Design, synthesis and biological evaluation of urea derivatives from o-hydroxybenzylamines and phenylisocyanate as potential FabH inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.06.049

FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-negative bacteria. A series of o-hydroxybe

Full text of DOI:10.1016/j.bmc.2011.06.049

Bioorganic & Medicinal Chemistry 19 (2011) 4413–4420
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of urea derivatives from
o-hydroxybenzylamines and phenylisocyanate as potential FabH inhibitors
⇑
Zi-Lin Li, Qing-Shan Li, Hong-Jia Zhang, Yang Hu, Di-Di Zhu, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
FabH, b-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central
to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-
negative bacteria. A series of o-hydroxybenzylamines 1–16 and its corresponding new urea derivatives
17–32 were synthesized and fully characterized by spectroscopic methods and elemental analysis. This
new urea derivatives class demonstrates strong antibacterial activity. Escherichia coli FabH inhibitory
assay and docking simulation indicated that the compounds 1-(5-bromo-2-hydroxybenzyl)-1-(4-fluoro-
phenyl)-3-phenylurea (18) and 1-(5-bromo-2-hydroxybenzyl)-1-(4-chlorophenyl)-3-phenylurea (20)
were potent inhibitors of E. coli FabH.
Received 29 April 2011
Revised 16 June 2011
Accepted 16 June 2011
Available online 25 June 2011
Keywords:
FabH
Inhibitors
Antibacterial
o-Hydroxybenzylamines
Urea derivatives
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction
O
The multi-drug resistance to most of all antibiotics has become a
serious medical problem1; therefore, there is an urgent need for
development of new antibacterial agents with divergent and unique
structure, which may act through novel target, surmounting the
problem of acquired resistance.2
S-CoA
Acetyl-CoA
CO2 CoA-SH
FabH
O
O
O
O
R
S-Acp
An attractive target is the fatty acid biosynthesis (FAB) in bacte-
ria, which is an essential metabolic process and is required for cell
viability and growth.3 The b-ketoacyl-acyl carrier protein synthase
III (FabH) is one of functional enzymes in FAB, which initiates the
FAB cycle by catalyzing the first condensation step between acet-
yl-CoA and malonyl-ACP (Fig. 1). The other bacterial condensing
enzymes FabB and FabF, functioning later in the cycle, differ signif-
icantly from FabH in that they use acyl-ACP rather than acetyl-CoA
as the primer for subsequent condensations and are hence nonre-
dundant. As a result, no other known enzyme in the pathway ap-
pears to be able to accomplish this essential reaction, and thus,
FabH appears to play a key role in the bacterial FAB cycle.4 More-
over, the key role and three-dimensional structure of the protein
are highly conserved across various Gram-positive and Gram-neg-
ative bacteria and therefore its inhibitors can be potent antibiotics
with broad-spectrum activity.5
β-ketoacyl-Acp
HO
S-Acp
Malonyl-CoA
Figure 1. FabH-catalyzed initiation reaction of fatty acid biosynthesis.
simulating the natural substrate of specific bacteria enzyme, o-hy-
droxy diphenyl ethers can damage and block the normal biosyn-
thesis of fatty acids.7–9 Its molecular fragment ‘o-hydroxyphenyl’
also shows favorable biological activity, which has a good affinity
to coenzyme NAD+ and plays a key role in the process of inhibiting
pathogens.10–12 Therefore, the ‘o-hydroxyphenyl’ fragment can be
used as core structure unit to design antibacterial drugs.13
Phenylisocyanate is a widely used intermediate in organic syn-
thesis,14 which can serve as the reactant to form urea derivatives.
It has been well established that urea derivatives have got a signifi-
cant place in modern medicinal chemistry. Urea derivatives have
been reported in the literature as anticancer agent,15 anticonvul-
sant,16 and CXCR3 antagonist.17 Furthermore, in the past years, a
large variety of urea derivatives were reported to function as
inhibitors on HIV protease enzyme,18 receptor tyrosine kinases
o-Hydroxy diphenyl ethers is widely used as effective broad-
spectrum antibiotic, which possess potent inhibiting effects on
Gram-positive and Gram-negative bacteria, yeast and viruses.6 By
⇑
Corresponding author. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.049

4414
Z.-L. Li et al. / Bioorg. Med. Chem. 19 (2011) 4413–4420
(RTKs),19 raf-1 kinases,20 NADH oxidase,21 g-secretase,22 and acyl-
coenzymeA-cholesterol acyltransferase (ACAT).23 However, to our
knowledge, few reports have been dedicated to the synthesis and
FabH inhibitory activity of urea derivatives.
Table 1
Chemical structures of 1–32
Compound
R1
R2
R3
R4
Compound
R1
R2
R3
R4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
H
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
F
H
F
H
Cl
H
Br
CH3
OCH3
H
F
H
Cl
H
Br
CH3
OCH3
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
H
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
F
H
F
H
Cl
H
Br
CH3
OCH3
H
F
H
Cl
H
Br
CH3
OCH3
Herein, in continuation of our earlier studies on urea deriva-
tives24,25 and an attempt to achieve potential antibacterial agents,
we synthesized a series of o-hydroxybenzylamines and besides,
through using these compounds as continuing point, designed
and synthesized a new series of urea derivatives. The antimicrobial
activity against two Gram-negative bacterial strains, Escherichia
coli and Pseudomonas fluorescence, and two Gram-positive bacterial
strains, Bacillus subtilis and Staphylococcus aureus, of these com-
pounds were also been determined. Docking simulations were per-
formed using the X-ray crystallographic structure of the FabH of
E. coli complexed with the most potent inhibitors (18 and 20) to
explore the binding modes of these compounds at the active site.
H
Cl
H
Br
H
H
H
F
H
Cl
H
Br
H
H
H
H
Cl
H
Br
H
H
H
F
H
Cl
H
Br
H
H
H
2. Results and discussion
2.1. Chemistry
compounds gave satisfactory analytical and spectroscopic data,
which in accordance with their depicted structures (Table 1).
A series of o-hydroxybenzylamines 1–16 and its corresponding
urea derivatives 17–32 were synthesized by the route outlined in
Scheme 1. The secondary amines (1–16) were obtained from the
corresponding Schiff bases after the reduction reactions with so-
dium borohydride in 60–90% yield.26 In the next step, the secondary
amines were condensed with phenylisocyanate in chloroform as
the solvent, affording the target compounds after 5 h for derivatives
with electron-donating substituent groups (17–22 and 25–30), and
after 10 h for the electron-withdrawing substituted derivatives
(23–24 and 31–32). The reactions were monitored by thin layer
chromatography (TLC) and the products were purified by recrystal-
lization with ethanol and hexyl hydride or by column chromatogra-
phy, with yields in the range of 50–80%. All of the synthetic
2.2. Biological activity
2.2.1. Antibacterial activity
All the synthetic compounds (1–32) were evaluated for their
antimicrobial activities against two Gram-negative bacterial
strains: E. coli and P. fluorescence and two Gram-positive bacterial
strains: B. subtilis and S. aureus by MTT method. The MICs (mini-
mum inhibitory concentrations) of the compounds against these
bacteria were presented in Table 2. Also included was the activity
of standard antibacterial agent kanamycin B under identical condi-
tions for comparison. The results revealed that most of the syn-
thetic compounds exhibited significant antibacterial activities.
Especially, the urea derivatives 17–32, whose MICs values range
R2
O
R2
H2N
R3
EtOH
H
reflux
N
R4
R1
OH
R1
OH
R2
R3
R4
NaBH4,EtOH
reflux
H
N
R1
OH
R3
R4
1-16
R2
PhNCO,CH3Cl
reflux
HN
O
N
R1
OH
R3
R4
17-32
Scheme 1. The synthetic routes of compounds 1–32.

Z.-L. Li et al. / Bioorg. Med. Chem. 19 (2011) 4413–4420
4415
Table 2
F, Cl, Br group on aniline component showed significant antibacte-
rial activity with MIC of 1.56–12.5 g/mL against E. coli ATCC3
Antimicrobial activity of the synthesized compounds
l
5218. Compounds 17, 19, 21, 25, 27 and 29 with o-substituted
electron-withdrawing group on aniline component exhibited less
potent activity with MIC of 12.5–50 lg/mL. These results demon-
Compound
Minimum inhibitory concentrations (
Gram-negative Gram-positive
P. fluorescence B. subtilis S. aureus
lg/mL)
E. coli
strated that the synthetic urea derivatives with p-substituted
halogen group on aniline component showed more potent antibac-
terial activity than those of o-substituted. Most significantly, the
antibacterial activity of these compounds enhanced slightly in
the order of substituent on aniline component: Br < F < Cl.
1
2
3
4
5
6
7
8
25
6.25
25
25
12.5
25
12.5
50
50
50
50
25
50
25
50
50
25
12.5
12.5
6.25
25
25
50
12.5
6.25
6.25
50
25
50
2.2.2. E. coli FabH inhibitory activity
50
50
50
The E. coli FabH inhibitory potency of the selected compounds
17–22 and 26–28 was examined and the results were summarized
in Table 3. As shown in Table 3, among the tested compounds,
compounds 18 and 20 showed potent inhibitory activity with
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
50
50
25
25
12.5
25
50
50
12.5
50
12.5
50
50
50
50
25
6.25
25
3.13
25
12.5
50
50
12.5
25
50
50
6.25
12.5
50
50
50
25
25
6.25
6.25
6.25
6.25
12.5
12.5
50
50
12.5
50
25
50
25
12.5
50
50
50
50
50
3.13
3.13
1.56
25
12.5
50
12.5
25
12.5
12.5
6.25
50
IC50 of 6.6 and 4.7
the positive control DDCP27 with IC50 of 2.1
pounds displayed moderate inhibitory activity with IC50 ranging
from 10.1 to 71.7 M. It suggested that the introduction of phenyl-
lM, respectively, which were comparable to
lM. Other tested com-
50
12.5
1.56
6.25
1.56
12.5
6.25
50
l
isocyanate and the formation of urea structure were essential for
the FabH inhibitory activity. It also can be seen from Table 3 that
the selected compounds displayed low hemolytic activity.
2.3. Molecular docking study of synthetic compounds and
binding model of compounds 18 and 20 and E. coli FabH
25
50
6.25
6.25
3.13
50
12.5
50
25
The binding affinity was evaluated by binding energy and inhib-
itory constant. Molecular docking of all the synthetic urea deriva-
tives and E. coli FabH was performed on the binding model based
12.5
6.25
25
50
25
25
50
50
50
50
1.56
25
6.25
50
Table 3
12.5
3.13
E. coli FabH inhibitory activity of the selected com-
pounds 17–22, 26–28
Kanamycin B
3.13
1.56
Compound
E. coli FabH
Hemolysis
LC30 (mg/mL)
from 1.56 to 50 lg/mL, displayed higher antibacterial potencies
than o-hydroxybenzylamines 1–16, with MICs values ranging from
a
IC50
(l
M)
17
18
19
20
21
22
26
27
37.4
6.6
21.8
4.7
56.4
41.0
71.7
51.5
10.1
2.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
6.25 to 50 g/mL, which showed the introduction of phenylisocya-
l
nate increased the hydrophobicity of o-hydroxybenzylamines and
lead to the increase of the antibacterial activity.
Out of the 16 urea derivatives, compounds 18 and 20 displayed
most potent activity with MIC values of both 1.56
E. coli ATCC35218, which were superior to the positive control
kanamycin B with corresponding MIC of 3.13 g/mL. Compounds
28 showed significant activity with MIC values of 3.13 g/mL
lg/mL against
l
28
DDCP
l
against E. coli ATCC35218, which was comparable to the positive
control kanamycin B. Besides, the data showed compounds 18
and 20 exhibited favorable activity with MIC of 6.25, 6.25,
a
Lytic concentration 30%.
3.13 lg/mL and 3.13, 6.25, 1.56 lg/mL against P. fluorescence, B.
subtilis, S. aureus, respectively, indicating that they possessing
broad-spectrum antibacterial activity.
Table 4
Docking parameters of synthetic compounds
Compound
Binding energy
Inhibitory
Based on the data obtained, we found that compounds with a
bromine atom on the 5-position of salicylaldehyde displayed higher
antibacterial activity against E. coli ATCC35218 than compounds
with two bromine atoms on the 3-position and 5-position of salicyl-
aldehyde. And the varieties of substitutes of aniline such as halogen,
methyl and methoxyl also lead to the different antibacterial activi-
ties of these urea derivatives. Among them, the derivatives which
have electron-withdrawing substituents (such as F, Cl, Br) exhibited
more potent activity against E. coli ATCC35218 than those have
electron-donating substituents (such as CH3, OCH3), and their MICs
4Gb (kcal/mol)
constant (fM)
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
ꢀ19.02
ꢀ21.88
ꢀ18.97
ꢀ21.08
ꢀ19.61
ꢀ19.46
ꢀ19.31
ꢀ18.97
ꢀ19.22
ꢀ19.3
38.36
1.61
12.42
1.03
7.06
5.48
57.82
35.61
8.1
7.13
8.43
values range from 12.5 to 50 lg/mL. We proposed that electron-
ꢀ19.2
ꢀ20.02
ꢀ16.53
ꢀ18.78
ꢀ18.57
ꢀ17.35
9.11
withdrawing halogen groups on aniline component were conducive
to the antibacterial activity and compounds with CH3, OCH3 substit-
uents on aniline component were not favorable for activity.
Moreover, among derivatives with electron-withdrawing sub-
stituents, compounds 18, 20, 22, 26, 28 and 30 with p-substituted
759.09
17.23
24.59
191.97

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Z.-L. Li et al. / Bioorg. Med. Chem. 19 (2011) 4413–4420
on the E. coli FabH-CoA complex structure (1HNJ.pdb).28 The FabH
active site generally contains a catalytic triad tunnel consisting of
Cys-His-Asn, which is conserved in various bacteria. This catalytic
triad plays an important role in the regulation of chain elongation
and substrate binding. Since the alkyl chain of CoA is broken by Cys
of the catalytic triad of FabH, interactions between Cys and sub-
strate appear to play an important role in substrate binding
(Fig. 1). Qiu et al. have refined three-dimensional structure of
E. coli FabH in the presence and absence of malonyl-CoA by X-ray
spectroscopy. Since, malonyl moiety is degraded by E. coli FabH,
molecular docking studies for FabH and malonyl-CoA was carried
out to identify a plausible malonyl-binding mode.28 They found
that in one of the binding modes appeared in the lower scored con-
formations, the malonyl carboxylate formed hydrogen bonds to the
backbone nitrogen of Phe304. Enlightened by this report, Kim and
co-workers designed pharmacophoremaps considering the interac-
tion with Phe304 and performed receptor-oriented pharmaco-
phore based in silico screening of E. coli FabH.3 YKAs3003, a
Figure 2. Binding model of compound 18 and E. coli FabH. H-bonds are displayed as dashed lines. Amino hydrogen of Gly306 forms hydrogen bond with oxygen atom of urea
structure of compound 18.
Figure 3. Binding model of compound 20 and E. coli FabH. H-bonds are displayed as dashed lines. Amino hydrogen of Asn247 forms hydrogen bond with oxygen atom of urea
structure of compound 20.

Z.-L. Li et al. / Bioorg. Med. Chem. 19 (2011) 4413–4420
4417
Schiff base condensed by 4-hydroxy salicylaldehyde and cyclohex-
4.2. General method for synthesis of o-hydroxybenzylamines
anamine, was hit by pharmacophore map I consisting of three fea-
tures (two hydrogen bond donors that involving the backbone
oxygen of Phe304 and Gly209, respectively, and one hydrophobic
interaction with Ile156, Phe157 and Met207).
1–16 and corresponding urea derivatives 17–32
Anilines (4.0 mmol) in ethanol (10 mL) was treated with equi-
molar quantities of substituted salicylaldehydes. The mixture was
refluxed for 4–6 h, and the reaction was monitored by TLC. The
products thus precipitated were filtered and recrystallized in etha-
nol. Without further purification, to an ethanolic solution of synthe-
sized Schiff bases, NaBH4 (4.0 mmol) was dissolved in ethanol and
slowly added in an ice bath with stirring. The mixture was refluxed
for 2–3 h. After that, the solvent was evaporated and water (15 mL)
was added. The product extraction was carried out with CH2Cl2
(3 ꢂ 20 mL). The organic layer was dried over anhydrous Na2SO4
and concentrated. The residue was purified by recrystallization
from ethanol to afford white or yellow crystals (1–16).
To chloroform solution of secondary amines (1–16), phenyliso-
cyanate (1:1) was slowly added with stirring. The mixture was re-
fluxed 5–10 h. The completion of reaction was checked by TLC. The
CHCl3 was removed under reduced pressure and the crude product
was purified by recrystallization with ethanol and hexyl hydride or
by column chromatography to afford urea derivatives (17–32).
The binding affinity of the synthetic urea derivatives and E. coli
FabH is depicted in Table 4. As shown in Table 4, the results of
binding energy and inhibitory constant almost have the same trend
with the structure–activity relationships. Structure–activity rela-
tionships in compounds 17–32 demonstrated that compounds with
different substituent group exhibit different bioactivity. Among
them, compounds 18 and 20 showed minimum antibacterial data.
Besides, in parameter of binding affinity, compounds 18 and 20 also
exhibit favorable data, with free binding energy of ꢀ21.88 and
ꢀ21.08 kcal/mol, inhibitory constant of 1.61 and 1.03 fM, respec-
tively. The estimated free binding energy of other compounds (17,
19, 21–32) are ranging from ꢀ20.02 to ꢀ16.53 kcal/mol and inhibi-
tory constants are ranging from 5.48 to 759.09 fM, respectively. This
molecular docking result is along with the biological assay data, sug-
gesting that compounds 18 and 20 are potential inhibitors of E. coli
FabH.
Based on these facts, compounds 18 and 20 with the most potent
inhibitory activity were selected for further molecular docking
study. The binding models of compounds 18, 20 and E. coli FabH
are depicted in Figures 2 and 3, respectively. In the binding model
of compound 18 and E. coli FabH, amino hydrogen of Gly306 forms
hydrogen bond (H–Oꢁ ꢁ ꢁH: 2.10 Å, 142.57°) with oxygen atom of urea
structure of compound 18. The urea structure of compound 18 pro-
jects into a hydrophobic interaction region of FabH, which contained
the side chains: Leu142, Leu189, Asn274 and Phe157. Amino hydro-
gen of Asn247 also forms hydrogen bond (H–Oꢁ ꢁ ꢁH: 1.91 Å, 159.87°)
with oxygen atom of urea structure of compound 20 and it exits a
hydrophobic interaction region, comprised of Phe213, Phe304,
Met207 and Gly152. The hydrophobic interactions between 18, 20
and FabH were important for their potent inhibitory activity.
4.2.1. 4-Bromo-2-(((2-fluorophenyl)amino)methyl)phenol (1)
White crystal, yield 69%, mp: 101–102 °C, 1H NMR (300 MHz,
CDCl3): 4.42 (s, 2H); 6.12 (s, 1H); 6.79 (d, J = 8.4 Hz, 1H); 6.84–
6.94 (m, 2H); 6.79 (d, J = 7.5 Hz, 2H); 7.35 (d, J = 2.6 Hz, 2H); 8.13
(s, 1H). ESI-MS
13H11BrFNO: C, 52.73; H, 3.74; N, 4.73. Found: C, 52.69; H, 3.72;
N, 4.72.
C
13H11BrFNO [M+H]+ 296.0. Anal. Calcd for
C
4.2.2. 4-Bromo-2-(((4-fluorophenyl)amino)methyl)phenol (2)
White crystal, yield 82%, mp: 107–108 °C, 1H NMR (300 MHz,
CDCl3): 4.36 (s, 2H); 6.13 (s, 1H); 6.76–6.80 (m, 3H); 6.95 (t,
J = 5.2 Hz, 2H); 7.29 (d, J = 1.5 Hz, 1H); 7.31 (d, J = 1.5 Hz, 1H);
8.45 (s, 1H). ESI-MS C13H11BrFNO [M+H]+ 296.0. Anal. Calcd for
C13H11BrFNO: C, 52.73; H, 3.74; N, 4.73. Found: C, 52.70; H, 3.71;
N, 4.75.
3. Conclusions
4.2.3. 4-Bromo-2-(((2-chlorophenyl)amino)methyl)phenol (3)
White crystal, yield 70%, mp: 112–113 °C, 1H NMR (300 MHz,
CDCl3): 4.37 (s, 2H); 6.12 (s, 1H); 6.73–6.78 (m, 3H); 7.01–7.04
(m, 2H); 7.27 (d, J = 1.5 Hz, 1H); 7.29 (d, J = 1.5 Hz, 1H); 8.17 (s,
In summary, a series of o-hydroxybenzylamines 1–16 and its
corresponding new urea derivatives 17–32 were prepared and
tested for their inhibitory activity against E. coli, P. fluorescence,
B.subtilis, and S. aureus. Many synthetic compounds showed potent
antibacterial and E. coli FabH inhibitory activities with compounds
18 and 20 being the most potent compounds. Preliminary struc-
ture–activity relationships and molecular modeling study provided
further insight into interactions between the enzyme and its li-
gand. The results provided valuable information for the design of
E. coli FabH inhibitors as antibiotics.
1H). ESI-MS
13H11BrClNO: C, 49.95; H, 3.55; N, 4.48. Found: C, 49.81; H,
3.56; N, 4.47.
C
13H11BrClNO [M+H]+ 312.0. Anal. Calcd for
C
4.2.4. 4-Bromo-2-(((4-chlorophenyl)amino)methyl)phenol (4)
White crystal, yield 78%, mp: 109–110 °C, 1H NMR (300 MHz,
CDCl3): 4.36 (s, 2H); 6.11 (s, 1H); 6.74–6.77 (m, 3H); 7.18–7.21
(m, 2H); 7.27 (d, J = 1.5 Hz, 1H); 7.31 (dd, J = 5.1, 1.5 Hz, 1H);
8.23 (s, 1H). ESI-MS C13H11BrClNO [M+H]+ 312.0. Anal. Calcd for
4. Experimental section
4.1. Chemistry
C13H11BrClNO: C, 49.95; H, 3.55; N, 4.48. Found: C, 49.86; H,
3.57; N, 4.46.
All chemicals (reagent grade) used were purchased from Sigma
Aldrich (USA) and Sinopharm Chemical Reagent Co., Ltd (China).
Melting points (uncorrected) were determined on a XT4 MP appa-
ratus (Taike Corp., Beijing, China). ESI-MS spectra were recorded on
a Mariner System 5304 Mass spectrometer., and 1H NMR spectra
were recorded on a Bruker DPX 300 model Spectrometer at 25 °C
with TMS and solvent signals allotted as internal standards. Chem-
ical shifts were reported in ppm (d). Elemental analyses were per-
formed on a CHN-O-Rapid instrument and were within 0.4% of
the theoretical values. TLC was run on the silica gel coated alumi-
num sheets (Silica Gel 60 Å GF254, E. Merk, Germany) and visual-
ized in UV light (254 nm).
4.2.5. 4-Bromo-2-(((2-bromophenyl)amino)methyl)phenol (5)
Yellow crystal, yield 83%, mp: 123–124 °C, 1H NMR (300 MHz,
CDCl3): 4.36 (s, 2H); 6.12 (s, 1H); 6.76–6.79 (m, 3H); 7.20–7.24
(m, 3H); 7.32 (d, J = 1.5 Hz, 1H); 8.32 (s, 1H). ESI-MS C13H11Br2NO
[M+H]+ 355.9. Anal. Calcd for C13H11Br2NO: C, 43.73; H, 3.11; N,
3.92. Found: C, 43.87; H, 3.08; N, 3.91.
4.2.6. 4-Bromo-2-(((4-bromophenyl)amino)methyl)phenol (6)
Yellow crystal, yield 81%, mp: 117–118 °C, 1H NMR (300 MHz,
CDCl3): 4.37 (s, 2H); 6.10 (s, 1H); 6.71 (dd, J = 6.8, 2.0 Hz, 2H);
6.76–6.79 (m, 1H); 7.30–7.37 (m, 4H); 8.32 (s, 1H). ESI-MS

4418
Z.-L. Li et al. / Bioorg. Med. Chem. 19 (2011) 4413–4420
C13H11Br2NO [M+H]+ 355.9. Anal. Calcd for C13H11Br2NO: C, 43.73;
H, 3.11; N, 3.92. Found: C, 43.79; H, 3.11; N, 3.93.
7.28–7.31 (m, 3H); 7.56 (d, J = 2.2 Hz, 1H); 9.55 (s, 1H). ESI-MS
13H10Br3NO [M+H]+ 433.8. Anal. Calcd for C13H10Br3NO: C,
C
35.82; H, 2.31; N, 3.21. Found: C, 35.98; H, 2.34; N, 3.20.
4.2.7. 4-Bromo-2-((p-tolylamino)methyl)phenol (7)
White crystal, yield 90%, mp: 97–98 °C, 1H NMR (300 MHz,
CDCl3): 2.27 (s, 3H); 4.36 (s, 2H); 6.12 (s, 1H); 6.74–6.76 (m, 3H);
7.05 (dd, J = 5.1, 0.4 Hz, 2H); 7.25 (d, J = 2.0 Hz, 1H); 7.05 (dd,
J = 5.2, 1.5 Hz, 1H); 8.13 (s, 1H). ESI-MS C14H14BrNO [M+H]+
292.0. Anal. Calcd for C14H14BrNO: C, 57.55; H, 4.83; N, 4.79.
Found: C, 57.63; H, 4.80; N, 4.77.
4.2.15. 2,4-Dibromo-6-((p-tolylamino)methyl)phenol (15)
White crystal, yield 80%, mp: 123–124 °C, 1H NMR (300 MHz,
CDCl3): 2.27 (s, 3H); 4.40 (s, 2H); 6.12 (s, 1H); 6.72 (d, J = 8.4 Hz,
2H); 7.06 (d, J = 8.2 Hz, 2H); 7.25 (d, J = 3.8 Hz, 1H); 7.58 (d,
J = 2.4 Hz, 1H); 9.56 (s, 1H). ESI-MS C14H13Br2NO [M+H]+ 369.9.
Anal. Calcd for C14H13Br2NO: C, 45.32; H, 3.53; N, 3.77. Found: C,
45.36; H, 3.56; N, 3.76.
4.2.8. 4-Bromo-2-(((4-methoxyphenyl)amino)methyl)phenol (8)
Lavender crystal, yield 65%, mp: 105–106 °C, 1H NMR (300 MHz,
CDCl3): 3.78 (s, 3H);4.37 (s, 2H); 6.11 (s, 1H); 6.76–6.79 (m, 1H); 6.83
(m, 4H);7.25 (d, J = 2.4 Hz, 1H); 7.29–7.33 (m, 1H); 8.16 (s, 1H). ESI-
MS C14H14BrNO2 [M+H]+ 308.0. Anal. Calcd for C14H14BrNO2: C,
54.56; H, 4.58; N, 4.55. Found: C, 54.47; H, 4.60; N, 4.57.
4.2.16. 2,4-Dibromo-6-(((4-methoxyphenyl)amino)methyl)
phenol (16)
Lavender crystal, yield 78%, mp: 119–120 °C, 1H NMR (300 MHz,
CDCl3): 3.76 (s, 3H);4.36 (s, 2H); 6.10 (s, 1H); 6.75–6.83 (m, 4H);
7.23 (d, J = 2.4 Hz, 1H); 7.57 (d, J = 2.4 Hz, 1H); 9.56 (s, 1H). ESI-
MS C14H13Br2NO2 [M+H]+ 385.9. Anal. Calcd for C14H13Br2NO2: C,
43.44; H, 3.39; N, 3.62. Found: C, 43.51; H, 3.36; N, 3.61.
4.2.9. 2,4-Dibromo-6-(((2-fluorophenyl)amino)methyl)phenol
(9)
Yellow crystal, yield 76%, mp: 114–115 °C, 1H NMR (300 MHz,
CDCl3): 4.42 (s, 2H); 6.17 (s, 1H); 6.70–6.80 (m, 2H); 6.97–7.05
(m, 2H); 7.32 (d, J = 2.4 Hz, 1H); 7.56 (d, J = 2.4 Hz, 1H); 8.20 (s,
4.2.17. 1-(5-Bromo-2-hydroxybenzyl)-1-(2-fluorophenyl)-3-
phenylurea (17)
White powder, yield 56%, mp: 140–141 °C, 1H NMR (300 MHz,
CDCl3): 4.68 (s, 2H); 6.11 (s, 1H); 6.69 (d, J = 1.5 Hz, 1H); 7.10–
7.12 (m, 1H); 7.17–7.20 (m, 2H); 7.24–7.30 (m, 4H); 7.47–7.53
(m, 3H); 7.60 (d, J = 1.5 Hz, 1H); 10.68 (s, 1H). ESI-MS
1H). ESI-MS
13H10Br2FNO: C, 41.63; H, 2.69; N, 3.73. Found: C, 41.67; H,
2.70; N, 3.76.
C
13H10Br2FNO [M+H]+ 374.0. Anal. Calcd for
C
C
20H16BrFN2O2 [M+H]+ 415.0. Anal. Calcd for C20H16BrFN2O2: C,
4.2.10. 2,4-Dibromo-6-(((4-fluorophenyl)amino)methyl)phenol
(10)
57.85; H, 3.88; N, 6.75. Found: C, 57.99; H, 3.87; N, 6.76.
Yellow crystal, yield 79%, mp: 125–126 °C, 1H NMR (300 MHz,
CDCl3): 4.24 (s, 2H); 6.12 (s, 1H); 6.53 (dd, J = 8.9, 4.5 Hz, 2H);
6.91 (t, J = 8.9 Hz, 2H); 7.32 (d, J = 2.0 Hz, 1H); 7.59 (d, J = 2.2 Hz,
1H); 9.63 (s, 1H). ESI-MS C13H10Br2FNO [M+H]+ 374.0. Anal. Calcd
for C13H10Br2FNO: C, 41.63; H, 2.69; N, 3.73. Found: C, 41.52; H,
2.70; N, 3.73.
4.2.18. 1-(5-Bromo-2-hydroxybenzyl)-1-(4-fluorophenyl)-3-
phenylurea (18)
White powder, yield 62%, mp: 139–140 °C, 1H NMR (300 MHz,
CDCl3): 4.64 (s, 2H); 6.10 (s, 1H); 6.70 (d, J = 2.4 Hz, 1H); 7.04–
7.06 (m, 1H); 7.21–7.24 (m, 2H); 7.27–7.29 (m, 4H); 7.49–7.52
(m, 3H); 7.61 (d, J = 1.5 Hz, 1H); 10.73 (s, 1H). ESI-MS
4.2.11. 2,4-Dibromo-6-(((2-chlorophenyl)amino)methyl)phenol
(11)
C
20H16BrFN2O2 [M+H]+ 415.0. Anal. Calcd for C20H16BrFN2O2: C,
57.85; H, 3.88; N, 6.75. Found: C, 57.81; H, 3.90; N, 6.77.
White crystal, yield 83%, mp: 124–125 °C, 1H NMR (300 MHz,
CDCl3): 4.45 (s, 2H); 5.78 (s, 1H); 6.67 (dd, J = 8.0, 1.3 Hz, 1H);
6.71–6.77 (m, 1H); 7.10–7.16 (m, 1H); 7.28–7.33 (m, 2H); 7.56
(d, J = 2.4 Hz, 1H); 9.61 (s, 1H). ESI-MS C13H10Br2ClNO [M+H]+
389.9. Anal. Calcd for C13H10Br2ClNO: C, 39.88; H, 2.57; N, 3.58.
Found: C, 43.00; H, 2.56; N, 3.59.
4.2.19. 1-(5-Bromo-2-hydroxybenzyl)-1-(2-chlorophenyl)-3-
phenylurea (19)
White powder, yield 71%, mp: 147–148 °C, 1H NMR (300 MHz,
CDCl3): 4.68 (s, 2H); 6.07 (s, 1H); 6.71 (d, J = 1.4 Hz, 1H); 7.05–
7.08 (m, 1H); 7.13–7.15 (m, 3H); 7.22–7.24 (m, 4H); 7.41–7.44
(m, 2H); 7.62 (d, J = 1.5 Hz, 1H); 10.65 (s, 1H). ESI-MS
4.2.12. 2,4-Dibromo-6-(((4-chlorophenyl)amino)methyl)phenol
(12)
C
20H16BrClN2O2 [M+H]+ 431.0. Anal. Calcd for C20H16BrClN2O2: C,
White crystal, yield 87%, mp: 130–131 °C, 1H NMR (300 MHz,
CDCl3): 4.23 (s, 2H); 6.36 (s, 1H); 6.51–6.54 (m, 2H); 7.07 (dd,
J = 6.8, 2.0 Hz, 2H); 7.27 (d, J = 2.4 Hz, 1H); 7.58 (d, J = 2.4 Hz, 1H);
9.59 (s, 1H). ESI-MS C13H10Br2ClNO [M+H]+ 389.9. Anal. Calcd for
55.64; H, 3.74; N, 6.49. Found: C, 55.69; H, 3.76; N, 6.48.
4.2.20. 1-(5-Bromo-2-hydroxybenzyl)-1-(4-chlorophenyl)-3-
phenylurea (20)
C13H10Br2ClNO: C, 39.88; H, 2.57; N, 3.58. Found: C, 39.79; H, 2.55;
White powder, yield 72%, mp: 157–158 °C, 1H NMR (300 MHz,
CDCl3): 4.71 (s, 2H); 6.06 (s, 1H); 6.72 (d, J = 2.6 Hz, 1H); 6.86 (d,
J = 8.6 Hz, 1H); 7.02–7.07 (m, 1H); 7.11–7.15 (m, 2H); 7.22–7.25
(m, 5H); 7.46–7.51 (m, 2H); 9.92 (s, 1H). ESI-MS C20H16BrClN2O2
[M+H]+ 431.0. Anal. Calcd for C20H16BrClN2O2: C, 55.64; H, 3.74;
N, 6.49. Found: C, 55.72; H, 3.75; N, 6.47.
N, 3.57.
4.2.13. 2,4-Dibromo-6-(((2-bromophenyl)amino)methyl)phenol
(13)
Yellow crystal, yield 85%, mp: 128–129 °C, 1H NMR (300 MHz,
CDCl3): 4.46 (s, 2H); 5.80 (s, 1H); 6.66–6.72 (m, 2H); 6.92 (s, 1H);
7.15–7.21 (m, 1H); 7.47–7.50 (m, 1H); 7.57 (d, J = 2.4 Hz, 1H); 9.56
(s, 1H). ESI-MS
13H10Br3NO: C, 35.82; H, 2.31; N, 3.21. Found: C, 35.64; H, 2.33;
N, 3.22.
C
13H10Br3NO [M+H]+ 433.8. Anal. Calcd for
4.2.21. 1-(5-Bromo-2-hydroxybenzyl)-1-(2-bromophenyl)-3-
phenylurea (21)
C
White powder, yield 51%, mp: 160–161 °C, 1H NMR (300 MHz,
CDCl3): 4.64 (s, 2H); 6.07 (s, 1H); 6.71 (d, J = 1.5 Hz, 1H); 7.04–
7.05 (m, 1H); 7.10–7.12 (m, 2H); 7.21–7.24 (m, 5H); 7.39–7.43
(m, 2H); 7.61 (d, J = 1.5 Hz, 1H); 10.72 (s, 1H). ESI-MS
4.2.14. 2,4-Dibromo-6-(((4-bromophenyl)amino)methyl)phenol
(14)
Yellow crystal, yield 89%, mp: 126–127 °C, 1H NMR (300 MHz,
CDCl3): 4.36 (s, 2H); 6.08 (s, 1H); 6.59 (dd, J = 6.8, 2.2 Hz, 2H);
C
20H16Br2N2O2 [M+H]+ 475.0. Anal. Calcd for C20H16Br2N2O2: C,
50.45; H, 3.39; N, 5.88. Found: C, 50.34; H, 3.40; N, 5.86.

Z.-L. Li et al. / Bioorg. Med. Chem. 19 (2011) 4413–4420
4419
4.2.22. 1-(5-Bromo-2-hydroxybenzyl)-1-(4-bromophenyl)-3-
4.2.29. 1-(2-Bromophenyl)-1-(3,5-dibromo-2-hydroxybenzyl)-
phenylurea (22)
3-phenylurea (29)
White powder, yield 58%, mp: 152–153 °C, 1H NMR (300 MHz,
CDCl3): 4.64 (s, 2H); 6.09 (s, 1H); 6.75 (d, J = 1.4 Hz, 1H); 7.07–
7.11 (m, 4H); 7.22–7.26 (m, 4H); 7.60 (d, J = 1.5 Hz, 1H); 7.64–
7.67 (m, 2H); 10.64 (s, 1H). ESI-MS C20H16Br2N2O2 [M+H]+ 475.0.
Anal. Calcd for C20H16Br2N2O2: C, 50.45; H, 3.39; N, 5.88. Found:
C, 50.32; H, 3.40; N, 5.87.
White powder, yield 74%, mp: 161–162 °C, 1H NMR (300 MHz,
CDCl3): 4.76 (s, 2H); 6.13 (s, 1H); 6.67 (d, J = 1.5 Hz, 1H); 7.06–
7.09 (m, 1H); 7.12–7.14 (m, 1H); 7.26–7.28 (m, 5H); 7.37–7.44
(m, 1H); 7.45–7.47 (m, 1H); 7.61 (d, J = 1.5 Hz, 1H); 10.72 (s, 1H).
ESI-MS
20H15Br3N2O2: C, 43.28; H, 2.72; N, 5.05. Found: C, 43.36; H,
C
20H15Br3N2O2 [M+H]+ 552.9. Anal. Calcd for
C
2.70; N, 5.06.
4.2.23. 1-(5-Bromo-2-hydroxybenzyl)-3-phenyl-1-p-tolylurea
(23)
4.2.30. 1-(4-Bromophenyl)-1-(3,5-dibromo-2-hydroxybenzyl)-
3-phenylurea (30)
White powder, yield 63%, mp: 144–145 °C, 1H NMR (300 MHz,
CDCl3): 3.14 (s, 3H); 4.77 (s, 2H); 6.14 (s, 1H); 7.00–7.04 (m, 3H);
7.05–7.09 (m, 2H); 7.17–7.19 (m, 2H); 7.27–7.32 (m, 4H); 7.59
(d, J = 1.5 Hz, 1H); 10.44 (s, 1H). ESI-MS C21H19BrN2O2 [M+H]+
411.0. Anal. Calcd for C21H19BrN2O2: C, 61.33; H, 4.66; N, 6.81.
Found: C, 61.55; H, 4.70; N, 6.86.
White powder, yield 76%, mp: 158–159 °C, 1H NMR (300 MHz,
CDCl3): 4.75 (s, 2H); 6.07 (s, 1H); 6.72 (d, J = 1.4 Hz, 1H); 7.05–
7.10 (m, 3H); 7.24–7.29 (m, 4H); 7.61 (d, J = 1.5 Hz, 1H); 7.65–
7.68 (m, 2H); 10.64 (s, 1H). ESI-MS C20H15Br3N2O2 [M+H]+ 552.9.
Anal. Calcd for C20H15Br3N2O2: C, 43.28; H, 2.72; N, 5.05. Found:
C, 43.43; H, 2.74; N, 5.06.
4.2.24. 1-(5-Bromo-2-hydroxybenzyl)-1-(4-methoxyphenyl)-3-
phenylurea (24)
4.2.31. 1-(3,5-Dibromo-2-hydroxybenzyl)-3-phenyl-1-(p-
tolyl)urea (31)
White powder, yield 66%, mp: 146–147 °C, 1H NMR (300 MHz,
CDCl3): 3.81 (s, 3H); 4.91 (s, 2H); 6.22 (s, 1H); 6.97–7.02 (m, 3H);
7.16–7.18 (m, 3H); 7.20–7.24 (m, 2H); 7.33–7.36 (m, 3H); 7.62
(d, J = 2.4 Hz, 1H); 10.90 (s, 1H). ESI-MS C21H19BrN2O3 [M+H]+
427.1. Anal. Calcd for C21H19BrN2O3: C, 59.03; H, 4.48; N, 6.56.
Found: C, 59.23; H, 4.45; N, 6.56.
White powder, yield 69%, mp: 145–146 °C, 1H NMR (300 MHz,
CDCl3): 3.30 (s, 3H); 4.78 (s, 2H); 6.07 (s, 1H); 6.71 (d, J = 1.5 Hz,
1H); 7.00–7.04 (m, 1H); 7.06–7.10 (m, 2H); 7.13–7.18 (m, 2H);
7.25–7.30 (m, 4H); 7.61 (d, J = 1.4 Hz, 1H); 10.44 (s, 1H). ESI-MS
C
21H18Br2N2O2 [M+H]+ 489.0. Anal. Calcd for C21H18Br2N2O2: C,
51.45; H, 3.70; N, 5.71. Found: C, 51.59; H, 3.73; N, 5.72.
4.2.25. 1-(3,5-Dibromo-2-hydroxybenzyl)-1-(2-fluorophenyl)-3-
phenylurea (25)
4.2.32. 1-(3,5-Dibromo-2-hydroxybenzyl)-1-(4-
methoxyphenyl)-3-phenylurea (32)
White powder, yield 69%, mp: 150–151 °C, 1H NMR (300 MHz,
CDCl3): 4.76 (s, 2H); 6.11 (s, 1H); 6.69 (d, J = 1.4 Hz, 1H); 7.05–
7.09 (m, 1H); 7.15–7.18 (m, 2H); 7.26–7.33 (m, 3H); 7.47–7.52
(m, 3H); 7.60 (d, J = 1.4 Hz, 1H); 10.60 (s, 1H). ESI-MS
White powder, yield 73%, mp: 142–143 °C, 1H NMR (300 MHz,
CDCl3): 3.77 (s, 3H); 4.80 (s, 2H); 6.17 (s, 1H); 6.98–7.02 (m, 3H);
7.14–7.17 (m, 2H); 7.21–7.26 (m, 2H); 7.35–7.38 (m, 3H); 7.63
(d, J = 2.4 Hz, 1H); 10.82 (s, 1H). ESI-MS C21H18Br2N2O3 [M+H]+
505.0. Anal. Calcd for C21H18Br2N2O3: C, 49.83; H, 3.58; N, 5.53.
Found: C, 49.96; H, 3.56; N, 5.54.
C
20H15Br2FN2O2 [M+H]+ 493.0. Anal. Calcd for C20H15Br2FN2O2: C,
48.61; H, 3.06; N, 5.67. Found: C, 48.48; H, 3.06; N, 5.65.
4.2.26. 1-(3,5-Dibromo-2-hydroxybenzyl)-1-(4-fluorophenyl)-3-
phenylurea (26)
4.3. Antibacterial activity
White powder, yield 73%, mp: 148–149 °C, 1H NMR (300 MHz,
CDCl3): 4.78 (s, 2H); 6.12 (s, 1H); 6.70 (d, J = 1.4 Hz, 1H);7.04–
7.06 (m, 1H); 7.20 (d, J = 4.3 Hz, 2H); 7.24–7.26 (m, 3H); 7.50–
7.55 (m, 3H); 7.60 (d, J = 1.5 Hz, 1H); 10.83 (s, 1H). ESI-MS
The antibacterial activity of the synthesized compounds was
tested against E. coli, P. fluorescence, B. subtilis, and S. aureus using
MH medium (Mueller-Hinton medium: casein hydrolysate 17.5 g,
soluble starch 1.5 g, beef extract 1000 mL), The MICs (minimum
inhibitory concentrations) of the test compounds were determined
by a colorimetric method using the dye MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl trtrazolium bromide). A stock solution of
C
20H15Br2FN2O2 [M+H]+ 493.0. Anal. Calcd for C20H15Br2FN2O2: C,
48.61; H, 3.06; N, 5.67. Found: C, 48.88; H, 3.07; N, 5.70.
4.2.27. 1-(2-Chlorophenyl)-1-(3,5-dibromo-2-hydroxybenzyl)-3-
phenylurea (27)
the synthesized compound (100 lg/mL) in DMSO was prepared
and graded quantities of the test compounds were incorporated
in specified quantity of sterilized liquid MH medium. A specified
quantity of the medium containing the compound was poured into
microtitration plates. Suspension of the microorganism was pre-
pared to contain approximately 105 cfu/mL and applied to microt-
itration plates with serially diluted compounds in DMSO to be
tested and incubated at 37 °C for 24 h. After the MICs were visually
White powder, yield 58%, mp: 151–152 °C, 1H NMR (300 MHz,
CDCl3): 4.76 (s, 2H); 6.10 (s, 1H); 6.70 (d, J = 1.4 Hz, 1H); 7.06–
7.09 (m, 1H); 7.18–7.20 (m, 2H); 7.24–7.27 (m, 4H); 7.41–7.45
(m, 2H); 7.61 (d, J = 1.5 Hz, 1H); 10.75 (s, 1H). ESI-MS
C
20H15Br2ClN2O2 [M+H]+ 508.9. Anal. Calcd for C20H15Br2ClN2O2:
C, 47.04; H, 2.96; N, 5.49. Found: C, 47.12; H, 2.98; N, 5.47.
determined on each of the microtitration plates, 50 lL of PBS
4.2.28. 1-(4-Chlorophenyl)-1-(3,5-dibromo-2-hydroxybenzyl)-3-
phenylurea (28)
(phosphate buffered saline 0.01 mol/L, pH 7.4: Na2HPO4ꢁ12H2O
2.9 g, KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2 g, distilled water 1000 mL)
containing 2 mg of MTT/mL was added to each well. Incubation
was continued at room temperature for 4–5 h. The content of each
White powder, yield 67%, mp: 146–147 °C, 1H NMR (300 MHz,
CDCl3): 4.76 (s, 2H); 6.07 (s, 1H); 6.72 (d, J = 1.4 Hz, 1H); 7.05–
7.08 (m, 1H); 7.14–7.17 (m, 2H); 7.24–7.28 (m, 5H); 7.50–7.52
(m, 1H); 7.61 (d, J = 1.5 Hz, 1H); 10.64 (s, 1H). ESI-MS
well was removed, and 100 lL of isopropanol containing 5% 1 mol/
L HCl was added to extract the dye. After 12 h of incubation at
room temperature, the optical density (OD) was measured with a
microplate reader at 550 nm.
C
20H15Br2ClN2O2 [M+H]+ 508.9. Anal. Calcd for C20H15Br2ClN2O2:
C, 47.04; H, 2.96; N, 5.49. Found: C, 47.17; H, 2.95; N, 5.47.

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Z.-L. Li et al. / Bioorg. Med. Chem. 19 (2011) 4413–4420
4.4. E. coli FabH purification and activity assay
References and notes
1. Leeb, M. Nature 2004, 431, 892.
2. Khalafi-Nezhad, A.; Rad, M. N. S.; Mohabatkar, H.; Asrari, Z.; Hemmateenejad, B.
Bioorg. Med. Chem. 2005, 13, 1931.
3. Lee, J.-Y.; Jeong, K.-W.; Lee, J.-U.; Kang, D.-I.; Kim, Y. Bioorg. Med. Chem. 2009,
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5. Puupponen-Pimiä, R.; Nohynek, L.; Meier, C.; Kähkönen, M.; Heinonen, A. H. J.
Appl. Microbiol. 2001, 90, 494.
Native E. coli FabH protein was overexpressed in E. coli DH10B
cells using the pET30 vector and purified to homogeneity in three
chromatographic steps (Q-Sepharose, MonoQ, and hydroxyapatite)
at 4 °C. The selenomethionine-substituted protein was expressed
in E. coli BL21(DE3) cells and purified in a similar way. Harvested
cells containing FabH were lysed by sonication in 20 mM Tris, pH
7.6, 5 mM imidazole, 0.5 M NaCl and centrifuged at 20,000 rpm
for 30 min. The supernatant was applied to a Ni-NTA agarose col-
umn, washed, and eluted using a 5–500 mM imidazole gradient
over 20 column volumes. Eluted protein was dialyzed against
20 mM Tris, pH 7.6, 1 mM DTT, and 100 mM NaCl. Purified FabHs
were concentrated up to 2 mg/mL and stored at ꢀ80 °C in 20 Mm
Tris, pH 7.6, 100 mM NaCl, 1 mM DTT, and 20% glycerol for enzy-
matic assay.
6. Herbert, P. S. FEMS Microbiol. Lett. 2001, 202, 1.
7. Rock, C. O.; Jackowski, S. Res. Commun. 2002, 292, 1155.
8. Roberts, C. W.; McLeod, R.; Rice, D. W. Mol. Biochem. Parasit. 2003, 126, 129.
9. McMurray, L. M.; Oethinger, M.; Levy, S. B. Nature 1998, 394, 531.
10. Perozzo, R.; Kuo, M.; Sidh, A. S. Biol. Chem. 2002, 277, 151.
11. Sharada, S.; Jacque, Z.; Alasdair, F. Bell.; Lizbeth, H.; Peter, J. T. Biochemistry
2003, 42, 4406.
12. Sharada, S.; Todd, J. S.; Francis, J.; Polina, N.; Guanglei, C.; Carlos, S.; Peter, J. J.
Med. Chem. 2004, 47, 509.
13. Heath, R. J.; White, S. W.; Rock, C. O. Appl. Microbiol. Biot. 2002, 58, 695.
14. Ozaki, S. Chem. Rev. 1972, 72, 457.
In a final 20
0.25 mM MgCl2, and 2.5
and H2O was added to 15
ture of 25 M acetyl-CoA, 0.5 mM NADH, and 0.5 mM NADPH
was added for FabH reaction for 25 min. The reaction was stopped
by adding 20 L of ice-cold 50% TCA, incubating for 5 min on ice,
and centrifuging to pellet the protein. The pellet was washed with
l
L reaction, 20 mM Na2HPO4, pH 7.0, 0.5 mM DTT,
M holo-ACP were mixed with 1 nM FabH,
L. After 1 min incubation, a 2 L mix-
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Acknowledgment
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This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239) and the Fundamental Research Fund for
the Central Universities (No. 1092020804).