Journal of Natural Products
Article
The 16S gene was cloned using primers 8−27f (5′-AGAGTTT-
GATCCTGGCTCAG-3′) and 1492r (5′-TACGGYTACCTTGT-
TACGA CTT-3′) and submitted to GenBank, accession number
JN982561. The taxonomically described relative with the closest
sequence match was Streptomyces carnosus.
Fermentation and Extraction. During the neurological activity
screening by DRG assay5 and chemistry screening by LS-DAD-MS,
the extract of Streptomyces sp. 1053U.I.1a.1b was not active, but was
shown to contain compounds with unusual MS ion features. In order
to characterize the structures of those compounds, the strain was
cultured at 30 °C with a Bioflo110 fermentor containing 10 L of the
medium ISP2 (0.4% yeast extract, 1% malt extract, 0.4% glucose, 2%
NaCl). After 8 days, the broth was centrifuged and the supernatant
was extracted with HP-20 resin for 4 h. The resin was filtered through
cheesecloth and washed with H2O to remove salts. The filtered resin
was eluted with MeOH to yield an extract.
precedent, in that the organization of substituents such as methyl,
hydroxy, and keto groups has not been previously observed. The
functionality is somewhat related to macrolides, such as
erythromycin and relatives,13 but overall there are no reported
compounds that are close to this organization. In addition, the
β-ketoamide-amino acid motif is reminiscent of many tetramic
acids.14 In the case of tetramic acid synthesis, the amino acid
undergoes a Dieckmann condensation with the side chain, leading
to termination of chain extension,15 but here the compounds are
further extended by amino acids. Finally, 4-O-methylglucose is
usually associated with eukaryotic metabolism, but it has been
found occasionally in actinomycete products such as glycosylated
nucleosides (tubericidin, dapiramicin).16
Totopotensamides were examined in a range of broad-net
phenotypic assays, at concentrations up to 100 μM. The
compounds did not exhibit activity against panels of bacterial,
fungal, or mammalian cell lines. They exhibited no effects on
receptors or ion channels, as gauged using mouse dorsal root
ganglion neurons and human receptor screens at the NIMH
Psychoactive Drug Screening Program. The compounds caused
no observable phenotypic effects on zebrafish larvae, nor did
they affect the producing bacteria as hormones or in any other
observable way. Thus, the biological activity of these unusual
products if any has yet to be determined.
Purification. The extract (500 mg) was separated into four
fractions (Fr1−Fr4) on a C18 column using step-gradient elution of
MeOH in H2O (40%, 60%, 70%, 80%). Fr2, eluting in 60% MeOH,
was passed through a LH-20 column (to remove diketopiperazines).
The early elution fractions from the LH-20 column were combined
according to the HPLC analysis and further purified by C18 HPLC
using 35% CH3CN in H2O with 0.1% TFA to obtain compounds 1
(3.5 mg, tR = 6.4 min) and 2 (1.0 mg, tR = 7.7 min).
Totopotensamide A (1): pale yellow solid; [α]25 +20 (c 0.1,
D
1
MeOH); UV (MeOH) λmax (log ε) 235 (2.11), 282 (1.45) nm; H
and 13C NMR (see Table 1); HRESIMS m/z 1146.5598 [M + H]+
The discovery of new molecules from a bacterium associated
with a new species of mollusk is perhaps only coincidence, but
we feel that linking exploration for biodiversity and natural
products can accelerate discovery in both areas. Complications
in making this assessment include the difficulty in determining
whether bacteria are truly symbiotic or whether they are casual
associates of the animals in question. For example, although we
have shown that actinobacteria are likely specific symbionts of
cone snails,17 in this particular case we do not know whether
the cultivated bacteria are symbionts or casual associates. The
lumun−lumun method, in providing access to animal diversity,
has already been shown to yield new animal species and new
animal venoms6 and could also provide a rich source of new
molecules from associated symbiotic bacteria. This report
represents an early-stage exploration of that potential.
(calcd for C52H84ClN7O19, 1146.5589).
Totopotensamide B (2): pale yellow solid; [α]25 +43 (c 0.1,
D
1
MeOH); UV (MeOH) λmax (log ε) 235 (2.10), 282 (1.35) nm; H
and 13C NMR (see Table 1); HRESIMS m/z 992.4793 [M + Na]+
(calcd for C45H72ClN7O14, 992.4723).
Acid Hydrolysis of Peptides. Compounds 1 and 2 (100 μg each)
were separately dissolved in 6 N HCl (500 μL) and heated in sealed
ampule vials at 110 °C for 16 h. The solvent was removed in vacuo.
LC-MS Analysis of D/L-FDLA Derivatives. The acid hydrolysates
of 1 and 2 were dissolved in H2O separately. To a 50 μL aliquot of
each were added 1 N NaHCO3 (20 μL) and 1-fluoro-2,4-
dinitrophenyl-5-L-leucinamide (1% solution in acetone, 100 μL), and
the mixture was heated to 40 °C for 50 min. The solutions were
cooled to room temperature, neutralized with 1 N HCl (20 μL), and
then dried in vacuo. The residues were dissolved in 1:1 CH3CN−H2O
and then analyzed by LC-MS. The analysis of the L- and D-FDLA
derivatives was performed on a Supelcosil LC-18 column (150 ×
4.6 mm, 5 μm) employing a linear gradient of from 25% to 70%
CH3CN in 0.01 M formic acid at 0.5 mL/min over 45 min. The retention
times of the D- and L-FDLA derivatives, respectively, were as follows: L-Ala:
29.00, 25.30 min, m/z 382 [M − H]−; D-allo-Thr: 21.29, 23.02 min, m/z
412 [M − H]−; D-Dab: 38.95, 44.35 min, m/z 705 [M − H]−; L-ClPhg:
39.22, 31.18 min, m/z 524 [M − H]−; L-Ile: 39.24, 31.16 min, m/z 424
[M − H]−.
LC-MS Analysis of L-FDAA Derivatives. L-FDAA was used to
derivatize the acid hydrolysates of 1 and 2 and three standard amino
acids (D-allo-Thr, D-Thr, and L-Ile). The reaction with L-FDAA was
performed with the same procedure as that used for FDLA. The
retention times of the L-FDAA derivatives were as follows: D-allo-Thr:
13.50 min, m/z 370 [M − H]−; D-Thr: 14.80 min, m/z 370 [M − H]−;
L-Ile: 24.32 min, m/z 382 [M − H]−.
Quantum Mechanical Energy Analysis of FDLA Derivertives
of Phg, DhPhg, and ClPhg. The conformational energetics of each
isomer (L-Phg-L-FDLA, D-Phg-L-FDLA, L-DhPhg-L-FDLA, D-DhPhg-L-
FDLA, L-ClPhg-L-FDLA, and D-ClPhg-L-FDLA) were studied using
quantum mechanical energy analysis. To understand which con-
formation was energetically preferred, L-Phg-L-FDLA, D-Phg-L-FDLA,
L-DhPhg-L-FDLA, and D-DhPhg-L-FDLA were each built in five likely
starting conformations. An additional hydrogen-bonding conformation
was also built for L-DhPhg-L-FDLA and D-DhPhg-L-FDLA. These
starting structures were optimized with Gaussian09 using density
functional theory.18 Specifically, the PBEPBE exchange and correlation
functional19,20 was employed with the 6-311G++(3df,3pd) basis set
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Jasco DIP-370 polarimeter. UV spectra were obtained
using a Perkin-Elmer Lambda2 UV/vis spectrometer. NMR data
were collected using either a Varian INOVA 500 (1H 500 MHz, 13C
125 MHz) NMR spectrometer with a 3 mm Nalorac MDBG probe or
a Varian INOVA 600 (1H 600 MHz, 13C 150 MHz) NMR spectrom-
1
eter equipped with a 5 mm H[13C,15N] triple resonance cold probe
with a z-axis gradient, utilizing residual solvent signals for referencing.
High-resolution mass spectra (HRMS) were obtained using a Bruker
(Billerica, MA, USA) APEXII FTICR mass spectrometer equipped
with an actively shielded 9.4 T superconducting magnet (Magnex
Scientific Ltd.), an external Bruker APOLLO ESI source, and a Synrad
50W CO2 CW laser. Supelco Discover HS (4.6 × 150 mm) and
semipreparative (10 × 150 mm) C18 (5 μm) columns were used for
analytical and semipreparative HPLC, respectively, as conducted on a
Hitachi Elite Lachrom System equipped with a diode array L-2455
detector.
Bacterial Material. Streptomyces sp. 1053U.I.1a.1b was cultivated
from Lienardia totopotens obtained by professional collectors near
Mactan Island, Cebu, Philippines, as previously described.5 Relevant
permission from local and national authorities in the Philippines was
obtained prior to beginning this study. The strain was cultured from
dissected hepatopancreas tissue and purified, and later the strain was
recovered from a glycerol stock and used for further chemical analysis.5
648
dx.doi.org/10.1021/np200886x | J. Nat. Prod. 2012, 75, 644−649