1556
Y. Usuki et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1553–1556
8
. Lammin, S. G.; Pedgrift, B. L.; Ratcliffe, A. J. Tetrahedron
Lett. 1996, 37, 6815.
9
. Schneider, S. E.; Bishop, P. A.; Salazar, M. A.; Bishop, O.
A.; Anslyn, E. V. Tetrahedron 1998, 54, 15063.
0. The identity and purity of all new compounds were
1
1
1
established by H NMR and MS: Spectral data for 4. H
NMR (400 MHz, CD OD) d 0.88 (3H, t, J 6.8), 1.18–1.42
3
(
26H, m), 1.57 (2H, m), 2.82 (3H, s), 3.14 (2H, t, J 7.2);
HRFABMS calcd for 298.3222. Found
98.3216.
18 40 3
C H N :
2
1
1
1. Shafi, M. S. J. Clin. Pathol. 1975, 28, 989.
2. Cells from an overnight culture were harvested by
centrifugation, washed with 50 mM succinate buffer (pH
6
1
.0), and suspended in the buffer to give a density of
0 cells/ml. The cell suspension was shaken with or
8
without each compound at 30 °C and the supernatant
obtained after removing cells by centrifugation was
+
+
assayed for its content of K ions using a K assay kit
HACH, Floriffoux, Bergium). The amount of UV-
(
absorbing materials was expressed as a value of the
absorption at a wavelength of 260 nm in cells of 1 cm
light-path.
Figure 5. Decrease in total glutathione content in S. cerevisiae cells
treated with 4 and NM, and the protective effect of PC. Cells were
incubated at 30 °C for 60 min in YPD medium containing 4 (100 lm)
or NM (10 lm), and PC at the concentrations indicated.
1
3. Cells from an overnight culture were inoculated into
freshly prepared YPD medium to give an initial cell
density of approximately 10 cells/ml. Cells were then
7
grown with or without each compound with vigorous
shaking and portions were withdrawn at various time
intervals to measure the cell growth by the turbidity-
dependent assay.
terminal guanidine, since the lactone ring structure of
the polyol macrolide compound is analogous to that
of AmB. The lower MIC values found for NM com-
pared to those for 4 indicate that the polyol lactone ring
augments the alkyl side chain’s interaction with plasma
membrane phospholipids.
7
1
4. After preincubation of the yeast cells (10 cells/ml) in YPD
0
0
medium with 40 lM 2 ,7 -dichlorodihydrofluorescein
diacetate (DCFH-DA) at 30 °C for 60 min, the cell
suspensions (1.0 ml) were withdrawn and further treated
with each chemical for the indicated times, washed, and
resuspended with 100 ll of phosphate-buffered saline. The
fluorescence intensity of the suspension (100 ll) containing
7
References and notes
10 cells was read using a Cytoflow 2300 fluorescence
spectrophotometer with excitation at 480 nm and emission
at 530 nm. Arbitrary units were directly given by the
fluorescence intensity.
1
2
. Blinov, N. O.; Golovkina, M. L.; Khokhlova, Y. M.;
Khlebarova, E. I.; Kleiner, E. M.; Georgieva, I. K.;
Sheikova, G. N.; Koreyako, A. I. Antibiotiki 1967, 12, 867.
. For a recent review on natural products with a guanidine
function, see: Berlinck, R. G. S. Nat. Prod. Rep. 2002, 19,
15. Cells from overnight cultures were further incubated in
7
YPD medium (10 cells/ml) at 30 °C with or without each
compound, collected, washed, resuspended in ice-cold
8 mM HCl containing 1.3% 5-sulfosalicylic acid, and
broken with glass beads by repeated vortexing. The
supernatant obtained after removing the beads and cell
debris by centrifugation (5000g) for 15 min at 4 °C was
used as the cytosol fraction. Glutathione was measured as
the total glutathione content, i.e., GSH and GSSG, by the
spectrophotometric monitoring of 2-nitro-5-thiobenzoate
formation from 5,5-dithiobis(2-nitrobenzoic acid).
16. For a recent review on the roles of glutathione in the
response to several stress situations in fungi, see: P o´ csi, I.;
Prade, R. A.; Penninckx, M. J. Adv. Microb. Physiol. 2004,
49, 1.
617.
3
. Grabley, S.; Hammann, P.; Raether, W.; Wink, J.; Zeeck,
A. J. Antibiot. 1990, 43, 639.
. Bassi, L.; Joos, B.; Gassmann, P.; Kaiser, H.-P.; Leuen-
berger, H.; Keller-Schierlein, W. Helv. Chim. Acta 1983,
4
66, 92.
5
6
7
. Nakayama, K.; Yamaguchi, T.; Doi, T.; Usuki, Y.;
Taniguchi, M.; Tanaka, T. J. Biosci. Bioeng. 2002, 94, 207.
. Poss, M. A.; Iwanowicz, E.; Reid, J. A.; Lin, J.; Gu, Z.
Tetrahedron Lett. 1992, 33, 5933.
. Levallet, C.; Lerpiniere, J.; Ko, S. Y. Tetrahedron 1997,
53, 5291.