Lipodepsipeptides with Antiprotozoal ActiVity
Journal of Natural Products, 2008, Vol. 71, No. 9 1549
L-Val with 2 mM CuSO4 (23.0 min; D-Val, 36.1 min), N-Me-L-Val
with 2 mM CuSO4 (15.5 min; N-Me-D-Val, 24.8 min), L-Pro with 2
mM CuSO4 (18.2 min; D-Pro, 32.3 min).
primers M13F/M13R and internal middle primers 359F and 781R as
previously described.21
Phylogenetic Analysis of Strain OSC3L. The bidirectional 16S
rRNA gene sequences of OSC3L were combined, and the resulting
consensus sequence was inspected both visually and by secondary
structure analysis using the CLC DNA Workbench 3. The GC content
was determined using the MBCF Oligo calculator from the Dana-Farber
.harvard.edu/docs/oligocalc.html). The 16S rRNA gene was aligned
together with related cyanobacterial strains representing 13 major genera
of the order Oscillatoriales (subsection III) obtained from GenBank
performed using ClustalX in MEGA 436 with standard gap opening
and extension penalties without gaps. The aligned 16S rRNA gene
sequences were used to generate phylogenetic trees in MEGA 4. The
phylogenetic relation of the cyanobacterial 16S rDNA genes were
compared by (1) the distance method by neighbor-joining (NJ), (2)
maximum parsimony (MP), and (3) the maximum-likelihood (ML)
method. The evolutionarily distant cyanobacterium Nostoc punctiforme
PCC73102 (GenBank acc. no. AF027655), from the order Nostocales,
was used as an outgroup. Selection of the phylogenetic method used
for the tree appearing in Figure 4 was based on tree topology and boot-
strap values.
For determination of the configuration of the N-Me-Ile residue, the
hydrolysate of 1 was divided into two portions and dissolved in 1 N
NaHCO3 (100 µL) followed by the separate addition of 50 µL of 3
mg/mL (acetone) L-FDAA and D-FDAA, respectively. The reaction
mixture was incubated at 80 °C for 3 min, then quenched by
neutralization with 50 µL of 2 N HCl. Next, 300 µL of 50% aqueous
CH3CN was added to the solution and the products were analyzed by
reversed-phase HPLC (LiChrosphere 100 C18, UV detection at 340 nm)
using a linear gradient of 9:1 50 mM triethylammonium phosphate
(TEAP) buffer (pH 3)/CH3CN to 1:1 TEAP/CH3CN over 60 min.
Because only N-Me-L-Ile and N-Me-L-allo-Ile were commercially
available, the D-Marfey’s reagent was used to make the N-Me-D-Ile
and N-Me-D-allo-Ile chromatographic equivalents. The retention times
(tR, min) of the Ile derivative from the hydrolysate of 1 matched that
of N-Me-L-Ile (40.7 min; N-Me-L-allo-Ile, 41.2 min; N-Me-D-Ile, 44.2
min; N-Me-D-allo-Ile, 44.7 min).
Preparation and Chiral Analysis of HMPA. L-Ile (100 mg, 0.75
mmol) was dissolved in 50 mL 0.2 N HClO4 at 0 °C. To this was
added a cold (0 °C) solution of NaNO3 (1.4 g, 20 mmol) in 20 mL of
H2O with rapid stirring. With continued stirring the reaction mixture
was allowed to reach room temperature, at which time evolution of N2
subsided (ca. 30 min). The solution was then boiled for 3 min, cooled
to room temperature, and then saturated with NaCl. This mixture was
extracted with Et2O and dried under vacuum. The three other stereoi-
somers (2R,3R)-HMPA, (2R,3S)-HMPA, and (2S,3R)-HMPA were
synthesized in a similar manner from D-Ile, D-allo-Ile, and L-allo-Ile,
respectively. A portion of the resultant oil was dissolved in aqueous 2
mM CuSO4 buffer for HPLC. The retention time (Chirex-D, linear
gradient 100:0 2 mM CuSO4/CH3CN to 95:5 over 20 min) of the natural
product hydrolysate matched that for 2S,3S-HMPA (18.7 min; 2R,3S-
HMPA, 15.2 min; 2S,3R-HMPA and 2R,3R-HMPA, 23.2 min).
Intracellular Trypanosoma cruzi Assay. The antitrypanosomal
activity was determined using a recombinant Tulahuen clone C4 of T.
cruzi that expresses ꢀ-galactosidase as a reporter enzyme.33 The method
is based on the growth inhibition effect of test samples on trypomas-
tigote, the intracellular form of the parasite, infecting Vero cells, as
previously described.34
Acknowledgment. We gratefully acknowledge the government of
Curac¸ao for permission and the Carmabi Research Station for assistance
in making these cyanobacterial collections. We thank R. C. Coates for
cyanobacterial culturing, and M. Balunas and C. Spatafora for assistance
with data management. Financial support for this work came from the
National Cancer Institute (CA52955) and the International Cooperative
Biodiversity Groups Program (grant no. 1U01 TW 006634-01).
Supporting Information Available: HPLC chromatograms of 1 and
1
2, full 1D and 2D NMR spectra for 1, H and 13C NMR spectra of 2,
and phylogenetic trees produced by alternative algorithms. This material
References and Notes
Antileishmanial Bioassay. Leishmaniasis bioassays were performed
using a method based on parasite (Leishmania mexicana) DNA
fluorescence, as previously described.35
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Morphological Analysis of Strain OSC3L. The morphological
characterization of OSC3L was performed using an Olympus BH-2
light microscope. The following parameters were selected to describe
its morphology: length, width, and length/width ratios of vegetative
cells, presence/absence of specialized cells such as heterocysts, akinetes,
or calyptra, the size and shape of trichomes, granulation, constrictions
at cross walls, morphology of terminal cells, and thallus growth
characteristics and coloration. Morphological identification was made
in accordance with traditional phycological20 and bacteriological
systems.18
DNA Extraction, 16S rRNA Gene PCR-Amplification, and
Cloning. Genomic DNA was extracted from 40 mg of cleaned algal
tissue using the Wizard Genomic DNA purification kit (cat. A1120)
following the manufacturer’s specifications (Promega, Madison, WI).
The isolated genomic DNA was further purified using a Genomic-tip
20/G kit from Qiagen (cat. 10223). The 16S rRNA gene was amplified
from isolated DNA using the cyanobacterial-specific primers 106F and
1509R, as previously described.21 The reaction volume was 25 µL
containing 0.5 µL of DNA (50 ng), 2.5 µL of 10 × PfuUltra IV reaction
buffer, 0.5 µL of dNTP mix (25 mM each of dATP, dTTP, dGTP, and
dCTP), 0.5 µL of each primer (10 µM), 0.5 µL of PfuUltra IV fusion
HS DNA polymerase (cat. 600760), and 20.25 µL of dH2O. The PCR
reaction was performed in an Eppendorf Mastercycler gradient as
follows: initial denaturation for 2 min at 95 °C, 30 cycles of
amplification: 20 s at 95 °C, 20 s at 50 °C, and 1.5 min at 72 °C, and
final elongation for 3 min at 72 °C. PCR products were subcloned using
the Zero Blunt TOPO PCR cloning kit (cat. K2800-20SC) from
Invitrogen, into the pCR-Blunt IV TOPO vector, and then transformed
into TOPO cells and cultured on LB-kanamycin plates. Plasmid DNA
was isolated using the QIAprep spin miniprep kit (cat. 27106) from
Qiagen and sequenced with pCR-Blunt IV TOPO vector specific
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