Journal of Natural Products
Note
previously described for DNA extraction from cyanobacteria of the
genus Arthrospira with a slight modification; freeze−thaw cycles were
replaced by grinding in liquid nitrogen.22 This extraction method
yielded a sufficient amount of the PCR-amplifiable template DNA.
The partial 16S rRNA gene was amplified from the isolated genomic
DNA using the cyanobacteria-specific primers 106F and 1509R.23 The
reaction volume was 26 μL containing 10.5 μL of nuclease-free H2O,
12.5 μL of PCR Master Mix (Promega M7502), 1 μL of each primer
(10 μM), and 1 μL of the template DNA. The PCR was performed in
a Bio-Rad C1000 thermal cycler as follows: initial denaturation for 30 s
at 98 °C, 35 amplification cycles of 10 s at 98 °C, 30 s at 53 °C, and 30
s at 72 °C, and a final extension for 10 min at 72 °C. PCR products
were purified using a MinElute PCR purification kit (Qiagen) and
sequenced using the cyanobacteria-specific primers 106F and 1509R as
well as the internal primer 359F. The resulting sequence was deposited
in the NCBI GenBank under the accession number JQ435860.
Extraction and Isolation. The cyanobacterial sample was
manually cleaned from debris and freeze-dried. The freeze-dried
sample (5 g) was extracted with the solvent mixture of CH2Cl2 and
MeOH (1:1) and concentrated in vacuo to yield the organic extract
(0.5 g). The resulting extract was fractionated using Diaion HP-20
resin and an increasing amount of iPrOH in H2O. The fraction eluting
at 40% iPrOH was found to contain a new nitrogen-containing peptide
with a molecular weight of 973.5 Da by LC-MS analysis and subjected
to reversed-phase HPLC (Varian C8 semipreparative column, 10 mm
× 250 mm, 3 mL/min) to afford stigonemapeptin (1, 4.1 mg, 0.08%).
inhibitory activity against these three enzymes. Stigonemapep-
tin (1) showed inhibition of elastase and chymotrypsin with
IC50 values of 0.26 and 2.93 μM, respectively, whereas no
inhibition was found against trypsin at the highest concen-
tration tested (10 μM). This result is in good agreement with
activity profiles of known Ahp-containing depsipeptides. Co-
crystallographic data of elastase with scyptolin A suggested that
the selectivity of this class of compounds for the inhibition of
serine proteases varies depending on the type of amino acids
located between Ahp and Thr due to different binding
preferences to the enzyme’s specificity pocket.20 Preferences
for the inhibition of chymotrypsin and elastase are conferred by
a bulky hydrophobic amino acid (Phe, Tyr, or Trp) and a small
neutral amino acid (Ala, Gly, or Val), respectively, whereas a
positively charged amino acid (Arg or Lys) is preferred for the
inhibition of trypsin. Therefore, the selective inhibitory activity
of 1 against elastase can be attributed to the presence of the
Abu residue between Ahp and Thr, as an Abu residue is
considered to be relatively small and neutral. Stigonemapeptin
(1) was also tested for its inhibitory activity against the 20S
proteasome and cytotoxicity against HT-29 cancer cells, but
showed no activities at the highest concentration tested (25
μM).
In summary, we have isolated a new Ahp-containing
depsipeptide, which possesses nonstandard amino acids Abu
and N-formylated Pro, from a bloom sample of the freshwater
cyanobacterium stigonema sp. (collection ID WI53). The
structure of stigonemapeptin (1) differed from other known
Ahp- and Abu-containing depsipeptides by the geometry of the
Abu residue and the amino acid composition of the branching
residues. Stigonemapeptin (1) showed selective inhibition of
elastase and chymotrypsin with 10-fold higher selectivity for
elastase.
Stigonemapeptin (1): colorless, amorphous powder; [α]25 −61
D
(c 0.15, MeOH); UV (MeOH) λmax (log ε) 203 (4.37), 223 (4.08),
272 (3.23) nm; IR (neat) νmax 3276, 2962, 2936, 1734, 1653, 1533,
1517 cm−1; 1H and 13C NMR, HMBC, and ROESY data, see Table 1;
HRESIMS m/z 996.4439 [M + Na]+ (calcd for C48H63N9NaO13,
996.4443).
Absolute Configuration of Amino Acids and Ahp by the
Advanced Marfey’s Method. Approximately 0.3 mg of 1 was
hydrolyzed with 6 N HCl (500 μL) for 16 h at 110 °C. The resulting
acid hydrolysate was separated into two equal portions for
derivatization with either L-FDLA or DL-FDLA. Each portion was
dissolved in 50 μL of H2O and mixed with 20 μL of 1 N NaHCO3 and
20 μL of L-FDLA or DL-FDLA (10 mg/mL in acetone). Then, acetone
was added to the final volume of 200 μL, and the reaction mixtures
were heated to 40 °C and stirred for 1 h. After cooling to room
temperature (rt), 20 μL of 1 N HCl was added, and the resulting
reaction mixtures were air-dried and redissolved in CH3CN. LC-MS
analysis was performed on a reversed-phase column (Alltima C18, 250
× 4.6 mm, 5 μm, 1.0 mL/min) with a linear gradient from 20% to 65%
aqueous CH3CN containing 0.1% formic acid for 50 min. The
selective ion chromatograms of L-FDLA and DL-FDLA for each amino
acid derivative were compared for the assignment of amino acid
configurations. Two peaks corresponding to the L- and D-FDLA
derivatives of each amino acid were observed as follows: Val 35.9 min
(L) and 44.2 min (D); Glu 27.4 min (L) and 28.9 min (D); NMeTyr
24.1 min (L) and 25.2 min (D); Phe 40.7 min (L) and 46.9 min (D);
Pro 30.4 min (L) and 34.3 min (D); Thr 24.6 min (L) and 30.7 min
(D). The L-FDLA derivative gave one peak for each amino acid at 35.9,
27.4, 24.1, 40.6, 30.5, and 24.6 min, confirming the L configuration for
Val, Gln, NMeTyr, Phe, Pro, and Thr. The presence of L-Thr was
further confirmed by chromatographic comparison of the L-FDLA
derivative of the acid hydrolysate with those of the amino acid
standards L-Thr, D-Thr, L-allo-Thr, and D-allo-Thr.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The optical rotation was
measured on a Perkin-Elmer 241 polarimeter. UV and IR spectra were
recorded on a Shimadzu UV spectrometer UV2401 and a Thermo
Nicolet 6700 FT-IR spectrometer, respectively. 1D and 2D NMR
1
spectra including H NMR, COSY, TOCSY, HSQC, HMBC, and T-
ROESY spectra were obtained on a Bruker Avance DRX 600 MHz
NMR spectrometer with a 5 mm CPTXI Z-gradient, whereas a Bruker
Avance II 900 MHz NMR spectrometer with a 5 mm ATM CPTCI Z-
gradient probe was used to acquire the DEPT-Q spectrum. 1H and 13
C
NMR chemical shifts were referenced to the DMSO-d6 solvent signals
(δH 2.50 and δC 39.51, respectively). A mixing time of 60 ms was set
for the TOCSY experiment and 200 ms for the T-ROESY experiment.
The HMBC spectrum was recorded with the average 3JCH of 8 Hz, and
1
the HSQC spectrum was measured with the average JCH of 145 Hz.
HRESIMS and LC-MS data were obtained on a Shimadzu IT-TOF
LC-MS spectrometer.
Sample Collection and Morphological Study. A bloom sample
of Stigonema sp. (collection ID WI53) was collected from North
Nokomis Lake in the Highland Lake District of northern Wisconsin in
August 2010 (N 45°50.4812′, W 89°26.4920′). A voucher specimen
has been retained at UIC under the collection ID WI53.
Morphological analysis was performed using a Zeiss Axiostar Plus
light microscope equipped with a Canon PowerShot A620 camera.
DNA Extraction and Phylogenetic Analysis of 16S rRNA
Gene Sequence. For genomic DNA isolation, a sample was re-
collected at the same site in August 2011, and the production of
stigonemapeptin (1) was confirmed by LC-MS analysis. The kit-based
DNA extraction method routinely used in our laboratory21 failed to
yield genomic DNA in a sufficient quantity and quality for PCR
amplification possibly due to the presence of a firm mucilaginous
sheath. Therefore, the CTAB-based method was employed as
For the absolute configuration of the Ahp residue, CrO3 oxidation
was carried out prior to acid hydrolysis as previously described.15,24
Briefly, approximately 0.4 mg of 1 was dissolved in 0.4 mL of glacial
AcOH, and CrO3 (2 mg) was added. The mixture was stirred at rt for
5 h and purified on a C18 SPE cartridge eluting with H2O and MeOH.
The resulting oxidized product was hydrolyzed and subjected to
advanced Marfey’s analysis in the same way as described above. LC-
MS analysis exclusively identified the presence of L-Glu, assigning the S
configuration to Ahp C-3.
Protease Inhibition Assays. The inhibitory activity of 1 against
three serine proteases including elastase, chymotrypsin, and trypsin
810
dx.doi.org/10.1021/np300150h | J. Nat. Prod. 2012, 75, 807−811