8724 Ke et al.
Asian J. Chem.
evaporated to alcohol. Acetohydrazide 1 was obtained in 82.2
%, m.p. 62-63 ºC ref. 67 ºC. Compound 1 mixed with 1.5 g of
isothiocyanatocyclopropane for refluxing ca. 3 h in alcohol.
After cooling, a good amount of solid was obtained.The solid
was filtered, drying, recrystallized from methanol to give
intermediate 2, white crystal, yield 88.5 %, m.p. 117-118 ºC,
1H NMR (400M, CDCl3): 0.49-0.52(m, 2H, cyclopropane-
CH2), 0.57-0.64 (m, 2H, cyclopropane-CH2), 1.79 (s, 3H, Me),
2.88-2.94 (m, 1H, cyclopropane-CH), 7.83 (br, 1H, NH), 9.15
(br, 1H, NH), 9.57 (br, 1H,NH). Compound 2 (10 mmol) mixed
with 15 mL, 2 N NaOH for refluxing ca. 4 h. After cooling, 4
N HCl was added, a good amount of solid was obtained. The
solid was filtered, drying, recrystallized from methanol to give
intermediate 3, white crystals, yield 86.3 %, m.p. 134-135ºC,
1H NMR (400 MHz, CDCl3): 0.94-0.99(m, 2H, cyclopropane-
CH2), 1.26-1.31 (m, 2H, cyclopropane-CH2), 2.94 (s, 3H, CH3),
3.05-3.10 (m, 1H, cyclopropane-CH), 10.86 (br, 1H, SH);. To
a stirred solution of 3 (1.5 g, 5.1 mmol) and K2CO3 (0.2 g, 5.6
mmol) in DMF (15 mL), a mixture of 1-(chloromethyl)-3-
fluorobenzene (5.6 mmol) was added dropwise. The resulting
mixture was stirred at room temperature for overnight. The
mixture was poured into water, The precipitate formed was
filtered off and recrystallized from petroleum ether/acetone
to give 4 in good yields. white crystal, yield, 85.6 %; m.p.
plant parts from each cup was measured and the mean was
calculated. The check test was carried out in distilled water
only. The inhibition rate was calculated from the plant height
using the following equation:
(CK − PT)
=
×100 %
Relative inhibition rate (%)
CK
where CK is the average plant height during the blank assay
and PT is the average plant height after treatment during testing.
Cloning, expression and purification of rice KARI:
The DNA sequence corresponding to mature KARI was
amplified by PCR using the oligonucleotide primers 5’-
aaaggatCCATGGTCGCGGCGC-3’and 5’-cccAaaTTtgaagctt
CTACG ATGACTGCCGGAG-3’. In these sequences, lower
case represents mismatched bases, underlining indicates the
location of introduced BamHI and HindIII restriction sites and
italics show the Met-54 codon or the reverse complement of
the TAG stop codon. The PCR product was digested with
BamHI and HindIII and ligated into the pET-30a plasmid that
had been digested with the same enzymes. The resultant
expression plasmid was used to transform Escherichia coli
BL21(DE3) cells.
A single colony of these cells was inoculated into 20 mL
of LB medium containing 50 mg/mL kanamycin. The culture
was incubated overnight at 37 ºC and was used to inoculate
each of two 500 mL volumes of LB medium containing 50
mg/mL kanamycin; the cultures were incubated at 37 ºC with
shaking. When an OD600 of 0.8 was reached, expression was
induced by adding 0.5 mM isopropyl β-D-thiogalactoside to
each culture. These were then incubated at room temperature
(22 ºC) for a further 4 h with shaking and the cells were har-
vested by centrifugation.
The frozen cell pellet was thawed, suspended in ice-cold
purification buffer [20 mM Tris–HCl (pH 7.9)/500 mM NaCl]
containing 5 mM imidazole and then treated with lysozyme
(10 mg/g of cells for 0.5 h at 0 ºC). The cells were disrupted
by sonication, insoluble material was removed by centrifu-
gation and the supernatant was passed through a 0.45 mm
filter. The cell extract was applied to a 7 mL column of His·Bind
resin (Novagen) that had been charged by using 50 mM NiSO4
then equilibrated with purification buffer containing 5 mM
imidazole. The loaded column was washed with 23 mL of the
same buffer, followed by 30 mL of purification buffer conta-
ining 25 mM imidazole and then KARI was eluted with 30
mL of purification buffer containing 400 mM imidazole. Frac-
tions containing the enzyme were pooled, concentrated to 2.5
mL by ultrafltration and exchanged into 20 mM Na-Hepes
buffer, pH 8.0 using a Pharmacia PD-10 column. The eluate
was snap-frozen in liquid nitrogen and stored at -70 ºC9.
Enzyme and protein assays: Gerwick et al.10, reported
that the inhibition of Escherichia coli KARI is time-depen-
dent. To characterise the steady-state inhibition constant,
Escherichia coli KARI was preincubated for 10 min with
NADPH, Mg2+ and the title compound, then the reaction was
initiated with hydroxypyruvate. Under these conditions, the
change in A340 was found to be linear with time.
1
100-102 ºC; H NMR (400 MHz, CDCl3), 1.14-1.18(m, 2H,
cycloprane-CH2), 1.21-1.27(m, 2H, cycloprane-CH2), 2.57(s,
3H, Het-CH3), 3.01-3.27(m, 1H, cyclopropane-CH), 4.96(s,
2H, CH2), 7.01-7.13(m, 1H, Ph-H), 7.22-7.61(m, 3H, Ph-H);
ESI-MS: 548.81[2M + Na]+, 526.68 [2M]+, 264.16 [M + H]+;
Anal. calcd. (%) for C13H14N3SF: C, 59.29; H, 5.36; N, 15.96.
Found (%): C, 58.98; H, 5.44; N, 15.67.
Herbicidal activity assay: In vivo herbicidal activity of
title compounds was determined by rape root and barnyardgrass
cup tests according to the literature1.
Inhibition of the root-growth of Rape (Brassica
campestris):The evaluated compounds were dissolved in water
and emulsified by 100 µL of N,N-dimethyl formamide with
0.1 µL of Tween 20. Rape seeds were soaked in distilled water
for 4 h before being placed on a filter paper in a 6 cm Petri
plate, to which 2 mL of inhibitor solution had been added in
advance. Usually, 15 seeds were used per plate. The plate was
placed in a dark room and allowed to germinate for 65 h at 28
1 ºC. The length of 10 rape roots selected from each plate
was measured and the mean was calculated. The check test
was carried out in distilled water only. The inhibition rate was
calculated from the root length using the following equation:
(CK − PT)
=
×100 %
Relative inhibition rate (%)
CK
where CK is the average root length during the blank assay
and PT is the average root length after treatment during testing.
Inhibition of the seedling-growth of Barnyard grass
(Echinochloa crusgalli): The evaluated compounds were
dissolved in water and emulsified if necessary. 10 barnyard
grass seeds were placed into a 50 mL cup covered with a layer
of glass beads and a piece of filter paper at the bottom, to
which 5 mL of inhibitor solution had been added in advance.
The cup was placed in a bright room and allowed to germinate
for 65 h at 28 1 ºC. The height of seedlings of above-ground
Structure determination: The cube-shaped single crystal
of the title compound was obtained by recrystallization from
EtOH. The crystal with dimensions of 0.24 mm × 0.20 mm ×