A. Tripathi et al. / Phytochemistry 72 (2011) 2369–2375
2373
drugs (Luesch et al., 2001; Taori et al., 2008; Lemmens-Grubert
et al., 2009).
NMR (100.62 MHz, CD3OD) data, see Table 1; HRESIMS m/z
[M+Na]+ 878.5225 (calcd for C46H73N5O10Na, 878.5250).
4.5. Advanced Marfey’s analysis of amino acid residues in 1
4. Experimental
A sample of compound 1 (1.0 mg) was treated with 6 N HCl
(1 mL) at 110 °C for 18 h. The resulting hydrolysate was concen-
trated to dryness, re-dissolved in H2O (0.6 mL) and divided into
two equal portions. Each portion was combined with either a 1%
4.1. General experimental procedures
Optical rotation was measured on a Bellingham Stanley ADP
440 polarimeter. UV and IR spectra were measured on a Varian
Cary 50 UV visible spectrophotometer and a Perkin–Elmer spec-
trum 100 FT-IR spectrophotometer, respectively. 1H, 13C, and 2D
NMR spectra were recorded in CD3OD on a 400 MHz Bruker NMR
spectrometer using the residual solvent signal (dH at 3.31 ppm
and dC at 49.1 ppm) as internal standards. HRESIMS and LC–MS/
MS data were obtained using a Bruker Daltonics MicrOTOF™ mass
spectrometer. HPLC isolation of lagunamide C (1) was conducted
on a Shimadzu LC-8A Preparative LC equipped with Shimadzu
SPD-M10A VP diode array detector, while an Agilent 1100 series
coupled with an Agilent LC/MSD trap XCT mass spectrometer
equipped with an ESI interface system was used for the detection
of the Marfey-derivatized alanine, N-methyl-phenylalanine, N-
methyl-alanine, and isoleucine as well as for Mosher’s derivatized
solution of 1-fluoro-2,4-dinitrophenyl-5-
Marfey’s reagent, 50 L) in acetone or a racemic mixture of a 1%
solution of 1-fluoro-2,4-dinitrophenyl-5-LD-alaninamide (LD-FDAA,
50 L) in acetone and 1 M NaHCO3 (25 L), and the two mixtures
were heated at 40 °C for 45 min. Both reaction mixtures were cooled
to r.t. and quenched by the addition of 2 N HCl (25 L), dried, and
redissolved in MeCN (500 L). The aliquots were subjected to
L-alaninamide (L-FDAA,
l
l
l
l
l
reversed-phase LC–MS (Agilent 1100 series) according to the
advanced Marfey’s method (Phenomenex, Luna, 150 ꢂ 2.0 mm,
5 lm, 100 Å; MeCN in 0.1% (v/v) aqueous HCOOH; at 0.20 mL/
min) using a linear gradient (10–50% MeCN over 60 min) (Marfey,
1984; Fuji et al., 1997a,b). An Agilent 1100 series MSD spectrometer
was used for detection in ESI (negative mode). The retention times
and ESIMS product ions (tR in min, m/z[MꢀH]ꢀ) of the
L-FDAA-deriv-
a-hydroxy acids in lagunamide C (1). Cell viability in 96-well
atized amino acids in the hydrolysate of the first portion were
observed to be Ile (45.1, 382.0), N-Me-Phe (48.6, 430.1), Ala
(28.3.0, 340.0), and N-Me-Ala (35.2, 354.0). The reaction with race-
mic LD-FDAA in the second portion gave rise to two peaks for each
corresponding amino acid moiety and the retention times and
ESIMS product ions (tR1/tR2, min, m/z [MꢀH]ꢀ) were observed to
be Ile (45.1/52.4, 382.0), N-Me-Phe (47.9/48.6, 430.1), Ala (28.3/
49.5, 340.0), and N-Me-Ala (35.2/36.1, 354.0). Consequently, the
absolute configurations of the moieties in the hydrolysate of 1 were
plates was measured using a Bio Rad Benchmark plus microplate
reader.
4.2. Marine cyanobacterial samples
Samples (about 1.5 L) of the filamentous marine cyanobacte-
rium, L. majuscula, were collected by hand from shallow waters
during low tides at Pulua Hantu Besar on June 25, 2007 and stored
in 70% aq. EtOH at ꢀ20 °C before workup. A voucher specimen of
this microalga is maintained at NIE under the code TLT/PHB/002.
confirmed as D-N-Me-Phe, L-Ala, and L-N-Me-Ala.
For the analysis of the Ile unit in 1, additional lagunamide C
(0.5 mg) was hydrolyzed in 6 N HCl at 110 °C for 20 h and was
4.3. Extraction and isolation
derivatized with Marfey’s reagent (
Two portions each of 0.5 mg standard
solved in 100 L of H2O. A 1.0% solution of
NaHCO3 (20 L) were added to one portion each of
Ile and to the other portions were added 1.0% solution of
(100 L) and 1 N NaHCO3 (20 L). All four mixtures were then
heated at 40 °C for 45 min. The solution were cooled to r.t., neutral-
ized with 2 N HCl (10 L) and evaporated to dryness. The residues
were then resuspended in MeCN (500 L). The aliquots were
L
-FDAA) as described above.
-Ile and -allo-Ile were dis-
-FDAA (100 L) and 1 N
-Ile and -allo-
-FDAA
L
L
The thawed cyanobacterial samples (ꢁ169 g dry wt.) were
exhaustively extracted using CHCl3/MeOH (1:1) to produce an or-
ganic extract (ꢁ2 g). The extract was then fractionated on normal
phase silica VFC using a stepwise gradient solvent system from hex-
ane, to EtOAc, and MeOH. Fractions eluted with EtOAc (100%) and
EtOAc:MeOH (9:1) were found to possess 100% toxicity (tested at
10 ppm) in the brine shrimp toxicity assay. Each fraction was passed
through a SEP-PAK RP-18 cartridge by eluting with 100% MeOH. The
cleaned fraction, which initially eluted with EtOAc:MeOH (9:1), was
l
l
L
l
L
L
D
l
l
l
l
subjected to reversed-phase LC–MS (Agilent 1100 series) according
to the Marfey’s method [column: Phenomenex, Luna, 150 ꢂ
subjected to C18 RP-HPLC [Phenomenex Sphereclone 5 lm ODS,
2.0 mm, 5 lm, 100 Å; mobile phase, MeCN in 0.1% (v/v) aqueous
250 ꢂ 10.00 mm, MeOH/H2O (81:19) at 3.0 mL/min, UV detection
at 230 nm] to obtain lagunamide C (1, 9.2 mg, 0.5 % of extract,
tR = 33.1 min). Additional source of compound 1 (6.9 mg, 0.35 % of
extract, tR = 43.1 min) was also obtained after the C18 RP-HPLC
HCOOH; flow rate, 0.20 mL/min] using a linear gradient (30–70%
MeCN over 40 min) (Marfey, 1984; Fuji et al., 1997a,b). An Agilent
1100 series MSD spectrometer was used for detection in API-ES
(negative mode). The derivatized Ile residue in the hydrolysate of
1 eluted at the same retention time as the derivatized standard
[Phenomenex Sphereclone 5
l
m ODS, 250 ꢂ 10.00 mm, MeOH/
H2O (78:22) at 3.0 mL/min, UV detection at 230 nm] of the fraction
initially eluted with 100% EtOAc. Both sources of lagunamide C were
pooled together (on the basis of 1H NMR spectra) and due to its
impurity were subjected to further C18 RP-HPLC [Phenomenex
L
-allo-Ile (14.2 min) but not that of
derivatized -Ile , 17.3 min) and
-allo-Ile, 17.8 min).
L
-Ile (13.0 min),
D-Ile (=D-FDAA
L
D
-allo-Ile (= -FDAA derivatized
D
L
Sphereclone 5
l
m ODS, 250 ꢂ 10.00 mm, MeOH/H2O (87:13) at
4.6. Preparation of isomers of 2-hydroxyisoleucic acid (Hila)
3.2 mL/min, UV detection at 230 nm] to yield pure lagunamide C
(1, 4.7 mg, 0.3 % of extract, tR = 19.2 min, refer to Supplementary
Information).
L-Isoleucine (100 mg, 0.75 mmol) was dissolved in 50 mL of
0.2 N perchloric acid (0 °C). To this was added a cold (0 °C) solution
of NaNO2 (1.4 g, 20 mmol) in 20 mL of H2O with rapid stirring. With
continued stirring the reaction mixture was allowed to reach r.t. un-
til evolution of N2 subsided (about 30 min). The solution was then
brought to boil for 3 min, cooled to r.t., and saturated with NaCl.
The mixture was then extracted with Et2O and dried under vacuum
to give 2S, 3S-Hila (L-Hila). The three other stereoisomers 2R,
4.4. Lagunamide C (1)
White, amorphous solid; ½a D25
ꢀ36 (c 0.5, MeOH); UV(MeOH)
ꢃ
kmax 220 nm (loge 2.89); IR (neat) 3316, 2914, 2486, 2251, 2054,
1694, 1439, 1276 cmꢀ1 1H NMR (400.13 MHz, CD3OD) and 13C
;