2
072
R. Montaser et al. / Phytochemistry 72 (2011) 2068–2074
chemical similarity was detected between this Guamanian sample
and another L. majuscula sample from Madagascar. The structural
similarity between the isolated secondary metabolites from both
samples suggests genetic similarity, including the putative gene
cluster encoding biosynthetic enzymes, and pitipeptolides could
be considered analogues of antanapeptins. Notably, those struc-
tural modifications uniquely granted the pitipeptolides cytotoxic
and antibacterial activities that were not found in the antanapep-
tins. Therefore, pitipeptolides can be potentially considered as lead
compounds and further structural optimization may selectively
enhance their cytotoxic or antibacterial activities.
Pitipeptolide D (4) was further purified from fraction 2 (eluting
between t 19.2 and 20.2 min using the conditions mentioned
above) using semi-preparative reversed-phase HPLC (Luna C18,
250 ꢁ 10 mm, 5 m, 2.0 mL/min; UV detection at 200/220 nm)
with a MeOH/H O linear gradient (75–100% aqueous MeOH over
20 min, then 100% MeOH for 10 min). The peak eluting at t
R
l
2
R
19.9 min was subjected to further purification on another semi-
preparative reversed-phase HPLC column (Phenomenex Phenyl-
hexyl, 250 ꢁ 10 mm, 5
200 nm) using a MeOH/H
MeOH over 35 min, and then 100% MeOH for 10 min) to afford
.1 mg pure compound 4 at t 20.1 min.
Pitipeptolides E (5) and F (6) co-eluted at t
lm, 2.0 mL/min; UV detection at 220/
2
O linear gradient (85–100% aqueous
1
R
R
20.6 min (fraction 3
4
. Experimental
from the first HPLC run mentioned above on the YMC-Pack ODS-AQ
column) to give an impure fraction (68 mg). An aliquot (5 mg) of
this mixture was further purified several times on an analytical re-
versed-phase HPLC column (Allure Restec C18, 250 ꢁ 4.6 mm,
4.1. General experimental procedures
Optical rotations were measured on a Perkin–Elmer 341 polar-
imeter, whereas UV and optical density were measured on a Spec-
5
lm, 1.0 mL/min; UV detection at 220/200 nm) using a MeOH/
H
2
O linear gradient (75–100% aqueous MeOH over 35 min, and
12.2 min
12.7 min (1.9 mg). Extrapolating
traMax M5 (Molecular Devices), and IR data were obtained on a
Perkin–Elmer Spectrum One FT-IR Spectrometer. 13C NMR spectra
then 100% MeOH for 10 min) to yield compound 5 at t
2.2 mg), and compound 6 at t
R
(
R
were recorded on a Bruker 500 MHz spectrometer operating at
1
those values suggests a total of about 30 mg of 5 and 26 mg of 6
in this sample.
1
25 MHz, whereas H and 2D NMR spectra were acquired on a
Bruker Avance II 600 MHz spectrometer. All spectra were obtained
in CDCl using residual solvent signals (d 7.26, d 77.16 ppm) as
internal standards. HSQC and HMBC experiments were optimized
3
H
C
4.4. Pitipeptolide C (3)
1
1
for
JCH = 145 and JCH = 7 Hz, respectively. HRMS data was re-
2
D
0
Colorless, amorphous solid; ½
a -121 (c 0.11, MeOH); UV
ꢂ
corded on an Agilent LC–TOF mass spectrometer equipped with
an APCI/ESI multimode ion source detector in positive ion mode,
whereas LC–MS data were obtained using an API 3200 triple quad-
rupole MS (Applied Biosystems) equipped with a Shimadzu LC sys-
tem. ESIMS fragmentation data were recorded on an API 3200 by
direct injection with a syringe driver. 2-Hydroxy isovaleric acid
(
MeOH) kmax (log e) 202 (4.57) nm; IR (film) mmax 3403, 2961,
2
7
932, 2874, 1727, 1653, 1511, 1464, 1415, 1371, 1188, 1030,
ꢃ1
1
13
36, 702 cm ; For H NMR, C NMR and HMBC spectroscopic
+
data, see Table 1; HRESI/APCIMS m/z 812.5178 for [M + H] (calcd
for C44 812.5168).
70 5 9
H N O
(
3
Hiva) standards were purchased from Sigma–Aldrich. 2-Hydroxy
-methyl pentanoic acid (Hmpa) standards were synthesized from
isoleucine (Van Draanen et al., 1991).
4.5. Pitipeptolide D (4)
2
0
Colorless, amorphous solid; ½
MeOH) kmax (log ) 202 (4.29) nm; IR (film)
935, 2876, 1735, 1654, 1513, 1455, 1370, 1180, 1039, 735,
a -112 (c 0.12, MeOH); UV
ꢂ
D
(
2
7
e
mmax 3410, 2966,
4
.2. Marine cyanobacterial sample
ꢃ1
1
13
01 cm ; For H NMR, C NMR and HMBC spectroscopic data,
The sample of the marine cyanobacterium Lyngbya majuscula
+
see Table 1; HRESI/APCIMS m/z 816.4524 for [M + Na] (calcd for
816.4518).
(
recollection of UOG strain VP627) was collected at Piti Bomb
43 63 5 9
C H N O
Holes, Guam, in February 2000. A voucher sample (voucher speci-
men number EC025) has been preserved at the Smithsonian Mar-
ine Station at Fort Pierce, FL.
4.6. Pitipeptolide E (5)
2
D
0
Colorless, amorphous solid; ½
a -105 (c 0.13, MeOH); UV
ꢂ
4
.3. Extraction and isolation
(MeOH) kmax (log
e
) 202 (4.34) nm; IR (film)
m
max 2967, 2372,
2
343, 2297, 1986, 1655, 1512, 1175, 804, 702 cm ; For 1H NMR,
ꢃ1
1
3
Freeze-dried L. majuscula (dry weight 269g) was extracted with
C NMR and HMBC spectroscopic data, see Table 2; HRESI/APCIMS
m/z 816.4521 for [M + Na] (calcd for C43
+
EtOAc–MeOH (2L ꢁ 3, 1:1, v/v) to afford the crude organic extract
35.5 g). The resulting extract was partitioned between hexanes
and MeOH–H O (80:20), the methanolic phase was concentrated
to dryness, and the residue was further partitioned between n-
BuOH and H O. After concentrating the n-BuOH extract in vacuo,
the resulting residue (7.2 g) was subjected to silica gel flash chro-
matography, eluting with CH Cl followed by increasing gradients
of i-PrOH in CH Cl , and finally with MeOH. The fraction eluting
with 4% i-PrOH in CH Cl was fractionated on a semipreparative re-
63 5 9
H N O Na 816.4518).
(
2
4.7. Pitipeptolide F (6)
2
0
2
Colorless, amorphous solid; ½
(MeOH) kmax (log ) 202 (4.34) nm; IR (film)
1726, 1646, 1506, 1413, 1370, 1275, 1177, 1094, 1032, 967, 805,
a -101 (c 0.10, MeOH); UV
ꢂ
D
e
mmax 3404, 2964,
2
2
ꢃ1
1
13
2
2
733, 701 cm ; For H NMR, C NMR and HMBC spectroscopic
+
2
2
data, see Table 2; HRESI/APCIMS m/z 816.4518 for [M + Na] (calcd
versed-phase HPLC column (YMC-Pack ODS-AQ, 250 ꢁ 10 mm,
63 5 9
for C43H N O Na 816.4518).
5
l
m, 2 mL/min; UV detection at 220/254 nm) using a MeOH/
2
H O linear gradient (75–100% aqueous MeOH over 30 min, and
4.8. Acid hydrolysis and enantioselective analysis
then 100% MeOH for 10 min) to yield 10 collected fractions. Pitip-
eptolides A (1) (378 mg) and B (2) (70.5 mg) eluted at t 21.3 min
fraction 4) and t 23.8 min (fraction 7), respectively. Pitipeptolide
C (3) (23 mg) eluted at t 25 min as a single peak (fraction 9). Fur-
ther purification of fractions 2 and 3 gave compounds 4, 5 and 6
see below).
R
A sample of compounds 3–6 (0.1 mg each) was hydrolyzed with
6 N HCl (0.5 mL) at 110 °C for 24 h. The hydrolysate was concen-
trated to dryness, resuspended in H O (100 lL), and then analyzed
2
by chiral HPLC. Amino acid units were analyzed by HPLC/MS chiral
analysis [column: Chirobiotic TAG (250 ꢁ 4.6 mm), Supelco;
(
R
R
(