Chloptosin
J . Org. Chem., Vol. 65, No. 2, 2000 463
of 200:1 and 150:1), two acetylated compounds showing Rf 0.64
and 0.50 (TLC-a) were eluted with CHCl3/MeOH (100:1).
Concentration of the former eluate gave diacetylchloptosin (2,
with MeOH (3 mL). From the MeOH-soluble fraction, valylpip-
erazic acid (5) (7 mg) was isolated by preparative TLC and
Sephadex LH-20 column chromatography as a colorless solid:
TLC-b Rf 0.34 (ninhydrin, Rydon-Smith pos); HVPE Rm 0.80;
55.7 mg) as colorless microcrystals: mp > 260 °C; [R]23 -81°
D
(c 0.4, CHCl3); UV λmaxMeOH (ꢀ) 213 nm (65660), 255 nm (16130),
mp 202-211 °C; [R]24 -6° (c 0.46, DMSO); EIMS m/z 229
D
0.1
N HCl-MeOH
(M+); 1H NMR (400 MHz, DMSO-d6) δ 8.0 (d, J ) 9 Hz,
ValNH2), 4.67, 4.59 (each dd, PipNH), 4.17 (dd, J ) 9, 5 Hz,
Valâ), 3.48 (dd, J ) 8.4, 3.4 Hz, PipR), 2.85, 2.74 (m, Pipδ),
2.13 (dqq, J ) 6.5, 6.5, 5 Hz, Valâ), 1.83, 1.66 (m, Pipâ), 1.66,
1.61 (m, Pipγ), 0.91 (d, J ) 6.5 Hz, Valγ′), 0.89 (d, J ) 6.5 Hz,
Valγ); 13C NMR (100 MHz, DMSO-d6) δ 173.3 (ValCO), 171.8
(PipCO), 57.9 (PipR), 57.1(ValR), 46.3 (Pipδ), 30.2 (Valâ), 27.2
(Pipâ), 22.7 (Pipγ), 19.3 (Valγ), 17.8 (Valγ′). Acid hydrolysis
of 5 (5.5 mg) with 6 N HCl (2 mL) at 105 °C for 19 h gave
(3S)-piperazic acid, which was changed to its DNP derivative,
DNP-(3S)-piperazic acid (1.1 mg), by the above-mentioned
procedure: TLC (CHCl3/MeOH, 5:2) Rf 0.17; FABMS (pos) m/z
319 (M + Na)+, 297 (M + H)+; FABMS (neg) m/z 295 (M -
298 nm (sh, 4810), λmax
(ꢀ) 213 nm (63360), 256
0.1
N NaOH-MeOH
nm (16840), 298 nm (sh, 4810), λmax
(ꢀ) 257 nm
(17010), 298 nm (sh, 6420); FABMS (pos) m/z 1627 (M + Na)+,
1605 (M + H)+, 803 (M/2 + H)+; H and 13C NMR data, see
1
Table 2. From the latter eluate, tetraacetylchloptosin (3, 153.8
mg) was obtained as colorless microcrystals: mp > 260 °C;
[R]23 -75° (c 1.0, CHCl3 ); UV λmax
(ꢀ) 225 nm (29980),
MeOH
D
0.1N HCl-MeOH
257 nm (17160), 305 nm (sh, 6080), λmax
(ꢀ) 218
0.1
N
nm (55070), 255 nm (20260), 300 nm (sh, 8020), λmax
-MeOH
NaOH
(ꢀ) 257 nm (21660), 300 nm (sh, 9710); FABMS (pos)
1
m/z 1711 (M + Na)+, 1689 (M + H)+; H and 13C NMR data,
n
see Table 2; J CH values, see Table 3.
Acid Hyd r olysis of 1. Crystalline 1 (10.1 mg) was hydro-
lyzed with 6 N HCl (1 mL) in a sealed tube at 105 °C for 16 h.
The hydrolysate was diluted with H2O (5 mL) and washed with
diethyl ether (5 mL twice). Evaporation of the aqueous layer
gave a residue (13.3 mg) containing several ninhydrin-positive
compounds (HVPE Rm 0.96, 0.87, 0.82, 0.76, and 0.45), and
the five known amino acids were isolated by preparative HVPE
and TLC-b. Concentration of the Rm 0.9 band yielded the
racemic piperazic acid (2.1 mg): Rm 0.98 (HVPE); Rf 0.37 (TLC-
b); [R]23D -5° (c 0.5, H2O). Evaporation of the Rm 0.7-0.8 band
gave a residue (7.2 mg) showing several ninhydrin-positive
spots by TLC-b. Further purification by preparative TLC-b
gave the following four amino acids:
H)-; [R]25 -232° (c 0.05, MeOH).
D
From the MeOH-insoluble residue, piperazic acid anhydride
(4) was obtained as colorless microcrystals (44 mg): TLC-b Rf
0.48 (ninhydrin neg, Mo-H2SO4 pos); mp 186-189 °C; [R]25
D
< -1° (c 0.25, MeCN-H2O, 1:1); EIMS m/z 224 (M+); FABMS
(pos) m/z 225 (M + H)+ (C10H16N4O2 requires 224). 1H NMR
(400 MHz, D2O/CD3CN, 1:1, 50 °C, TSP δ ) 0) δ 4.18 (dd, J )
12.4, 2.4 Hz, H4a, H8a), 3.06 (ddd, J ) 13.0, 3.6, 3.6 Hz, H2eq,
H6eq), 2.72 (ddd, J ) 13.0, 13.0, 3.6 Hz, H2ax, H6ax), 2.28
(m, H4eq, H8eq), 1.90 (ddddd, J ) 14.0, 4.4, 4.4, 3.6, 3.6 Hz,
H3eq, H7eq), 1.81 (ddddd, J ) 14.0, 13.0, 12.4, 4.4, 3.6 Hz,
H3ax, H7ax); 13C NMR (100 MHz, D2O/CD3CN, 1:1, 50 °C) δ
164.7 (C9, C10), 60.6 (C4a, C8a), 49.0 (C2, C6), 31.9 (C4, C8),
26.3 (C3, C7). Chemical shifts were assigned by 1H-1H COSY
and HMQC experiments.
D-Valine (1.2 mg) showing Rm 0.86 and Rf 0.46 was obtained,
and its chirality was determined by application onto a chiral
HPTLC plate (D Rf 0.40, L Rf 0.50).
D-Threonine (0.7 mg) showing Rm 0.81 and Rf 0.33 was
identified by Avicel TLC (Funacel SF Cellulose, Funakoshi)
developed by an upper layer of BuOH/Me2CO/28% NH4OH/
H2O (8:1:1:6) (threonine Rf 0.43, allothreonine Rf 0.35) and by
chiral HPTLC (D-threonine Rf 0.44, L-threonine Rf 0.49).
L-Serine (0.6 mg) showing Rm 0.88 and Rf 0.25 was isolated,
and its chirality was determined by chiral HPTLC (L Rf 0.41,
D Rf 0.38). O-Methylserine (1 mg) showing Rm 0.77 and Rf 0.34
was identical with authentic O-methyl-DL-serine (Sigma). Acid
hydrolysis of the authentic sample generated serine under the
hydrolysis conditions. Therefore, the chirality of O-meth-
ylserine was deduced to be L.
Mild Acid Hyd r olysis of 1. Crystalline 1 (799 mg) was
hydrolyzed with 6 N HCl (10 mL) in a sealed tube at 105 °C
for 4 h and concentrated to give a hydrolysate (1.01 g) that
was then purified by column chromatography on ODS silica
gel RP-18 (SSC-ODS-7515-12A, Senshu Scientific, 200 mL).
The column was developed with 10% MeCN (500 mL, fractions
1-45) and then with 20% MeCN in H2O (500 mL, fractions
46-86).
Cell Via bility. AsPC-1 cells were cultured in Roswel Park
Memorial Institute 1640 medium supplemented with 10% fetal
bovine serum, 100 µg/mL kanamycin, 100 units/mL penicillin
G, and 30 µg/mL glutamine in a 5% CO2/95% air atmosphere.
Cells (1 × 105) were seeded in 24-well culture plates. After 24
h, chemicals or microbial culture broths were added to the
cells, and then the cells were incubated for another 24 h. Cell
viability was determined by cell counting with a hemocytom-
eter after staining with trypan blue. Surviving cells were
expressed as a percentage of the total cell population. The EC50
values are means of triplicate determinations.
Nu clea r F r a gm en ta tion . Cells (2 × 105) were plated on
coverslips in 12-well culture plates and treated with 1 for 24
h. Then, the cells were fixed for 15 min with 3% paraformal-
dehyde solution, and the nuclei were stained for 5 min with
the dye Hoechst 33258. The coverslips were examined under
a fluorescence microscope.
An tim icr obia l a ctivity. In vitro antibacterial activity
(minimum inhibitory concentrations, MICs) were determined
by the 2-fold agar dilution method with Bacto Mueller-Hinton
Medium (Difco) at 37 °C for 18 h, according to the standard
method of J apan Society of Chemotherapy. The standard
bacterial strains and clinical isolates (60 strains) of methicillin-
resistant Staphylococcus aureus (MRSA) were used from the
stock cultures of Institute of Microbial Chemistry.
A solid (248 mg) obtained from fractions 13 and 14 was
further purified by column chromatography on Dowex 50W-
X2 (200-400 mesh, pyridine form 300 mL) eluted with 0.1 M
pyridine-formate buffer (pH 2.7, 850 mL). The eluate of
fractions 56-64 (each 10 mL) was concentrated to give a
colorless hygroscopic solid (99.7 mg) of piperazic acid (race-
mic): HVPE Rm 0.98 (brownish purple by ninhydrin). TLC-b:
Rf 0.37; [R]23 +2° (c 0.1, H2O). Piperazic acid (46.8 mg) was
Ack n ow led gm en t. The authors wish to thank Dr.
Katsuharu Iinuma, Meiji Seika Kaisha, Ltd., for the
large-scale preparation of chloptosin, and Miss Kumi
Nakazawa, Keio University, for her technical assistance.
This work was financially supported in part by grants
from the Ministry of Education, Science, Culture and
Sports of J apan, and the Science Research Promotion
Fund of the Promotion and Mutual Aid Corporation for
Private Schools of J apan.
D
dissolved in a mixture of H2O (1.8 mL) and EtOH (2 mL)
containing NaHCO3 (148 mg). The mixture was added to 2,4-
dinitrofluorobenzene (185 mg, Wako Pure Chemical) in EtOH
(5 mL) and kept overnight at room temperature. The DNP
derivative was purified by diethyl ether extraction, Sephadex
LH-20 column chromatography (Pharmacia, MeOH), and then
preparative TLC (CHCl3/MeOH, 5:2, Rf 0.17) to give a yellow-
ish solid (10.9 mg): FABMS (pos) m/z 319 (M + Na)+, 297 (M
+ H)+; FABMS (neg) m/z 295 (M - H)-; [R]23 +30° (c 1.0,
D
MeOH) (a racemic mixture). DNP-(3R)-piperazic acid obtained
from polyoxypeptin A by a similar procedure showed [R]25
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra of 1-3 and selected 2D NMR spectra. This material
D
+211° (c 1.0, MeOH) (lit.10 +307°).
Evaporation of the eluate in fractions 21-25 (ODS silica
gel column) gave a solid (76 mg) that was separated into
MeOH-soluble and MeOH-insoluble fractions by treatment
J O991314B