Syntheses, structures and antibiotic activities of LpxC inhibitors based on the diacetylene scaffold

Article abstract of DOI:10.1016/j.bmc.2010.12.017

Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and biochemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-butadiyne) threonyl-hydroxamate scaffold. These studies provide a molecular interpretation for the differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute stereochemical requirement at the C2, but not C3, position of the threonyl group.

Full text of DOI:10.1016/j.bmc.2010.12.017

Bioorganic & Medicinal Chemistry 19 (2011) 852–860
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Syntheses, structures and antibiotic activities of LpxC inhibitors
based on the diacetylene scaffold
Xiaofei Liang a,b, Chul-Jin Lee c,d, Xin Chen b, Hak Suk Chung c, Daina Zeng c, Christian R. H. Raetz c,
b,c,d,
Yaoxian Li a, Pei Zhou b,c,d, , Eric J. Toone
⇑
⇑
a Department of Chemistry, Jilin University, Changchun, Jilin 130021, PR China
b Department of Chemistry, Duke University, Durham, NC 27708, USA
c Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
d Structural Biology and Biophysics Program, Duke University, Durham, NC 27710, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics
against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and bio-
chemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-bu-
tadiyne) threonyl-hydroxamate scaffold. These studies provide a molecular interpretation for the
differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute
stereochemical requirement at the C2, but not C3, position of the threonyl group.
Received 14 October 2010
Revised 1 December 2010
Accepted 2 December 2010
Available online 9 December 2010
Keywords:
LpxC
Ó 2010 Elsevier Ltd. All rights reserved.
Inhibitor
Antibiotic
Diphenyl-diacetylene
1,4-Diphenyl-1,3-butadiyne
Hydroxamate
1. Introduction
nosuppressed individuals, the discovery of potent antibiotic
activity of CHIR-090 against P. aeruginosa has generated significant
Lipid A, the hydrophobic membrane anchor of lipopolysaccha-
ride, is the major lipid component of the outer leaflet of the outer
membrane of Gram-negative bacteria.1,2 Because lipid A is essen-
tial for the viability of the vast majority of Gram-negative bacteria,
the zinc-dependent UDP-3-O-(acyl)-N-acetylglucosamine deace-
tylase (LpxC), which catalyzes the first irreversible step in lipid A
biosynthesis, has become an attractive target for the development
of novel therapeutics for treatment of multidrug-resistant Gram-
negative infections.3 Indeed, a large number of compounds with
vastly different chemical scaffolds targeting LpxC have been
reported in both the scientific4,5 and patent6–14 literature.
Among the well-characterized compounds, CHIR-090 based on
the diphenyl-acetylene scaffold (Table 1) is the most potent LpxC
inhibitor reported to date, and it is the first reported compound
that effectively kills both Escherichia coli and Pseudomonas aerugin-
osa in bacterial disk diffusion assays.15 Because P. aeruginosa is a
predominant cause of morbidity and mortality in cystic fibrosis pa-
tients and can cause a variety of infections, particularly in immu-
interest in further developing LpxC inhibitors as novel antibiotics
for treatment of multidrug-resistant Pseudomonas and other
Gram-negative infections. However, CHIR-090 is about 600-fold
less effective against LpxC orthologs from the Rhizobiaceae family
than against E. coli LpxC, which raises concerns surrounding rapid
evolution of antibiotic resistance for CHIR-090 sensitive strains
through point mutations.16 Our recent structural and biochemical
studies of the LpxC/CHIR-090 complex suggest that van der Waals
(vdW) clashes between the phenyl ring distal to the hydroxamate
group of CHIR-090 and LpxC residues at the substrate-binding pas-
sage result in a large decrease in CHIR-090 activity against LpxC
orthologs of the Rhizobiaceae family.17 This study has motivated
us to investigate the antibiotic profiles of LpxC inhibitors based
on a chemical scaffold of reduced radius, such as the diacetylene
(1,3-butadiyne) backbone. Recently, we showed that the 1,4-di-
phenyl-1,3-butadiyne (referred to as diphenyl-diacetylene below)
derived LPC-009 (Table 1), which is one of the many structures
listed in the international patent WO 2004/062601 A2,6 effectively
diminishes the resistance to CHIR-090 by 20-fold for LpxC en-
zymes from the Rhizobiaceae family of bacteria and enhances the
antibiotic activity against E. coli and P. aeruginosa by 2–4-fold over
CHIR-090.18 Although diacetylene-based LpxC inhibitors have
appeared in a number of international patents,6,8 there is a lack
⇑
Corresponding authors. Tel.: +1 919 668 6409 (P.Z.); tel.: +1 919 681 3484
(E.J.T.).
E-mail addresses: peizhou@biochem.duke.edu (P. Zhou), toone@chem.duke.edu
(E.J. Toone).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.017

X. Liang et al. / Bioorg. Med. Chem. 19 (2011) 852–860
853
Table 1
MICs of LpxC inhibitors
Name
Structure
MIC (lg/mL)
E. coli wild type
E. coli W3110RL
E. coli W3110PA
P. aeruginosa PAO1
OH
O
R
S
NHOH
N
H
CHIR-090
0.2
125
1.3
1.6
O
O
N
OH
O
R
S
NHOH
N
H
O
LPC-009
0.05
6.3
0.7
0.7
OH
O
R
S
NHOH
N
H
O
LPC-011
0.03
3.9
0.5
0.5
H2N
OH
O
R
S
NHOH
N
H
O
NHOH
OH
LPC-012
0.06
6.3
1.3
1.3
H2N
OH
O
R
S
N
H
O
LPC-013
0.5
>25
8.3
12.5
NH2
O
O
O
S
S
NHOH
N
H
O
LPC-053
LPC-054
LPC-055
0.07
12.5
0.8
0.9
H2N
H2N
H2N
OH
R
R
NHOH
N
H
O
6.3
>25
>25
>25
OH
S
R
NHOH
N
H
O
3.1
>25
>25
>25

854
X. Liang et al. / Bioorg. Med. Chem. 19 (2011) 852–860
of synthetic details for these compounds. The potency, spectrum of
inhibition, and structure–activity relationship of these compounds
have not been systematically characterized. In addition, the diphe-
nyl-diacetylene compound LPC-009 has limited solubility in aque-
ous solution. In order to improve the solubility and probe the
stereochemical requirement of the threonyl group of LPC-009,
we have carefully selected from published patents6,8 a set of
LPC-009 amino derivatives and designed additional compounds
based on structural insights from the LpxC/LPC-009 complexes.18
These compounds were synthesized using optimized procedures.
Through detailed biochemical assays and structural characteriza-
tions of these LpxC–inhibitor interactions, we reveal the molecular
basis underlying the observed structure–activity relationship of
LPC-009 amino derivatives as well as the stereochemical require-
ment of the threonyl head group.
hydroxamic acid (LPC-053) by treatment with hydroxylamine un-
der basic conditions.
Diastereoisomer LPC-054, which incorporates the R absolute
configuration at both C2 and C3 began from D-threonine. D-Threo-
nine was refluxed with thionyl chloride in MeOH to produce the cor-
responding methyl ester hydrochloride, and then coupled with
diacetylene acid 6, resulting in diacetylene amide methyl ester
(92% yield). Treatment with hydroxylamine under basic conditions
converted the methyl ester to the corresponding hydroxamic acid
LPC-054 (93% yield; Scheme 2). By employing identical procedures,
enantiomer LPC-055 with 2R and 3S configurations was obtained
starting from allo-D-threonine (Scheme 2). The overall yield
(ꢀ48%) for the last two steps was slightly lower than for LPC-054.
Neither racemization nor epimerization was observed by LC–MS
or NMR during the synthesis of LPC-011-013 and 053-055 when
using 25% sodium methoxide in methanol solution as the base for
converting the methyl esters into the corresponding hydroxamic
acids.
2. Results
2.1. Synthesis
2.2. Structure–activity relationship
Synthesis of LPC-011 began with acylation of
L-threonine
The antibiotic activities of LPC-011-013 and 053-055 were eval-
uated by measuring the minimum inhibitory concentrations
(MICs) against wild-type E. coli (W3110), P. aeruginosa (PAO1),
and modified E. coli strains with the native lpxC gene replaced by
that of Rhizobium leguminosarum (W3110RL) or P. aeruginosa
(W3110PA) (Table 1).
methyl ester using sodium 4-ethynylbenzoate 1 (Scheme 1). Under
modified Glaser coupling conditions,19,20 the resulting 4-ethynyl-
benzamide 2 was coupled with 4-ethynylbenzenamine to afford
diacetylene methyl ester 3a. Finally, the methyl ester was
converted to the corresponding hydroxamic acid (LPC-011) by
treatment with hydroxylamine under basic conditions. Regioiso-
mers LPC-012 and LPC-013 were synthesized through the same
procedure. 2-Ethynylbenzenamine was very sluggish to proceed
the Glaser coupling with 4-ethynylbenzamide 2; the isolated yield
was only 26% even after 80 h reaction at room temperature.
Elevated temperature did not improve the yield.
In order to study the effect of threonyl chirality on LPC-011
inhibitory activity, three diastereoisomers of LPC-011 were syn-
thesized (Scheme 2). For convergence of the synthesis, 4-[(40-ami-
nophenyl)buta-1,3-diyn-1-yl]benzoic acid 66 was prepared in four
steps: Sonogashira coupling21 of methyl 4-iodobenzoate with (tri-
methylsilyl)-acetylene, followed by treatment with potassium car-
bonate to afford methyl 4-ethynylbenzoate 4. Employing a similar
procedure for 3a–c, 4 was coupled with 4-ethynylbenzenamine to
give diacetylene methyl ester 5. Upon hydrolysis with NaOH,
diacetylene acid 6 was obtained from 5 in 90% yield.
2.2.1. Amino substitution of the distal phenyl ring
In general, addition of an amino group to the distal phenyl ring
of LPC-009 enhanced aqueous solubility. However, these substitu-
tions have very different effects on antibiotic activities. The most
effective compound, LPC-011 with an amino substitution at the
para-position of the distal phenyl ring, shows enhancement in anti-
biotic activity over CHIR-090 against all of the tested bacterial
strains: compared to CHIR-090, LPC-011 is roughly 7-fold more
potent against wild-type E. coli W3110, 32-fold more potent
against E. coli W3110RL, and ꢀ3-fold more potent against E. coli
W3110PA and P. aeruginosa PAO1. These results are consistent
with the notion that LpxC inhibitors with the narrow diacetylene
scaffold effectively overcome the resistance mechanism observed
for R. leguminosarum LpxC,17,18 and have a superior antibiotic pro-
file compared to CHIR-090. Compound LPC-011 also displays en-
hanced antibiotic activity (ꢀ1.5-fold) toward wild-type E. coli and
P. aeruginosa compared to the parent compound LPC-009. In con-
trast, amino substitution at the meta-position (LPC-012) has little
or slightly negative effect, and amino substitution at the ortho-
position (LPC-013) results in a significant decrease in antibiotic
activity (>10-fold).
For the preparation of allo-
S absolute configuration at C3 position,
L
-threonine methyl ester 11 with the
-threonine methyl ester
L
(8) was acylated with benzoyl chloride to generate N-benzoyl ami-
no ester 9. Compound 9 was treated with thionyl chloride17 on ice
to give oxazoline 10, establishing the S absolute configuration at
C5.22,23 Acid hydrolysis of 10 in boiling 6 N HCl aqueous solution
resulted in allo-L-threonine. This compound was converted to the
To probe the molecular basis for the distinct antibiotic profiles
for compounds with amino substitutions at different positions
(ortho, meta and para), we determined the crystal structures of
E. coli LpxC in complex with the para-amino substituted compound
corresponding methyl ester hydrochloride 11 (Scheme 2). Under
standard amide coupling conditions (EDC/HOBt/DIEA), 11 was re-
acted with diacetylene acid 6 to give diacetylene amide 7 in 90%
yield. Finally, the methyl ester was converted to the corresponding
OH
OH
O
O
R
O
OH
S
O
NHOH
N
H
CO2Me
N
H
ONa
ii
i
iii
N
H
CO2Me
O
LPC-011: 4-NH2
LPC-012: 3-NH2
LPC-013: 2-NH2
3a: 4-NH2
3b: 3-NH2
3c: 2-NH2
H2N
H2N
1
2
Scheme 1. Reagents and conditions: (i)
acetylene, Cu(OAc)2, pyridine, MeOH, room temperature; (iii) H2NOHꢁHCl, 25% NaOMe/MeOH, THF, 0 °C, 2 h, then room temperature, 16 h.
L
-threonine methyl ester hydrochloride, EDCꢁHCl, HOBt, DIEA, DMF, 0 °C, 1 h, then room temperature, 20 h, 48%; (ii) substituted

X. Liang et al. / Bioorg. Med. Chem. 19 (2011) 852–860
855
OH
O
CO2Me
ii
CO2H
N
H
CO2Me
CO2Me
i
iii
4
5
6
7
H2N
H2N
H2N
vi
v
iv
OH
OH
OH
O
O
S
O
R
S
R
S
R
NHOH
NHOH
NHOH
N
N
H
N
H
H
O
O
O
LPC-053
LPC-055
LPC-054
H2N
CO2Me
H2N
H2N
OH
CO2Me
O
OH
OH
CO2Me
O
N
ix
viii
vii
N
H2N
CO2Me
H
H2N
9
10
11
8
Scheme 2. Reagents and conditions: (i) 4-ethynylbenzenamine, Cu(OAc)2, pyridine, MeOH, room temperature, 48 h, 35%; (ii) 3 N NaOH, MeOH, reflux, 1 h, 90%; (iii) 11,
EDCꢁHCl, HOBt, DIEA, DMF, 0 °C, 1 h, then room temperature, 18 h, 92%; (iv) H2NOHꢁHCl, 25% NaOMe/MeOH, THF, 0 °C, 2 h, then room temperature, 14 h, 70%;
(v) (1)
D
-threonine, SOCl2, MeOH, reflux, 1 h, 99%; (2) 6, EDCꢁHCl, HOBt, DIEA, DMF, 0 °C, 1 h, then room temperature, 18 h, 92%; (3) H2NOHꢁHCl, 25% NaOMe/MeOH, THF, 0 °C,
2 h, then room temperature, 14 h, 93%; (vi) (1) allo-
D
-threonine, SOCl2, MeOH, reflux, 1 h, 99%; (2) 6, EDCꢁHCl, HOBt, DIEA, DMF, 0 °C, 1 h, then room temperature, 18 h, 69%;
(3) H2NOHꢁHCl, 25% NaOMe/MeOH, THF, 0 °C, 2 h, then room temperature, 14 h, 69%; (vii) benzoyl chloride, Et3N, MeOH, 0 °C, 2 h, 92%; (viii) SOCl2, 0 °C, 5 days, 94%; (ix) (1)
6 N HCl, reflux, 5 h; (2) SOCl2, MeOH, reflux, 1 h, 98%.
LPC-011 and meta-amino substituted compound LPC-012 (crystal-
lographic statistics shown in Table S1 in Supplementary data). The
overall structure of E. coli LpxC in these inhibitor-bound complexes
is essentially identical to that in the E. coli LpxC/LPC-009 com-
plex:18 the threonyl-hydroxamate head group occupies the LpxC
active site, the proximal phenyl group locates at the entrance of
the hydrophobic substrate-binding passage, the diacetylene group
penetrates through the passage, and the distal phenyl ring inter-
acts with a cluster of hydrophobic residues, including I198,
M195, F212 and V217 in E. coli LpxC (Fig. 1A and B). Additional
electron density, which was interpreted as a buffer sulfate anion,
was observed in the active site, mediating hydrogen bonds with
the inhibitor threonyl group and K239 in E. coli LpxC.
para-amino substituted distal phenyl ring with F212 of E. coli
LpxC.
Amino substitution at the meta-position generated different
results for distinct LpxC orthologs. In the E. coli LpxC/LPC-012
complex, the meta-amino forms a hydrogen bond with the car-
bonyl oxygen of the side group of Q202 (Fig. 1B), but this hydrogen
bond apparently does not contribute significantly to the overall
binding energy, as the activity of the meta-amino substituted com-
pound is comparable to the less soluble, unsubstituted compound
LPC-009.
In contrast to para- or meta-substitutions, an amino group at
the ortho-position is clearly detrimental: structural modeling sug-
gests that regardless of the orientation of the distal phenyl ring,
amino substitution at the ortho-position generates vdW clashes
with Q202 and F212 of E. coli LpxC and the corresponding residues
in other LpxC orthologs (Fig. 1D and E). Consequently, the ortho-
amino substituted compound LPC-013 is substantially less effec-
tive than the parent compound LPC-009 for all of the bacterial
strains tested (>10-fold).
Of all three possible positions of the distal phenyl ring, substitu-
tion at the para-position is best tolerated, as this position remains
solvent exposed and does not generate vdW clashes with nearby
residues. In addition, para-amino substitution of the distal phenyl
group, due to its proximity to the F212 of E. coli LpxC, likely en-
hances the edge-to-face p–p interaction between the distal phenyl
ring of LPC-011 and F212 of E. coli LpxC, modestly increasing the
activity of the para-amino compound LPC-011 relative to
LPC-009 for E. coli (Fig. 1A). To obtain more quantitative measure-
ments of the inhibitory effect of LPC-011, we performed detailed
enzyme kinetic studies. Analogous to LPC-009 but unlike the slow
tight-binding inhibitor CHIR-090,15,16,18 we observed a similar
fractional inhibition of product accumulation with or without
LPC-011 pre incubation (1–3 h) with enzyme, suggesting that
LPC-011 does not inhibit E. coli LpxC in a time-dependent fashion.
A KaI pp value of 0.20 0.02 nM and a corresponding KI value of
0.067 0.007 nM were calculated for LPC-011 based on the
assumption of competitive inhibition and a measured KM value of
2.2.2. Stereochemistry of the threonyl group
We also investigated the stereochemical requirement for the
threonine head group in LPC-011 on its antibiotic activity using
three diastereoisomers of LPC-011. The MIC results show that
the S-configuration at C2 position of the threonine moiety in
LPC-011 is absolutely required for effective antibiotic activity.
When the absolute configuration in C2 position is inverted from
S to R, the resulting (2R,3R)-diastereomer LPC-054 and (2R,3S)-
enantiomer LPC-055 exhibited significantly decreased activity
compared to LPC-011 (over 100-fold for E. coli). On the other hand,
the absolute configuration at C3 position is less critical than the
stereochemistry at the C2 position. For example, the (2S,3S)-diaste-
reomer LPC-053 showed only a slight decrease (1.6–3.2-fold) in
antibiotic activity compared to the (2S,3R)-diastereomer LPC-011
against all of tested bacterial strains.
2.5 0.2 lM (Fig. 1C, see Experimental Section for details). A
2.7-fold reduction of KaI pp and KI values of LPC-011 in comparison
with LPC-009 (KaI pp = 0.55 0.09 nM and KI = 0.18 0.03 nM) fur-
ther supports the favorable energetic interaction between the

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X. Liang et al. / Bioorg. Med. Chem. 19 (2011) 852–860
Figure 1. Structural and biochemical characterization of LPC-009 derivatives with amino substitutions at the distal phenyl ring. (A) Structure of the E. coli LpxC/LPC-011
complex. (B) Structure of the E. coli LpxC/LPC-012 complex. LpxC is shown in the ribbon diagram. Inhibitors, LpxC residues important for inhibitor binding, and a sulfate
molecule in the active site are shown in the stick model. Blue meshes represent the Fo–Fc omit map (contoured at 3.4r) surrounding the inhibitors. The active site zinc ion is
shown in a space-filling model. Hydrogen bonds are denoted as dashed lines. (C) Inhibition curve of LPC-011 against E. coli LpxC. (D) Sequence alignment of the Insert II
substrate-binding passage. Conserved hydrophobic residues and the two Gly residues at the exit of the substrate-binding passage (G209 and G210 in E. coli LpxC) are colored
in orange. S214 in R. leguminosarum LpxC (corresponding to G210 in E. coli LpxC), which is the main cause of CHIR-090 resistance, is colored in purple. Residues predicted to
clash with LPC-013 are boxed. Sequence alignment includes LpxC orthologs from Escherichia coli, Pseudomonas aeruginosa, Helicobacter pylori, Aquifex aeolicus, and Rhizobium
leguminosarum. (E) Docking model of LPC-013 bound to E. coli LpxC based on the LPC-011 complex structure, illustrating vdW clashes between the ortho-amino group with
residues in the Insert II substrate-binding passage.
The nearly identical activity for the C3 diastereomeric com-
pound LPC-053 is surprising, as the methyl group of LPC-011 forms
a critical vdW interaction with the highly conserved F192 in E. coli
LpxC and the hydroxyl group of LPC-011 participates in hydrogen
bonds with K239 and H265. To seek a molecular understanding of
the binding mode of the C3-S diastereometeric compound LPC-
053, we determined its complex structure with E. coli LpxC
(Fig. 2A, crystallographic statistics shown in Table S1 in Supple-
mentary data). The structure reveals intact vdW interactions be-
tween the methyl group of LPC-053 and F192, highlighting their
significant energetic contribution to the binding affinity. In con-
trast to the C3-R compound LPC-011, the hydroxyl group in LPC-
053 points away from the active site and is unable to form direct
hydrogen bonds with K239 and H265. Interestingly, the loss of
hydrogen bonds in LPC-011 (2S,3R) is compensated by the forma-
tion of a water-mediated hydrogen bond between the hydroxyl
group in LPC-053 (2S,3S) and the carbonyl oxygen of F192
(Fig. 2A). Similar to the LPC-011 and LPC-012 complex, additional
density was observed in the active site, which was interpreted as a
sulfate anion from the buffer. The energetic contribution of the
Figure 2. Stereochemical requirement of the threonyl group of LpxC inhibitors in the active site. (A) Structure of LPC-053 bound to E. coli LpxC. LpxC in shown in the ribbon
diagram. Blue meshes represent the Fo–Fc omit map (contoured at 3.4 ) surrounding the inhibitor. The active site zinc ion is shown in a space-filling model. LPC-053, LpxC
r
residues important for inhibitor binding, and a sulfate molecule in the active site are shown in the stick model. Hydrogen bonds are denoted as dashed lines. Structural
models of LPC-054 and LPC-055 complexes (based on the structures of LPC-011 and LPC-053 complexes) are shown in (B) and (C), respectively, illustrating vdW clashes
between the side-chain of the inhibitor threonyl group and backbone residues in the Insert I loop of LpxC.

X. Liang et al. / Bioorg. Med. Chem. 19 (2011) 852–860
857
sulfate group to LPC-053 binding to LpxC is unclear, but the sulfate
4. Experimental section
group is well-positioned to form hydrogen bonds with the
hydroxyl group of LPC-053 and K239 of E. coli LpxC. Thus the
change of the stereochemistry at the C3 position only has a limited
effect (<3.5-fold) on the overall antibiotic activity. In contrast, the
stereochemistry at the C2 position is critical: with the same overall
binding mode, alteration of the C2 stereochemistry from S to R—
regardless of the rotameric state of the threonyl side-chain—gener-
ates vdW clashes with residues (such as C63 in E. coli LpxC) in the
Insert I loop (Fig. 2B and C), resulting in significantly reduced anti-
biotic activities for compounds LPC-054 and LPC-055.
LC/MS analysis was conducted on an Agilent 1200 HPLC with a
quadrupole mass analyzer. LC chromatography used an Agilent
XDB-C18 column (4.6 ꢂ 50 mm, 1.8
lm) with a water/acetonitrile
(each with 0.2% (v/v) formic acid) gradient at a flow rate of
0.5 mL/min. HRMS analyses were performed at the Duke MS
Center. Proton (1H) and carbon (13C) NMR spectra were recorded
at 300 and 75 MHz, respectively, on a Varian spectrometer.
Column chromatography was conducted using either silica gel
(Silicycle 40–64 lm) or prepacked RediSep columns (Teledyne
Isco Inc., Lincoln, NE) on an Isco CombiFlash Rf instrument. All
moisture-sensitive reactions were carried out using dry solvents
and under a slight pressure of ultra-pure argon. Glassware was
dried in an oven at 140 °C for at least 12 h prior to use, and then
assembled quickly while hot, sealed with rubber septa, and al-
lowed to cool under a stream of argon. Reactions were stirred
magnetically using Teflon-coated magnetic stirring bars. Commer-
cially available disposable syringes were used for transferring
reagents and solvents.
3. Discussion and conclusion
Most of the LpxC inhibitors reported in the literature are limited
by their spectrum of antibiotic activities. The structural prediction
and experimental validation of broad-spectrum antibiotic activi-
ties of LpxC inhibitors based on the diacetylene moiety—a chemical
scaffold initially reported in the international patent WO 2004/
062601 A2—are an important step forward in the search for better
antibiotics targeting LpxC. To improve the solubility of LPC-009 in
aqueous solution and to probe the stereochemical requirement of
the threonyl group, we investigated the structures and antibiotic
profiles of LPC-009 derivatives. Although all three amino substitu-
ents of the distal phenyl ring (LPC-011-013) have improved solu-
bility in aqueous solution, they have very different antibiotic
profiles. The para-amino substituent LPC-011 slightly enhances
the antibiotic activity of the parent compound LPC-009, and has
a 2.7-fold lower KI value than that of LPC-009 against E. coli LpxC.
The para-position is largely exposed to solvent, suggesting that
even more extensive functionality can be tolerated. Therefore, it
4.1. Methyl (2S,3R)-2-[(40-ethynylphenyl)formamido]-3-hydro-
xybutanoate (2)
To a stirred mixture of sodium 4-ethynylbenzoate 1 (2.52 g,
15 mmol) and L-threonine methyl ester hydrochloride (3.05 g,
18 mmol, 1.2 equiv) in anhydrous DMF (100 mL) was added
N-ethyl-N0-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDCꢁHCl) (3.45 g 18 mmol, 1.2 equiv) and 1-hydroxybenzotria-
zole hydrate (HOBt) (2.45 g 18 mmol, 1.2 equiv) at room temper-
ature. The mixture was cooled in an ice-bath, and
diisopropylethylamine (DIEA) (10.45 mL, 60 mmol, 4.0 equiv)
was added. The whole reaction mixture was stirred under argon
at 0 °C for 1 h, then allowed to warm to ambient temperature,
and the stirring was continued for additional 20 h. The resulting
yellow solution was evaporated to dryness under reduced pres-
sure, and the resulting residue was treated with water (100 mL)
and extracted with EtOAc (3 ꢂ 100 mL). The combined extracts
were washed with 1 N HCl (2 ꢂ 70 mL) and brine (100 mL), and
dried (anhydrous Na2SO4). Evaporation of the solvent afforded
the crude product (3.5 g), which was crystallized from EtOAc/hex-
ane to give 2 (1.9 g, 48% yield) as light yellow crystal. 1H NMR
(300 MHz, CDCl3): d 1.28 (d, J = 6.3 Hz, 3H), 3.21 (s, 2H), 3.79 (s,
3H), 4.41–4.49 (m, 1H), 4.80 (dd, J = 2.4, 2.4 Hz, 1H), 6.94 (d,
J = 9.0 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.7 Hz, 2H);
13C NMR (75 MHz, CDCl3): d 20.34, 52.95, 57.81, 68.42, 79.94,
82.89, 126.06, 127.39, 132.55, 133.86, 167.28, 171.74; MS (ESI,
positive): m/z 284 [M+Na]+.
might be possible to replace the para-amino group with
a
fluorophore or an azide group without significantly affecting its
antibiotic activity. This may allow the development of fluores-
cence-based inhibitor binding assays or new tools based on click
chemistry via Cu(I)-catalyzed azide-alkyne cycloaddition24 for
affinity purification and identification of human metalloproteins
that tightly associate with LpxC inhibitors in cells—a particular
concern for the unintended off-target activity of hydroxamate-
containing compounds.
Stereochemistry is an important factor of inhibitor design. We
show that the R-stereoisomer at the C2 position of the threonyl
group is detrimental to the antibiotic profiles of LPC-011 deriva-
tives. In contrast, the chirality at the C3 position of the threonyl
group is less stringent, and compounds with either S- or R-config-
urations at the C3 position show similar antibiotic activities for
tested bacterial strains. Our structural investigation reveals an
invariant threonyl methyl-F192 interaction regardless of the ste-
reochemistry at the C3 position. However, the loss of favorable
hydrogen bonds between the hydroxyl group in the 3R configura-
tion of LPC-011 and K239 and H265 of E. coli LpxC is compensated
by a water-mediated hydrogen bond with the carbonyl group of
F192 of E. coli LpxC involving the same hydroxyl group in the 3S
configuration of LPC-053 pointing away from the active site to-
ward the solvent. Previous structural studies of the LpxC/UDP com-
plex have revealed a UDP-binding pocket that is located adjacent to
the active site occupied by the threonyl head group of CHIR-
090.17,25,26 Recently, uridine-based compounds have been shown
to inhibit LpxC in vitro, presumably by competing for the same
UDP-binding pocket with the LpxC substrate UDP-3-O-(acyl)-
N-acetylglucosamine.27 Because the hydroxyl group in the 3S
compound LPC-053 is solvent exposed, it may be a convenient
functional group for further derivatization to extend the current
compound binding interface to the adjacent UDP-binding pocket
and generate more potent inhibitors based on the well-known
ligand additivity effect.28
4.2. General procedure for Glaser coupling of 4-ethynylbenz-
amide 2 with ethynylbenzenamine
Copper(II) acetate (0.36 g, 2 mmol, 2.0 equiv) was added at
room temperature to a stirred solution of 2 (0.26 g, 1 mmol) and
substituted acetylene (5 mmol, 5 equiv) dissolved in anhydrous
pyridine (4 mL) and MeOH (4 mL), and the reaction mixture was
stirred at room temperature for 18 h (80 h for 3-ethynylbenzen-
amine and 2-ethynylbenzenamine). The resulting blue solution
was concentrated to dryness with a rotavapor, and the residue
was treated with water (50 mL) and extracted with EtOAc
(3 ꢂ 50 mL). The combined organic extracts were washed with
water (50 mL) and brine (50 mL), and dried (anhydrous Na2SO4).
The crude product was purified by the CombiFlash system (eluting
with 50–75% EtOAc in hexane) to afford diacetylene methyl ester
3a–c as yellow solid.

858
X. Liang et al. / Bioorg. Med. Chem. 19 (2011) 852–860
4.2.1. Methyl (2S,3R)-2-{40-[(400-aminophenyl)buta-10,30-diynyl]-
benzamido}-3-hydroxy-butanoate (3a)
71.82, 75.76, 79.52, 83.29, 116.72, 118.17, 121.69, 121.76, 125.47,
127.61, 129.18, 132.30, 134.22, 148.19, 168.02, 168.41; HRMS:
calcd for C21H19N3O4 377.1376; found 377.1369 (M+).
67% Yield; 1H NMR (300 MHz, CDCl3): d 1.28 (d, J = 6.3 Hz, 3H),
3.79 (s, 3H), 3.94 (br, s, 1H), 4.47–4.44 (m, 1H), 4.80 (dd, J = 8.7,
2.4 Hz, 1H), 6.59 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 7.33
(d, J = 8.7 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H);
13C NMR (75 MHz, CDCl3: d 20.35, 52.96, 57.79, 68.44, 72.01,
79.89, 84.53, 110.51, 114.84, 126.32, 127.44, 132.72, 133.62,
134.43, 148.01, 167.21, 171.72; MS (ESI, positive): m/z 399
[M+Na]+.
4.3.3. (2S,3R)-2-{40-[(200-Aminophenyl)buta-10,30-diynyl]benz-
amido}-1,3-dihydroxy-butanamide (LPC-013)
74% Yield; 1H NMR (300 MHz, CD3OD): d 1.23 (d, J = 6.3 Hz, 3H),
4.18–4.22 (m, 1H), 4.44 (d, J = 5.1 Hz, 1H), 6.59 (m, 1H), 6.74 (dd,
J = 8.1, 0.6 Hz, 1H), 7.09–7.15 (m, 1H), 7.26 (dd, J = 1.2 Hz, 1H),
7.60 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 6.9 Hz, 2H); 13C NMR (75 MHz,
CD3OD): d 19.16, 57.92, 67.26, 75.86, 77.75, 80.21, 80.99, 105.01,
114.53, 117.03, 125.61, 127.38, 127.63, 130.87, 132.03, 132.19,
132.80, 134.14, 151.15, 168.03, 168.39; HRMS: calcd for
4.2.2. Methyl (2S,3R)-2-{40-[(300-aminophenyl)buta-10,30-diynyl]-
benzamido}-3-hydroxy-butanoate (3b)
62% Yield; 1H NMR (300 MHz, CDCl3): d 1.26 (d, J = 6.3 Hz, 3H),
3.76 (s, 3H), 4.42–4.44 (m, 1H), 4.78 (dd, J = 9.0, 2.4 Hz, 1H), 6.68 (d,
J = 8.1 Hz, 1H), 6.80 (s, 1H), 6.92 (d, J = 7.5 Hz, 1H), 7.09 (t,
J = 15.6 Hz, 1H), 7.54 (d, J = 8.7 Hz), 7.77 (d, J = 8.4 Hz, 2H); 13C
NMR (75 MHz, CDCl3): d 20.35, 52.94, 58.00, 68.35, 73.19, 80.33,
83.54, 116.79, 118.66, 122.28, 123.24, 125.88, 127.51, 129.64,
132.84, 133.89, 146.60, 167.34, 171.73; MS (ESI, positive): m/z
399 [M+Na]+.
C
21H19N3O4 377.1376; found 377.1374 (M+).
4.4. General procedure for synthesis of LPC-053, 054 and 055
To a stirred mixture of acid 6 (120 mg, 0.46 mmol) and
allo-L-threonine methyl ester hydrochloride 11 (94 mg, 0.55 mmol,
1.2 equiv) in anhydrous DMF (5 mL) was added EDCꢁHCl (106 mg
0.55 mmol, 1.2 equiv), HOBt (75 mg, 0.55 mmol, 1.2 equiv) at room
temperature. The mixture was chilled to 0 °C with an ice-bath, and
DIEA (0.32 mL, 1.84 mmol, 4 equiv) was added. The reaction mix-
ture was stirred under argon at 0 °C for 1 h, then allowed to warm
to ambient temperature with the stirring continued for additional
18 h. The resulting yellow solution was condensed to dryness with
a rotavapor, and the residue was treated with water (20 mL), ex-
tracted with EtOAc (3 ꢂ 50 mL). The combined extracts were
washed with brine (40 mL), and dried. Evaporation of the solvent
afforded the crude product, which was purified by CombiFlash
(eluting with 1–3% MeOH in DCM) to afford methyl (2S,3S)-2-{40-
[(400-aminophenyl)buta-10,30-diynyl]benzamido}-3-hydroxybutano-
ate 7 (159 mg, 92% yield) as yellow solid. 1H NMR (300 MHz,
DMSO-d6): d 1.16 (d, J = 6.3 Hz, 3H), 3.62 (s, 3H), 3.98–4.08 (m,
1H), 4.37 (t, J = 14.4 Hz, 1H), 5.06 (d, J = 6.0 Hz, 1H), 5.83 (br s,
2H), 6.53 (d, J = 8.7 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.63 (d,
J = 8.4 Hz, 2H), 7.87 (d, J = 8.7 Hz, 2H), 8.64 (d, J = 7.8 Hz, 1H); 13C
NMR (75 MHz, DMSO-d6): d 21.06, 52.34, 60.14, 67.08, 71.78,
77.41, 80.78, 86.43, 105.83, 114.27, 124.96, 128.63, 132.64,
134.55, 134.70, 151.56, 166.39, 171.94; MS (ESI, positive): m/z
377 [M+H]+.
4.2.3. Methyl (2S,3R)-2-{40-[(200-aminophenyl)buta-10,30-diynyl]-
benzamido}-3-hydroxy-butanoate (3c)
26% Yield; 1H NMR (300 MHz, CDCl3): d 1.27 (d, J = 6.3 Hz, 3H),
3.77 (s, 3H), 4.35 (br, 1H), 4.41–4.46 (m, 1H), 4.79 (dd, J = 8.7,
2.4 Hz, 1H), 6.64–6.69 (m, 2H), 7.06 (d, J = 8.7 Hz, 1H), 7.12–7.18
(m, 1H), 7.33 (dd, J = 8.1, 1.5 Hz, 1H), 7.55 (d, J = 8.7 Hz, 2H), 7.79
(d, J = 8.4 Hz, 2H); 13C NMR (75 MHz, CDCl3): d 20.35, 52.95,
57.98, 68.37, 76.70, 98.95, 80.33, 81.81, 105.95, 114.70, 118.20,
125.87, 127.54, 131.16, 132.72, 133.38, 133.90, 149.98, 167.29,
171.73; MS (ESI, positive): m/z 399 [M+Na]+.
4.3. General procedure for preparing hydroxamic acids (LPC-
011, 012, 013) from the corresponding methyl esters 3a–c
To an ice-cold solution of 3 (120 mg, 0.32 mmol) dissolved in
anhydrous MeOH (1.5 mL) and THF (1.5 mL) was added hydroxyl-
amine hydrochloride (110 mg, 1.60 mmol, 5 equiv) followed by
25% sodium methoxide in methanol solution (0.53 mL, 2.20 mmol,
7 equiv). The reaction mixture was stirred under argon at 0 °C for
2 h, then allowed to warm to ambient temperature and stirring
was continued overnight (16 h). The resulting yellow suspension
was concentrated to dryness with a rotavapor, and the residue
was treated water (50 mL). The mixture was extracted with EtOAc
(3 ꢂ 50 mL). The combined organic layers were washed with brine
(30 mL) and dried. Evaporation of the solvent afforded the crude
product, which was purified by CombiFlash (eluting with MeOH
in DCM 7–10%) to afford hydroxamic acid as yellow solid.
Following the similar procedure, methyl (2R,3R)-2-{40-[(400-ami-
nophenyl)buta-10,30-diynyl]benzamido}-3-hydroxybutanoate (14)
and methyl (2R,3S)-2-{40-[(400-aminophenyl)buta-10,30-diynyl]benz-
amido}-3-hydroxybutanoate (16) were obtained.
4.4.1. Methyl (2R,3R)-2-{40-[(400-aminophenyl)buta-10,30-diynyl]-
benzamido}-3-hydroxy-butanoate (14)
1H NMR (300 MHz, DMSO-d6): d 1.13 (d, J = 6.0 Hz, 3H), 3.64 (s,
3H), 4.14–4.19 (m, 1H), 4.48 (m, 1H), 4.94 (d, J = 7.5 Hz, 1H), 5.83
(br s, 2H), 6.53 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 7.2 Hz, 2H), 7.65 (d,
J = 6.9 Hz, 2H), 7.90 (d, J = 7.2 Hz, 2H), 8.37 (d, J = 7.5 Hz, 1H); 13C
NMR (75 MHz, DMSO-d6): d 20.92, 52.59, 59.76, 67.08, 71.77,
77.42, 80.77, 86.44, 105.82, 114.27, 125.00, 128.56, 132.71,
134.70, 151.56, 166.74, 171.69; MS (ESI, positive): m/z 377 [M+H]+.
4.3.1. (2S,3R)-2-{40-[(400-Aminophenyl)buta-10,30-diynyl]benz-
amido}-1,3-dihydroxy-butanamide (LPC-011)
61% Yield; 1H (300 MHz, CD3OD): d 1.23 (d, J = 6.6 Hz, 3H),
4.21–4.17 (m, 1H), 4.43 (d, J = 5.1 Hz, 1H), 6.62 (d, J = 6.9 Hz, 2H),
7.24 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 7.87 (d, J = 8.4 Hz,
2H); 13C NMR (75 MHz, CD3OD): d 57.90, 67.23, 70.73, 76.63,
78.97, 84.57, 108.32, 114.22, 126.06, 127.56, 132.07, 133.81,
150.22, 168.08, 168.41; HRMS: calcd for C21H19N3O4 377.1376;
found 377.1376 (M+).
4.4.2. Methyl (2R,3S)-2-{40-[(400-aminophenyl)buta-10,30-diynyl]-
benzamido}-3-hydroxy-butanoate (16)
1H NMR (300 MHz, DMSO-d6): d 1.15 (d, J = 6.3 Hz, 3H), 3.63 (s,
3H), 4.00–4.06 (m, 1H), 4.36 (t, J = 13.2 Hz, 1H), 5.06 (d, J = 4.2 Hz,
1H), 5.83 (br s, 2H), 6.53 (d, J = 6.9 Hz, 2H), 7.24 (d, J = 6.9 Hz, 2H),
7.63 (d, J = 6.6 Hz, 2H), 7.87 (d, J = 8.1 Hz, 2H), 8.64 (d, J = 6.9 Hz,
1H); 13C NMR (75 MHz, DMSO-d6): d 21.07, 52.33, 60.14, 67.06,
71.76, 77.40, 80.79, 86.43, 105.81, 114.26, 124.95, 128.63, 132.64,
134.54, 134.70, 151.56, 166.38, 171.94; MS (ESI, positive): m/z
377 [M+H]+.
4.3.2. (2S,3R)-2-{40-[(300-Aminophenyl)buta-10,30-diynyl]benz-
amido}-1,3-dihydroxy-butanamide (LPC-012)
66% Yield; 1H NMR (300 MHz, CD3OD): d 1.23 (d, J = 6.3 Hz, 3H),
4.18–4.21 (m, 1H), 4.43 (d, J = 5.1 Hz, 1H), 6.73–6.77 (m, 1H), 6.80–
6.84 (m, 2H), 7.07 (m, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.88 (d,
J = 8.4 Hz, 2H); 13C NMR (75 MHz, CD3OD): d 19.16, 57.91, 67.24,

X. Liang et al. / Bioorg. Med. Chem. 19 (2011) 852–860
859
To an ice-cold solution of 7 (100 mg, 0.26 mmol) dissolved in
anhydrous MeOH (1 mL) and THF (1 mL) was added hydroxyl-
amine hydrochloride (92 mg, 1.33 mmol, 5 equiv) followed by
25% sodium methoxide in methanol solution (0.47 mL, 2.0 mmol,
7.5 equiv). The reaction mixture was stirred under argon at 0 °C
for 2 h, then allowed to warm to ambient temperature with the
stirring continued overnight (14 h). The resulting yellow suspen-
sion was condensed to dryness with a rotavapor, and the residue
was treated water (20 mL), extracted with EtOAc (3 ꢂ 50 mL).
The combined extracts were washed with brine (20 mL), and dried.
Evaporation of the solvent afforded the crude product, which was
purified by CombiFlash (eluting with 1–8% MeOH in DCM) to afford
(2S,3S)-2-{40-[(400-aminophenyl)buta-10,30-diynyl]benzamido}-1,3-
dihydroxy-butanamide (LPC-053) (70 mg, 70% yield) as yellow
solid. 1H NMR (300 MHz, DMSO-d6): d 1.09 (d, J = 6.3 Hz, 3H),
3.97 (br s, 1H), 4.23 (t, J = 16.5 Hz, 1H), 4.94 (br s, 1H), 5.82 (s,
2H), 6.54 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.7 Hz, 2H), 7.60 (d,
J = 8.1 Hz, 2H), 7.87 (d, J = 8.7 Hz, 2H), 8.42 (d, J = 8.7 Hz, 1H),
8.81 (br s, 1H), 10.58 (br s, 1H); 13C NMR (75 MHz, DMSO-d6) d
21.09, 57.95, 66.84, 71.79, 77.26, 80.83, 86.34, 105.83, 114.27,
124.70, 128.62, 132.55, 134.69, 135.02, 151.54, 165.93, 167.69;
HRMS: calcd for C21H19N3O4H 378.1454; found 378.1454 [M+H]+.
Following the similarprocedure, (2R,3R)-2-{40-[(400-aminophenyl)-
buta-10,30-diynyl] benzamido}-1,3-dihydroxybutanamide (LPC-054)
and (2R,3S)-2-{40-[(400-aminophenyl) buta-10,30-diynyl]benzamido}-
1,3-dihydroxybutanamide (LPC-055) were synthesized.
15 lg/mL of L-161,240 and 10 mM sodium citrate. The resulting col-
onies were purified three times on this media. Genomic DNA from
resistant colonies was isolated, and the region around lpxC was
amplified with the primers 300-up-lpxC and 300-down-lpxC, and
sequenced with paLpxC-361-50 and paLpxC-581-30. The colony that
harbored the P. aeruginosa lpxC knock-in was named as W3110PA.
4.6. Protein purification, enzyme inhibition and MIC tests
The wild-type E. coli LpxC, UDP-3-O-[(R)-3-hydroxymyristoyl]-
N-acetylglucosamine, and [a-
32P] UDP-3-O-[(R)-3-hydroxymyri-
stoyl]-N-acetylglucosamine were prepared following established
procedures.17,29,30 Assays of LpxC activity and extraction of KaI pp
and KI values were performed as described previously,18 except
that the LPC-011 concentrations were varied from 0.8 pM to
51 nM. MICs were determined according to the NCCLS protocol
adapted to 96-well plates and LB media in the presence of 5%
DMSO as described previously.18,31
4.7. X-ray crystallography
Purified E. coli LpxC (1-300) samples at a final concentration of
10 mg/mL were mixed with 4-fold molar excess of individual com-
pounds. Crystals of E. coli LpxC in complex with LPC-011, LPC-012
or LPC-053 were obtained using the hanging drop vapor diffusion
method at 4 °C in solutions containing 0.1 M HEPES pH 7.5, 1.5–
1.7 M Li2SO4 and 10 mM DTT. The crystals were cryoprotected with
perfluoropolyether (PFO-X175/08) before being flash-frozen in liquid
nitrogen. Diffraction data were collected in-house at 100 K using a
Rigaku MicroMax-007 HF rotating anode generator and R-Axis IV++
detector. X-ray diffraction data were processed with HKL2000.32
Molecular replacement with phaser was used to obtain the initial
phases of the LpxC-inhibitor complex structures.33 The structure of
the E. coli LpxC/LPC-009 complex (PDB entry 3P3E) was used as the
search model. Water molecules were added using phenix34 and ver-
ified with coot.35 Additional electron density at the protein packing
interface is observed in all three E. coli LpxC–inhibitor complex crys-
tals. Because this electron density cannot be fitted with the protein or
the inhibitor, it likely represents a buffer molecule (e.g., HEPES) or
impurity from the chemical synthesis. The final models were ob-
tained after iterative cycles of manual model building with coot
and refinement using phenix. molprobity36 was used to evaluate
the quality of the refined structures. The statistics for the LpxC–inhib-
itor complexes are shown in Table S1. Structures factors and coordi-
nates for E. coli LpxC in complex with LPC-011, LPC-012, and LPC-053
have been deposited in the RCSB Protein Data Bank with the acces-
sion codes 3PS1, 3PS2, 3PS3, respectively.
Spectral data for LPC-054: 1H NMR (300 MHz, DMSO-d6): d 1.07
(d, J = 5.4 Hz, 3H), 3.98–4.04 (m, 1H), 4.24 (d, J = 12.9 Hz, 1H), 4.87
(d, J = 5.7 Hz, 1H), 5.82 (br s, 2H), 6.53 (d, J = 8.4 Hz, 2H), 7.24 (d,
J = 8.4 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.90 (d, J = 6.6 Hz, 2H),
8.13 (d, J = 7.8 Hz, 1H), 8.84 (br s, 1H), 10.65 (br s, 1H); 13C NMR
(75 MHz, DMSO-d6): d 21.00, 58.80, 67.09, 71.79, 77.29, 80.84,
86.36, 105.84, 114.27, 124.74, 128.59, 132.58, 134.69, 135.02,
151.54, 166.28, 167.62; HRMS: calcd for C21H19N3O4H 378.1454;
found 378.1453 [M+H]+.
Spectral data for LPC-055: 1H NMR (300 MHz, DMSO-d6): d 1.09
(d, J = 6.0 Hz, 3H), 3.95–3.98 (m, 1H), 4.23 (t, J = 16.2 Hz, 1H), 4.94
(d, J = 3.9 Hz, 1H), 5.82 (br s, 2H), 6.53 (d, J = 7.8 Hz, 2H), 7.24 (d,
J = 7.2 Hz, 2H), 7.61 (d, J = 8.1 Hz, 2H), 7.87 (d, J = 7.5 Hz, 2H),
8.42 (d, J = 8.7 Hz, 1H), 8.81 (br s, 1H), 10.58 (br s, 1H); 13C NMR
(75 MHz, DMSO-d6): d 21.09, 57.95, 66.84, 71.79, 77.26, 80.83,
86.34, 105.84, 114.27, 124.70, 128.62, 132.55, 134.69, 135.03,
151.55, 165.93, 167.70; HRMS: calcd for C21H19N3O4H 378.1454;
found 378.1460 [M+H]+.
4.5. Construction of E. coli W3110PA
P. aeruginosa lpxC was used to replaceE. coli chromosomal lpxC. A
linear PCR product containing the P. aeruginosa lpxC with flanking
sequences complementary to the upstream 50 region of E. coli lpxC
and to the downstream 30 region of E. coli lpxC, was amplified from
a plasmid carrying P. aeruginosa lpxC using primers pa-LpxC-50and
pa-LpxC-30 (Table S2). The PCR product was gel purified and electro-
porated into E. coli DY330 cells, which carry k-red recombinases.
While DY330 cannot survive on the LB/agar plate supplemented
Acknowledgements
This research was supported by National Institutes of Health
Grants AI055588 (to P.Z.), and GM-51310 (to C.RH.R). X.L. is sup-
ported by the China Scholarship Council. We thank Dr. Nathan I.
Nicely for assistance with collection of X-ray diffraction data at
the Duke University Crystallography Shared Resource.
with 15 lg/mL of L-161,240, cells wherein E. coli lpxC replaced with
P. aeruginosa lpxC can survive on this media. Transformants were
therefore selected directly using L-161,240. Genomic DNA from
resistant colonies was isolated, and the region around lpxC was
amplified with primers 300-up-lpxC and 300-down-lpxC and se-
quenced with primers paLpxC-361-50 and paLpxC-581-30
(Table S2). One clone in which P. aeruginosa lpxC had replaced chro-
mosomal E. coli lpxC was selected and grown at 30 °C. This strain
was used to generate P1vir lysate, which was used to transduce
chromosomal P. aeruginosa lpxC into the chromosome of E. coli
W3110. Transduced cells were plated on LB/agar containing
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.017.
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