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CH-Leu), 26.4 (b-CH2-Glu), 31.4 (g-CH2-Glu), 39.8 (b-CH2-
Leu), 51.4 (a-CH-Leu), 54.4 (a-CH-Glu), 63.0 (CH2), 174.1,
175.3, 176.2 (CO). MS (ES+) m/z 317, 318 [M+H]+. HPLC
(B) Rt: 23 min; purity: >99%.
(6)1H NMR (400 MHz) (D2O) d: 1.03 (d, 3H, J=5.9, d-
CH3-Leu), 1.08 (d, 3H, J=5.6, d-CH3-Leu), 1.70–1.90 (m, 3H,
b-CH2- and g-CH-Leu), 2.20–2.40 (m, 2H, b-CH2-Glu), 2.60–
2.75 (m, 2H, g-CH2-Glu), 3.92 (m, 1H, a-CH-Glu), 4.04 (2H,
m, CH2), 4.48 (m, 1H, a-CH-Leu). MS (ES+) m/z 333
[M+H]+. HPLC (B) Rt: 17.1 min; purity: >99%.
14. All compounds were initially tested as substrates and/or
inhibitors at 0.5 mM using recombinant wild-type or C79A
mutant C. fasciculata glutathionylspermidine synthetase-ami-
dase. The relative activity as a substrate is the activity expres-
sed as the % of activity measured with 0.5 mM glutathione.
The % inhibition was determined according to the following
equation:
ꢀ
ꢁ
vi
% inhibition ¼ 1 À
:100
vo
Initial velocity measurements were made at 8–10 inhibitor
concentrations, ranging from 0 to 2.5 mM, and 0.5 mM glu-
tathione. Spermidine (2 mM), Mg2+ (10 mM) and ATP (2
mM) were present in saturating conditions. The assays were
conducted in 100 mM HEPES buffer pH 7.3 at 25 ꢁC with 40
nM GspS. Assay mixtures were incubated for 5 min prior to
initiation with spermidine. IC50 values were determined
according to the following two parameter equation, where the
lower data limit is 0, that is the data are background corrected,
and the upper data limit is 100, that is the data are range
corrected.
12. Odake, S.; Okayama, T.; Obata, M.; Morikawa, T.; Hat-
tori, S.; Hori, H.; Nagai, Y. Chem. Pharm. Bull. 1990, 38,
1007.
13. All compounds gave satisfactory spectroscopic data: We
report the NMR, MS and HPLC data obtained with the most
active compounds. HPLC on RP18 with A: H2O–TFA (0.1%)
and B: CH3CN–TFA (0.1%) in a 35 min linear gradient (sys-
tem I: from 10 to 90% B or system II: from 0 to 20% B).
1
(3) H NMR (400 MHz) (CD3OD) d: 0.90 (d, 3H, J=5.63,
d-CH3-Leu), 0.94 (d, 3H, J=5.63, d-CH3-Leu), 1.54–1.74 (m,
3H, b-CH2- and g-CH-Leu), 2.05–2.16 (m, 1H, b-CH2-Glu),
2.17–2.28 (m, 1H, b-CH2-Glu), 2.45–2.61 (m, 2H, g-CH2-Glu),
3.68–3.87 (m, 1H, a-CH-Glu), 4.31–4.45 (m, 1H, a-CH-Leu),
4.60–4.80 (2H, m, CH2). 13C NMR (CD3OD) d: 21.7 (d-CH3-
Leu), 23.5 (d-CH3-Leu), 25.9 (g-CH-Leu), 27.5 (b-CH2-Glu),
32.5 (g-CH2-Glu), 41.5 (b-CH2-Leu), 53.5 (a-CH-Leu), 54.9
(a-CH-Glu), 58.3 (CH2), 130.0 (C), 175.0, 175.4 (CO). MS
(FAB+) m/z 342 [M+H]+. HPLC (A) Rt: 6.3 min; purity:
95%.
100
ꢀ
ꢁ
y ¼
s
x
1 þ
IC50
In this equation, s is a slope factor. The equation assumes that
y falls with increasing x.
Initial velocity measurements were determined at three
inhibitor concentrations and a serial dilution of glutathione,
ranging from 0.03 to 1 mM. From these data, Michaelis–
Menten and Lineweaver–Burk plots were constructed, and
kinetic inhibitory values were determined by non-linear least
squares regression analysis using the Grafit1 programme.
Inhibitors gave the best fit with mixed or competitive type
inhibition. The indicated errors are the standard errors on the
fit.
1
(4) H NMR (400 MHz) (D2O + two drops (CD3)2CO) d:
0.80 (d, 3H, J=5.36, d-CH3-Leu), 0.84 (d, 3H, J=5.98, d-
CH3-Leu), 1.42–1.69 (m, 3H, b-CH2- and g-CH-Leu), 1.92–
2.18 (m, 2H, b-CH2-Glu), 2.32–2.45 (m, 2H, g-CH2-Glu),
3.61–3.72 (m, 1H, a-CH-Glu), 4.25–4.35 (m, 1H, a-CH-Leu),
4.55–4.78 (m, 2H, CH2). 13C NMR (D2O + two drops
(CD3)2CO) d: 20.9 (d-CH3-Leu), 22.4 (d-CH3-Leu), 24.5 (g-
15. Oza, S. L.; Ariyanayagam, M. R.; Fairlamb, A. H. Bio-
chem. J. 2002, 364, 679.