Notes
Journal of Natural Products, 2009, Vol. 72, No. 12 2175
Table 3. Preliminary Screening of Marine Natural Products for Their Ability to Inhibit the Specific Binding of [3H]-BQ-123 and
[3H]-NPY to the ETA and Y1 Receptors, Respectivelya
ETA
Y1
no.
name
100 µg/mL
10 µg/mL
100 µg/mL
10 µg/mL
1
2
3
4
5
6
7
8
5-bromo-N,N-dimethyltryptamine19
89 ( 0 (2)b
79 ( 0 (2)
96 ( 0 (2)
62 ( 5 (2)
100 ( 0 (2)
68 ( 1 (2)
79 ( 0 (2)
56 ( 6 (3)
68 ( 2 (2)
79 ( 7 (2)
88 ( 0 (2)
68 ( 7 (2)
80 ( 0 (2)
97 ( 8 (2)
98 ( 2 (2)
99 (1 (2)
76 ( 3 (2)
86 ( 1 (2)
18 ( 2 (2)
17 ( 3 (2)
29 ( 7 (2)
4 ( 1 (2)
40 ( 11 (2)
78 ( 3 (2)
81 ( 4 (2)
81 ( 6 (2)
100 ( 0 (2)
56 ( 0 (2)
12 ( 2 (2)
100 ( 0 (2)
100 ( 0 (2)
100 (0 (2)
100 ( 0 (2)
82 ( 2 (2)
73 ( 2 (2)
90 ( 2 (2)
100 ( 0 (2)
100 ( 0 (2)
100 ( 0 (2)
100 ( 0 (2)
100 ( 0 (2)
95 ( 1 (2)
2-(13-carboxy-14,15-diacetoxyhexadecanyl)-2-penten-4-olide20
NorKA (2)
5,6-dibromo-N,N-dimethyltryptamine19
3-hydroxydihydrodiscorhabdin C21
discorhabdin C21
discorhabdin A21
plakortide N22
100 ( 0 (2)
100 ( 0 (2)
94 ( 1 (2)
100 ( 0 (2)
85 ( 4 (3)
78 ( 9 (2)
100 ( 0 (2)
100 ( 0 (2)
100 ( 0 (2)
100 ( 0 (2)
9
stevensine24
10
11
12
13
oridin24
plakortide F23
plakortide F (free acid)23
sceptrin24
a For the neuropeptide Y Y1 receptor binding assay, the endogenous ligand NPY and the selective nonpeptide Y1 antagonist BIBP3226 were used as
positive controls, giving IC50 values of 1.2 and 8.8 nM, respectively. For the endothelin ETA receptor binding assay, the endogenous ligands
endothelin-1 and endothelin-2 were used as positive controls, giving IC50 values of 47 and 288 pM, respectively. b The results are expressed in
percentage of specific radioligand binding (the number of independent experiments is given in parentheses).
5-OHKF (1): colorless, amorphous solid; [R]2D5 -12 (c 1.15, MeOH);
UV (MeOH) λmax 204 nm.1H NMR (Table 1, 600 MHz, DMSO-d6);
13C NMR (Table 1, 150 MHz); HRESIMS m/z 1494. 9313 [M + H]+
(calcd for C75H124N14O17, 1494.9347).
with 0.5 mL of PBS buffer. To measure total cell binding of the
radioligand, 0.2 mL of 1 M NaOH was added to the 24 wells and left
for 30 min. Then 0.3 mL of distilled H2O was added, and the resulting
solution was placed into scintillation vials with scintillation liquid
(Ready Safe, Beckman Coulter) and the radioactivity counted on a
Beckman Coulter model LS 6500.
NorKA (2): colorless, amorphous solid; [R]2D5 -16 (c 0.25, MeOH);
1
UV (MeOH) λmax 204 nm; H NMR (Table 1, 600 MHz, DMSO-d6);
13C NMR (Table 1, 150 MHz); HRESIMS m/z 880.4750 [M + H]+
(calcd for C45H65N7O11, 880.4742).
Acknowledgment. Financial support for this project was provided
by NIAID 5RO1AI1036596, an NIH research facilities improvement
grant C06 RR-14503-01, and the National Secretary of Science,
Technology and Innovation (SENACYT) from the Panamanian Gov-
ernment (COL06-006). We are grateful to the administrative staff of
the Smithsonian Tropical Research Institute.
Marfey’s Analysis of Kahalalides.18 A sample of 0.1 to 0.2 mg of
peptide was hydrolyzed by treatment with 2 mL of 6 N HCl and left
at 120 °C for 24 h in a sealed ampule. The hydrolysate was dried under
N2. In an Eppendorf tube, 3.6 µmol of a 1% acetone solution of FDAA
(N-(5-flouro-2,4-dinitrophenyl)-L-alaninamide) and 20 µmol of a 1 M
solution of NaHCO3 were added to 2.5 µmol of amino acid. The reaction
mixture was heated with frequent shaking over a hot plate at 40 °C for
1 h and then cooled to RT. Then, 20 µmol of 2 M HCl and 1 mL of
MeOH were added to the reaction mixture. The samples were analyzed
by LC-MS, by which molecular weights and retention times were
compared with those of standard amino acids. Acetonitrile/water
containing 0.05% HCOOH was used as the mobile phase under a linear
gradient elution mode (MeCN, 10-60%, 40 min for 5-OHKF, 70 min
for norKA) at a flow rate of 0.6 mL/min. Different conditions were
used to analyze Phe and Leu in norKA (2): MeOH/water containing
0.05% HCOOH was used as the mobile phase under a linear gradient
elution mode (MeOH, 20-85%, 90 min for norKA) at a flow rate of
0.8 mL/min. A mass range of m/z 100-3000 was covered with a scan
time of 1 s, and data were collected in the positive ion mode. UV
detection at 200-400 nm was performed by photodiode array detection.
Acid Hydrolysis of 5-OHKF (1) and KF (3) to Acquire Fragment
1. 5-OHKF (5 mg) was hydrolyzed with 4 N HCl/EtOH (1:1) at 70 °C
for 8 h, and the fragments were separated by HPLC with solvents A
(H2O + 0.1% HCOOH) and B (MeCN + 0.1% HCOOH) and gradients
thereof: A 70% to 60% in 10 min, linear 60% in 10 min. The separation
yielded fragment 1. Fragment 1 is a peptide, KF-[5-MeHex-Val-5]
(HRESIMS m/z [M + H]+, 1266.7834; calcd for C63H104N13O14,
1266.7826). The mild acid hydrolysis of KF (3) under the same
condition produced fragment 2, which has the same molecular weight
as fragment 1.
Supporting Information Available: HRESIMS and 1D and 2D
NMR spectra of 1 and 2. This material is available free of charge via
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