2
062 Journal of Natural Products, 2008, Vol. 71, No. 12
Notes
Carriebowmide (1): colorless, amorphous powder; [R]25
-40.0
Next, the absolute configuration of the amino acid units was
D
determined. The configuration of all R-amino acid residues in 1
was determined by chiral HPLC analysis, comparing the amino
acid content in two acid hydrolysates with standards. Compound 1
(c 0.12, MeOH); UV (MeOH) λmax(log ε) 218 (4.04), 258 (2.81) nm;
IR (KBr film) νmax 2957, 2870, 1738, 1650, 1646, 1538, 1453, 1410,
-
1
1
13
1
385, 1367, 1315, 1279, 1240, 1215, 1186, 1083 cm ; H and
NMR data, see Table 1, assignments were made by interpretation of
D DQF COSY, edited-HSQC, HMBC, and NOESY data; HRESI/
C
14
was separately subjected to desulfurization with Raney-Ni in order
to convert Met(O) to 2-aminobutyric acid (Aba) prior to a second
acid hydrolysis. Retention times established an L-configuration for
Ala, N-Me-Leu, Phe, and Met(O) and the D-configuration for N-Me-
Phe. The configuration of the hydroxy acid was determined using
a different chiral column, and its retention time indicated the
presence of R-Hmba. The configuration of the Amha residue was
2
+
TOFMS m/z 881.4838 [M + H] (calcd for C46
69 6 9
H N O S, 881.4847).
Acid Hydrolysis and Chiral HPLC Analysis. Compound 1 (0.1
mg) was suspended in 6 N HCl (0.3 mL) and heated at 115 °C for
1
8 h in a sealed tube. The hydrolysate was concentrated to dryness.
The residue was reconstituted in 0.2 mL of H O and analyzed by chiral
2
HPLC, comparing the retention times with those of authentic standards
[Phenomenex Chirex (D) Penicillamine, 4.6 × 250 mm, 5 µm]; solvent
2.0 mM CuSO and mixtures of 2.0 mM CuSO -MeCN (95:5, 90:10,
6
determined as 2R,3R by Marfey analysis by using two standards
(
2R,3R and 2S,3R) of the four possible stereoisomers. Amha
4
4
residues with 3S configuration have not been reported to date to
occur in cyanobacterial metabolites. These data established the
residue sequence of cyclo[2R,3R-Amha-L-Ala-N-Me-L-Leu-L-Phe-
L-Met(O)-N-Me-D-Phe-R-Hmba] for carriebowmide (1).
When tested against a natural assemblage of reef fish, only the
lipophilic extract reduced feeding on agar food pellets (p < 0.001).
The fish consumed 3.35 ( 0.11 (mean ( SE) agar cubes of the
control food, 2.25 ( 0.31 agar cubes of the food containing the
hydrophilic extract, and 0.85 ( 0.22 of the agar cubes that contained
the lipophilic extract. Unfortunately, not enough carriebowmide
remained after structure elucidation to determine its feeding-
deterrent properties.
or 85:15); detection at 254 nm. Using 2.0 mM CuSO
and 2.0 mM CuSO -MeCN (95:5) for D,L-N-Me-leucine, with a flow
rate of 0.8 mL/min, the retention times (t , min) for authentic standards
were L-Ala (11.0) and D-Ala (15.2), and N-Me-L-Leu (50.2) and N-Me-
D-Leu (62.1), respectively. Similarly, using 2.0 mM CuSO -MeCN
90:10) for D,L-N-Me-phenylalanine and 2.0 mM CuSO -MeCN (85:
5) for D,L-phenylalanine, with a flow rate of 1.0 mL/min, the t min
for authentic standards were N-Me-L-Phe (59.5) and N-Me-D-Phe (69.8),
and L-Phe (39.7) and D-Phe (41.8), respectively. The t (min) of the
amino acids in the hydrolysate under the respective conditions were
1.0 (100:0), 50.2 (95:5), 69.8 (90:10), and 39.7 (85:15), indicating
4
for D,L-alanine
4
R
4
(
4
1
R
R
1
the presence of L-Ala, N-Me-L-Leu, N-Me-D-Phe, and L-Phe in the
hydrolysate. The stereochemistry of the R-hydroxy acid was determined
using a different chiral column for the HPLC analysis [Chiralpak MA
Experimental Section
(
+) (4.6 × 50 mm), Diacel Chemical Industries, Ltd.; solvent, 2.0 mM
CuSO
The t
4
-MeCN (90:10); flow rate, 1.0 mL/min; detection at 254 nm].
(min) for authentic standards were R-Hmba (10.3) and S-Hmba
General Experimental Procedures. Optical rotation was recorded
on a Perkin-Elmer model 343 polarimeter. UV spectral data were
acquired on a Hitachi U-3010 spectrophotometer. IR spectral data were
obtained on a Bruker Vector 22 FT-IR spectrometer. NMR data
were collected on a JEOL ECA-600 spectrometer operating at 600.17
R
(
13.8). The retention time of the R-hydroxyamino acid in the hydrolysate
under these conditions was 10.3, indicating the presence of R-Hmba
in the hydrolysate.
Advanced Marfey Analysis. A portion of the acid hydrolysate (0.05
6
1
13
MHz for H and 150.9 MHz for C. The edited-gHSQC experiment
was optimized for JCH ) 140 Hz, and the gHMBC spectrum was
mg) and the standards (2R,3R)-Amha and (2S,3R)-Amha were sepa-
rately derivatized by treatment with 1 M NaHCO (50 µL) and a 1%
3
2
/3
1
optimized for
residual CH
of data from 2D DQF COSY and gHMQC experiments. Similarly,
NMR chemical shifts (referenced to CD OD observed at δ 49.0) were
J
CH ) 8 Hz. H NMR chemical shifts (referenced to
3
OH observed at δ 3.30) were assigned using a combination
solution of 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide in acetone (50
µL). After heating at 45 °C for 1 h, the reaction mixtures were cooled,
acidified with 2 N HCL (25 µL), and diluted with MeCN (100 µL).
These solutions were subjected to reversed-phase HPLC analysis [C18
(3.0 × 250 mm), 5 µ, Atlantis; flow rate 1.0 mL/min, detection at 340
nm] using 50 mM aqueous NH COOCH -MeCN (70:30). The t (min)
1
3
C
3
assigned on the basis of multiplicity-edited HSQC experiments. The
HRMS data were obtained using an Agilent LC-TOF mass spectrometer
equipped with an APCI/ESI multimode ion source detector at the Mass
Spectrometer Facility at the University of California, Riverside, CA.
Silica gel 60 (EMD Chemicals, Inc. 230-400 mesh) and Varian
BondElut octadecyl (C18) were used for column chromatography. All
solvents used were of HPLC grade (Fisher Scientific).
Collection, Extraction, and Isolation. The sample of L. polychroa
was collected on August 19, 2005, from Carrie Bow Cay fore-reef,
Belize, at a depth of 10 m. The sample was identified by one of us
4
3
R
of L-FDAA derivatives of (2R,3R)-Amha and (2S,3R)-Amha were 53.5
and 59.5, respectively. The t (min) of the Amha derivative in the
R
hydrolysate under these conditions was 53.5, indicating the presence
of (2R,3R)-Amha in the hydrolysate.
Desulfurization, Acid Hydrolysis, and Chiral HPLC Analysis.
Compound 1 (0.2 mg) was dissolved in EtOH (0.5 mL) and treated
2
with an excess of fresh Raney-Ni (2400) as a slurry in H O (0.1 mL)
(
V.J.P.), and a voucher specimen is maintained at the Smithsonian
and refluxed for 0.5 h. The product was filtered, concentrated, and
subjected to acid hydrolysis as above at 115 °C for 12 h in a sealed
tube. The hydrolysate was concentrated to dryness. The residue was
Marine Station, Fort Pierce, FL. Filament width: 47.5 ( 5.53 µm (mean
(
SD); cell width: 37.8 ( 5.33 µm; cell length: 7.00 ( 1.97 µm.
The freeze-dried material (57.3 g) was first extracted with
reconstituted in 0.2 mL of H
comparing the retention times with those of authentic standards
Phenomenex Chirex (D) penicillamine, 4.6 × 250 mm, 5 µm]; solvent
.0 mM CuSO ; detection at 254 nm. The t (min) for authentic
standards were L-Aba (16.8) and D-Aba (27.7). The t (min) of the
2
O and analyzed by chiral HPLC,
2
EtOAc-MeOH (1:1) and then with EtOH-H O (1:1). Concentration
of the extracts by rotary evaporation at 45 °C under reduced pressure
furnished 7.1 g (12.3% yield) of the organic extract and 6.2 g (10.8%
yield) of a polar extract. The fish feeding assays conducted at Golden
Reef, Belize, in June 2006 indicated the less polar fraction significantly
deterred feeding by a natural assemblage of reef fish. Therefore, the
less polar EtOAc-MeOH (1:1)-soluble organic fraction was selected
for chemical analysis. The EtOAc-MeOH (1:1)-soluble fraction (4.0
g) was chromatographed on a column of Si gel (30.0 g). The column
was prepared in hexanes and eluted with a hexanes-EtOAc-MeOH
step gradient system to give seven subfractions. Fraction 6 (0.90 g),
[
2
4
R
R
amino acid in the hydrolysate under this condition was 16.8, indicating
the presence of L-Aba in the hydrolysate.
Preparation and Identification of the Standard Samples of
(
3
2S,3R)-Amha and (2R,3R)-Amha. Samples (0.2 mg) of lyngbyastatin
and ulongamide A from our pure sample repository were separately
subjected to acid hydrolysis as above. The hydrolysates were dried,
1
and each was reconstituted in H O (100 µL) for separation. Each
which eluted with EtOAc-25% MeOH, showed a H NMR pattern
2
hydrolysate was separated by reversed-phase HPLC (Phenomenex
analytical, 250 × 4.6 mm, 5 µm, phenyl-hexyl) using MeCN-90%
corresponding to a peptide. This fraction was rechromatographed on a
column of C18 using a MeOH-H
2
O step gradient system to give six
subfractions. The subfraction 4 (18 mg), which eluted with MeOH-25%
2
H O. Lyngbyastatin 3 and ulongamide A hydrolysates furnished six
H
2
O, was further separated by reversed-phase HPLC (semiprep, 5 µm,
fractions and three fractions, respectively. The retention times of the
standards identified the common amino acids glycine, N-Me-valine,
N-Me-leucine, and N-Me-alanine in the lyngbyastatin 3 hydrolysate
and N-Me-valine and phenylalanine in the ulongamide A hydrolysate.
A portion from the hydrolysates of lyngbyastatin 3 and ulongamide A
RP-18) using MeOH to give three subfractions. Subfraction 2 (4.0 mg)
was further purified by reversed-phase HPLC using a MeOH-10%
H O mixture to give 2.2 mg of carriebowmide (1, yield, 0.004% dry
2
wt).