A R T I C L E S
Wilson et al.
leading to the truncated saliniketals and mature rifamycins
CHCl
3
(1:9). Silica fractions were further purified by HPLC on an
(
Figure 7). We are presently probing the multifunctional
RP C18 column (Phenomenex Luna, 10 mm ×250 mm, 5 µm,
MeCN/H O 56:44, v/v).
4a-Deoxyrifamycin W (7). HPLC ESI-MS: [M + Na] 662.3;
H NMR (600 MHz, DMSO-d ) δ 12.40 (s, 8-OH), 10.54 (br s,
-OH), 9.59 (s, NH), 7.47 (s, H-3), 6.32 (dd, J ) 15.5, 11.2 Hz,
Sare1259 to explore its preferred substrates, its reaction profile,
and its molecular mechanism that likely will lead to new P450
catalyzed chemistry.
2
+
3
1
6
6
H-18), 6.16 (d, J ) 10.8 Hz, H-17), 5.99 (dd, J ) 15.6, 6.9 Hz,
H-19), 4.76 (d, J ) 6.5 Hz, 23-OH), 4.35 (s, 21-OH), 4.31 (d, J )
5.8 Hz, 27-OH), 3.84 (d, J ) 5.6 Hz, H-27), 3.80-3.73 (m, H-25),
3.68 (d, J ) 6.5 Hz, 25-OH), 3.35-3.28 (m, H-23), 2.40 (dd, J )
15.1, 7.3 Hz, H-28), 2.20 (dd, J ) 14.6, 7.2 Hz, H-20), 2.13 (s,
H-14), 2.00 (s, H-30), 1.89 (s, H-13), 1.65 (br d, J ) 6.8 Hz, H-22),
1.59-1.51 (m, H-24), 1.25-1.15 (m, H-26), 0.95 (d, J ) 7.0 Hz,
H-34a), 0.91 (d, J ) 6.9 Hz, H-32), 0.81 (d, J ) 6.8 Hz, H-31),
0.55 (d, J ) 6.7 Hz, H-33), 0.18 (d, J ) 6.9 Hz, H-34).
Experimental Section
General Methods. Low-resolution LC/MS was carried out on
a Hewlett-Packard series 1100 LC/MS system in positive ion mode
with a linear gradient of 10-90% MeCN at a flow rate of 0.7 mL/
min over 24 min on an RP C18 column (Phenomenex Luna, 4.6
1
mm ×100 mm, 5 µm). H, heteronuclear multiple bond correlation
(
gHMBC) and heteronuclear single quantum coherence (gHSQC)
NMR spectral data were obtained on a Bruker DRX600 spectro-
13
photometer equipped with a 1.7 mm cryoprobe. C NMR spectral
data were obtained on a Varian VX-500 instrument equipped with
an XSens cold probe. Rifamycin SV (4) sodium salt was purchased
from Sigma Aldrich (St. Louis, MO). Saliniketal A (1) was
Chemical Complementation of S. arenicola Mutants. Chemical
complementation studies were carried out in 50 mL cultures of
the S. arenicola rifK::aprR mutant in A1 production media.
Compounds 4-8 (2-6 mg) were individually added in triplicate
1
R
graciously provided by William H. Fenical. Rifamycin W (7), 27-
to 1 day old production cultures of rifK::apr . The cultures were
O-demethyl-24-O-desacetylrifamycin SV (6, DMDARSV), and 27-
O-demethylrifamycin SV (5, DMRSV) were isolated and confirmed
based on literature precedence.
then allowed to grow for 4 more days before being extracted as
described above. Bioconversion of the rifamycin intermediates
was monitored by HPLC-MS. Retention times, m/z ratios, and
UV profiles of the biotransformation products were compared
to authentic standards.
1
3,16,17
Bacterial Strains, Culture Conditions, and Extraction of
Natural Products. Salinispora arenicola strain CNS-205 and
AmycolatopsismediterraneistrainsS699,MT45025H,andMT1601KH
Synthesis of 15N-AHBA. AHBA was synthesized by reacting
1
3,20
3
,5-dihydroxybenzoic acid (2.0 g, 13 mmol) with NH
4
Cl (1.7 g,
were obtained as described previously.
All S. arenicola seed
3
2 mmol) and aqueous NH OH (14.8N, 6 mL, 89 mmol) in a sealed
high-pressure reaction tube at 180 °C for 40 h.
4
cultures were grown in A1 liquid media (10 g of starch, 5 g of
yeast extract, and 2 g of peptone per liter of seawater), and
production cultures were grown in A1BFeC liquid media (10 g of
starch, 5 g of yeast extract, 2 g of peptone, 100 mg of KBr, 40 mg
3
9,40
After cooling,
the volatile components of the reaction mixture were removed in
vacuo and the remaining solid was redissolved in 100 mL of 6 N
HCl. The resulting solution was heated under reflux for 16 h,
filtered, extracted with EtOAc (3 × 40 mL) to remove unreacted
starting material, and concentrated to 25 mL. The desired product
crystallized from this solution yielding gray crystals, which were
collected and recrystallized from 6 N HCl to furnish pure AHBA
hydrochloride as white crystals in 68% overall yield (1.67 g, 8.8
of Fe
2
(SO
4
)
3
·4H
2 3
O, 1 g of CaCO per 1 L of seawater) at 28 °C
2
0
and 225 rpm. Amycolatopsis mediterranei S699 seed and produc-
3
5
tion cultures were both grown in YMG liquid media at 28 °C
and 225 rpm, unless otherwise noted. Extraction of saliniketal and
rifamycin compounds was achieved by acidifying the culture broth
to pH 2-3 with 1 N HCl and extracting the cultures 3× with equal
volumes of EtOAc. The organic fraction was dried under vacuum,
resuspended in MeOH, and analyzed by LC/MS. Escherichia coli
15
mmol). The [ N]AHBA was prepared following the same synthetic
1
5
route but substituting NH
4
Cl with NH
4
Cl. The statistically
1
5
3
6
37
expected N-enrichment of 25% in the product was verified by
mass spectrometric analysis. H NMR (500 MHz, CD
m, 1H), 7.48 (m, 1H), 7.51 (m, 1H). HPLC ESI-MS: AHBA [M
strains EPI300, BW25113/pKD20, and S17-1 used for mu-
tagenesis experiments were grown in Luria-Bertani (LB) media with
appropriate antibiotics.
1
3
OD) δ 7.04
(
+
15
+
+
H] 154.1; [ N]AHBA [M + H] 154.1 (75%), 155.2 (25%).
13
Genetic Manipulations. Inactivation of rifamycin and saliniketal
3
Stable Isotope Labeling Experiments of 1. [U- C ]Propionate
biosynthetic genes was performed using REDIRECT PCR targeting
2
0,38
incorporation experiments were carried out in 2 × 1 L cultures of
technology as described previously for S. arenicola.
E. coli
S. arenicola CNS-205 grown in A1BFeC liquid media at 225 rpm
EPI300 (Epicenter) carrying fosmids BPPW5227, BPAF1230, and
BPAF1361 were provided by the Joint Genome Institute, Walnut
Creek, CA. Each of the genes targeted for inactivation was replaced
with an apramycin resistance (acc(3)IV) cassette by double
crossover homologous recombination on the fosmid containing the
gene of interest. The mutant fosmid was then transformed into E.
coli S17-1 and transferred to S. arenicola CNS-205 via conjugation.
Exconjugates were confirmed by PCR analysis and restriction
digest.
1
3
and 30 °C for 7 days. Sodium [U- C
3
]propionate (50 mg/L)
(
Cambridge Isotopes Laboratories, Incorporated) was aseptically
added to each liter of culture after 48 h. The cultures were extracted
1
with EtOAc, and 1 was isolated as described previously and
13
analyzed by C NMR (Figures S5 and S6, Supporting Information).
1
5
[
N]AHBA incorporation studies were carried out in 50 mL
R
cultures of rifK::apr in A1BFeC at 225 rpm and 30 °C for 5 days.
1
5
[
N]AHBA (3 mg) was added to each culture after 24 h and
R
extracted as described above on day five. Crude extracts were
Isolation of 34a-Deoxyrifamycin W (7) from the sare1259::apr
1
5
R
analyzed by HPLC-MS. Incorporation of N into AHBA and 1
41
Mutant. The sare1259::apr mutant was cultured in 8 × 1 L
was calculated from m/z. HPLC ESI-MS: saliniketal A (1) [M +
Fernbach flasks of A1BFeC media for 7 days at 28 °C while shaking
at 225 rpm. XAD-7 resin (30 g) was added to each flask on day 7
and continued to shake for 5 h before collecting the resin by
filtration. The resin was then extracted with 3 × 1 L of acetone
and concentrated under vacuum. The residue was separated by silica
gel flash chromatography under isocratic conditions with MeOH/
+
15
+
Na] 418.3; [ N]saliniketal A (1) [M + Na] 418.2 (75%), [M +
+
Na] 419.2 (25%).
Acknowledgment. We thank William H. Fenical and Paul R.
Jensen for kindly providing authentic standards of saliniketal A
(
39) Becker, A. M.; Rickards, R. W.; Brown, R. F. C. Tetrahedron 1983,
39, 4189.
(
(
35) Kim, C. G.; Yu, T. W.; Fryhle, C. B.; Handa, S.; Floss, H. G. J. Biol.
Chem. 1998, 273, 6030.
(40) Wang, Z.; Silverman, R. B. Bioorg. Med. Chem. 2006, 14, 2242.
(41) Biemann, K. Mass spectrometry: organic chemical applications;
McGraw-Hill: New York, 1962.
(42) Oppolzer, W.; Prelog, V.; Sensi, P. Experientia 1964, 20, 336.
(43) White, R. J.; Martinelli, E.; Lancini, G. Proc. Natl. Acad. Sci. U.S.A.
1974, 71, 3260.
36) Datsenko, K. A.; Wanner, B. L. Proc. Natl. Acad. Sci. U.S.A. 2000,
9
7, 6640.
(
(
37) Simon, R.; Priefer, U.; Puhler, A. Bio-Technol 1983, 1, 784.
38) Gust, B.; Challis, G. L.; Fowler, K.; Kieser, T.; Chater, K. F. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 1541.
1
2764 J. AM. CHEM. SOC. 9 VOL. 132, NO. 36, 2010