation.
After centrifugation to pellet the cells and resin, the supernatants were
decanted and the pellets were extracted with methanol. Extracts concen-
trated to 10× were analyzed by LC-MS.
Synthesis of 4,6-D2-3-Hydroxyanthranilic Acid. The 4,6-D2-3-
hydroxyanthranilic acid was synthesized according to the published
protocol.14 In brief, 3-hydroxyanthranilic acid (1.08 g, 7.1 mmol) was
placed in a thick-walled screw-capped tube, and deuterated water (D2O,
20 mL) was added to the tube after flushing with nitrogen. Potassium tert-
butoxide (1.54 g, 13.7 mol) was added to the tube; the cap was screwed
on tightly and the tube was heated at 100 °C for 3 days. The reaction
mixture was then cooled to room temperature and acidified to pH 6 with
6 N HCl. The colorless precipitate, which appeared during acidification,
was separated by filtration and dried under vacuum, to yield 4,6-D2-3-
NP800376N