Journal of Natural Products
ARTICLE
1
4,15
LC-MS Analysis of D/L-FDLA Derivatives.
. The acid hy-
drolysates of 1-4 were dissolved in H O (100 μL) separately. To a 50
2
μL aliquot of each were added 1 N NaHCO
3
(20 μL) and 1% 1-fluoro-
2,4-dinitrophenyl-5-L-leucinamide (L-FDLA or D/L-FDLA solution in
acetone, 100 μL) and then heated to 40 °C for 50 min. The solutions
were cooled to room temperature, neutralized with 1 N HCl (20 μL),
and then dried in vacuo. The residues were dissolved in 1:1
CH CN-H O and then analyzed by LC-MS. The analysis of the L-
3 2
and D/L-FDLA (mixture of D- and L-FDLA) derivatives was performed
on a Supelcosil LC-18 column (150 ꢀ 4.6 mm, 5 μm) employing a linear
3
gradient of 25% CH CN-75% 0.01 M formic acid to 70%
CH
3
CN-30% 0.01 M formic acid at 0.5 mL/min over 45 min. On
the basis of the elution order of the D- and L-FDLA derivatives for each
1
4,15
residue,
it is possible to assign whether the configuration of the R
carbon is D or L: amino acids in which the L-FDLA analogue elutes first
have an L configuration; those for which the D-FDLA analogue elutes
first have a D configuration. The retention times of the D/L-FDLA
mixtures (with the L-FDLA t
R
underlined) were as follows: D-3-OHLeu:
-
2
3.93, 29.94, m/z 440 [M - H] ; D-3-OMeAla: 23.10, 27.95, m/z 412
-
-
[
2
3
M - H] ; L-NMeThr 20.63, 23.27, m/z 426 [M - H] ; L-Ala 24.25,
- -
Figure 2. Effects of mirabamides E-H in HIV-1 neutralization assays
8.37, m/z 382 [M - H] ; L-ClHpr 30.30, 31.30, m/z 456 [M - H] ;
(standard deviations averaged 15%).
-
,4-DiMe-L-Gln 23.41, 25.26 m/z 468 [M - H] ; L-Dab 37.02, 42.85
-
m/z 705 [M - H] . Based on these determinations and analysis of the
NMR data, the following absolute configurations were assigned: 2R,3R-
OHLeu, 2R-3-OMeAla, 2S,3R-NMeThr, 2S-Ala, 2S,4S-ClHpr,
out using a Waters Micromass Q-tof micro integrated LC-MS system
employing negative ion ESI mode with an ion source temperature of
1
30 °C, a desolvation temperature of 400 °C, and desolvation with
2
S,3S,4R-DiMeGln, 2S,3S-Dab.
Absolute Configuration of Rhamnose. Compounds 1 and 2
nitrogen gas at a flow rate of 600 L/h. Analytical and semipreparative
HPLC was accomplished utilizing a Beckman System Gold 126 solvent
module equipped with a 168 PDA detector.
Sponge Material. Stelletta clavosa Ridley, 1884 was collected by
epibenthic sled from interlagoon seabed areas in the Torres Strait and
frozen immediately after collection. The sample was identified by M.K.H.
A voucher specimen is maintained at the Queensland Museum under
accession number G329301.
(0.2 mg each) were separately dissolved in 1 N HCl (250 μL) and heated
at 80 °C for 4 h with stirring. After cooling, the solvent was removed in
vacuo and the residue was dissolved in 1-(trimethylsilyl)imidazole (40
μL) and pyridine (160 μL). The solution was stirred at 60 °C for 20 min.
The solvent was removed by blowing with nitrogen. The residue was
2 2 2 2 2
partitioned with 1:1 CH Cl -H O in 1 mL. The CH Cl layer was
analyzed by GC-MS. A 30 m ꢀ 0.25 mm i.d. Restek RT-bDEXm column
was used for enantioselective GC with the starting temperature at 75 °C
and final temperature at 230 °C at a rate of 5 °C/min. A Waters GCT
Premier time-of-flight mass spectrometer was employed for analysis.
The retention time of the trimethylsilyl derivative of the L-rhamnose
standard was 14.69 min. The trimethylsilyl derivatives of L-rhamnose in
the hydrolysates of 1 and 2 had retention times of 14.68 and 14.68 min,
respectively.
Extraction and Isolation. The frozen sponge (365 g wet wt) was
exhaustively extracted with MeOH to yield 7.3 g of extract. The extract
was separated on HP20SS resin using a gradient of H O to IPA in 25%
2
steps, and a final wash of 100% MeOH, to yield five fractions. The third
fraction (50/50 H O/IPA) was further fractionated on Sephadex LH-20
2
with 1:1 CH
3
Cl-MeOH to give six fractions (Fr3.1-3.6). Fr3.2 was
chromatographed by HPLC using a Phenomenex Luna C18 column
(
250 ꢀ 10 mm) employing 40% CH CN-60% 0.2 M NaCl in H O at 4
3
2
HIV Infectivity Assays. Single-round HIV-1 infectivity assays
mL/min to yield compound 1 (7.0 mg, t
10.0 mg, t
compound 4 (15.0 mg, t
R
= 10.1 min), compound 2
1
9,20
were performed as described previously
using viruses pseudotyped
(
R
= 12.8 min), compound 3 (10.0 mg, t
= 23.9 min), and mirabamide C (3.5 mg, t
4.6 min) after desalting by C18 solid-phase extraction.
Mirabamide E (1):. colorless, amorphous powder; [R]
.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.30), 236 (4.41), 276
R
= 18.0 min),
with HIV-1 YU2-V3 Envelope, a CCR5-using strain. Briefly, virus stocks
were added to wells in a 96-well plate format in the presence of buffer,
vehicle, or increasing amounts of depsipeptide, followed by addition of
TZM-bl target cells that constitutively express HIV receptors CD4 and
R
R
=
1
2
0
D
-4 (c
0
1
13
CCR5. Plates were incubated at 37 °C, 5% CO , for 18 h, after which
2
(3.51) nm; H and C NMR data Table 1; HRESIMS m/z 1578.7705
þ
35
113 ClN13O24, 1578.7710).
fresh media was added to each of the wells. Following incubation for 24
h, cells were lysed and luciferase activity was measured using a BrightGlo
kit (Promega, Madison, WI) with a Synergy 2 luminescence plate reader
(BioTek, Winooski, VT). Inhibition curves were fit to eq 1 using
Kaleidagraph software, and results normalized to positive and negative
controls and plotted.
[M þ H] (calcd for C72
H
2
0
Mirabamide F (2):. colorless, amorphous powder; [R]
-6 (c
D
0
.1, MeOH); UV (MeOH) λ (log ε) 218 (4.15), 236 (4.26), 276
max
1
13
(3.35) nm; H and C NMR data Tables 2 and 3; HRESIMS m/z
þ
35
113 ClN13O23, 1562.7761).
1562.7765 [M þ H] (calcd for C72
H
20
Mirabamide G (3):. colorless, amorphous powder; [R]
D
þ13 (c
0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.11), 236 (4.21), 276
% infection ¼100=ð1þK ½inhibitorꢁÞ
ð1Þ
a
1
13
(3.32) nm; H and C NMR data Tables 2 and 3; HRESIMS m/z
þ
35
103 13 20
1432.7136 [M þ H] (calcd for C H
ClN O , 1432.7131).
66
20
Mirabamide H (4):. colorless, amorphous powder; [R]
D
þ12 (c
0.1, MeOH); UV (MeOH) λ (log ε) 218 (3.95), 236 (4.06), 276
’ ASSOCIATED CONTENT
Supporting Information.
and wgNOESY spectra for compounds 1-4. Ion chromatograms
of the D/L-FDLA derivatives and the L-FDLA derivatives of the
hydrolysis product of 1, papuamide A, and mirabamide C in
max
1
13
(3.29) nm; H and C NMR data Tables 2 and 3; HRESIMS m/z
1
13
þ
35
103 ClN13
S
H NMR, C NMR, HMBC,
1416.7180 [M þ H] (calcd for C66
H
O
19, 1416.7182).
b
Acid Hydrolysis of Peptides. Compounds 1-4, 300 μg each,
were separately dissolved in degassed 6 N HCl (600 μL) and heated in
sealed glass vials at 110 °C for 17 h. The solvent was removed in vacuo.
1
92
dx.doi.org/10.1021/np100613p |J. Nat. Prod. 2011, 74, 185–193