Acyl-sulfamates target the essential glycerol-phosphate acyltransferase (PlsY) in Gram-positive bacteria

Article abstract of DOI:10.1016/j.bmc.2012.06.029

PlsY is the essential first step in membrane phospholipid synthesis of Gram-positive pathogens. PlsY catalyzes the transfer of the fatty acid from acyl-phosphate to the 1-position of glycerol-3-phosphate to form the first intermediate in membrane biogenesis. A series of non-metabolizable, acyl-sulfamate analogs of the acyl-phosphate PlsY substrate were prepared and evaluated as inhibitors of Staphylococcus aureus PlsY and for their Gram-positive antibacterial activities. From this series phenyl (8-phenyloctanoyl) sulfamate had the best overall profile, selectively inhibiting S. aureus phospholipid biosynthesis and causing the accumulation of both long-chain fatty acids and acyl-acyl carrier protein intermediates demonstrating that PlsY was the primary cellular target. Bacillus anthracis was unique in being more potently inhibited by long chain acyl-sulfamates than other bacterial species. However, it is shown that Bacillus anthracis PlsY is not more sensitive to the acyl-sulfamates than S. aureus PlsY. Metabolic profiling showed that B. anthracis growth inhibition by the acyl-sulfamates was not specific for lipid synthesis illustrating that the amphipathic acyl-sulfamates can also have off-target effects in Gram-positive bacteria. Nonetheless, this study further advances PlsY as a druggable target for the development of novel antibacterial therapeutics, through the discovery and validation of the probe compound phenyl (8-phenyloctanoyl) sulfamate as a S. aureus PlsY inhibitor.

Full text of DOI:10.1016/j.bmc.2012.06.029

Bioorganic & Medicinal Chemistry 20 (2012) 4985–4994
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Acyl-sulfamates target the essential glycerol-phosphate acyltransferase (PlsY)
in Gram-positive bacteria
Philip T. Cherian a, , Jiangwei Yao b, , Roberta Leonardi b, Marcus M. Maddox a, Vicki A. Luna c,
Charles O. Rock b, Richard E. Lee a,
⇑
a Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
b Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
c Center for Biological Defense, University of South Florida, 3602 Spectrum Blvd, Tampa, FL 33612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
PlsY is the essential first step in membrane phospholipid synthesis of Gram-positive pathogens. PlsY cat-
alyzes the transfer of the fatty acid from acyl-phosphate to the 1-position of glycerol-3-phosphate to
form the first intermediate in membrane biogenesis. A series of non-metabolizable, acyl-sulfamate ana-
logs of the acyl-phosphate PlsY substrate were prepared and evaluated as inhibitors of Staphylococcus
aureus PlsY and for their Gram-positive antibacterial activities. From this series phenyl (8-phenylocta-
noyl) sulfamate had the best overall profile, selectively inhibiting S. aureus phospholipid biosynthesis
and causing the accumulation of both long-chain fatty acids and acyl–acyl carrier protein intermediates
demonstrating that PlsY was the primary cellular target. Bacillus anthracis was unique in being more
potently inhibited by long chain acyl-sulfamates than other bacterial species. However, it is shown that
Bacillus anthracis PlsY is not more sensitive to the acyl-sulfamates than S. aureus PlsY. Metabolic profiling
showed that B. anthracis growth inhibition by the acyl-sulfamates was not specific for lipid synthesis
illustrating that the amphipathic acyl-sulfamates can also have off-target effects in Gram-positive
bacteria. Nonetheless, this study further advances PlsY as a druggable target for the development of novel
antibacterial therapeutics, through the discovery and validation of the probe compound phenyl
(8-phenyloctanoyl) sulfamate as a S. aureus PlsY inhibitor.
Received 8 May 2012
Revised 11 June 2012
Accepted 16 June 2012
Available online 23 June 2012
Keywords:
Antibacterial
Phospholipid
Membrane
PlsY
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
new therapeutic targets that are essential for cell viability and suit-
able for therapeutic intervention.
The creeping rise in antibacterial drug resistance combined
with the failure of current discovery programs to produce new
antibacterial targets and lead compounds to meet this growing
threat is of great concern to the whole medical community.1 This
comes at a time when increasingly virulent and resistant strains
of bacteria such as methicillin resistant Staphylococcus aureus
(MRSA) lineage USA300 are becoming dominant in our clinics
causing much morbidity and mortality. Current efforts at develop-
ing new Gram-positive antibacterial agents are largely focused
around standard antibacterial classes including b-lactams, oxazo-
lidinones and fluoroquinolones for which class specific resistance
mechanisms preexist.2 Thus, there is an urgent need to develop
One such alternative target is the recently discovered PlsX and
PlsY mediated phospholipid biosynthetic pathway.3 PlsX catalyzes
the formation of acyl-PO4 from the acyl–acyl carrier protein (ACP)
end-products of fatty acid biosynthesis. PlsY transfers the acyl
group from acyl-PO4 to the 1-position of glycerol-PO4 to form the
first intermediate in membrane phospholipid synthesis. Targeting
this pathway appears desirable because it is essential in all signif-
icant Gram-positive human pathogens3 and disrupting bacterial
membrane biogenesis is a proven strategy for the development
of potent antibacterials.4 Furthermore, humans synthesize the
phospholipids using acyl-CoA-dependent glycerol-PO4 acyltrans-
ferases and PlsY homologs are not found in mammalian genomes.
Despite these clear advantages, targeting PlsY presents challenges.
PlsY is a small (23 kDa) integral membrane protein with 5 trans-
membrane helicies.5 Although the residues critical for catalysis
are known, structural information is not available and the enzyme
is most active in its native membrane environment. Thus, our first
approach to inhibitor design was to synthesize five classes of
nonhydrolyzable acyl-PO4-based bioisosteric inhibitors of PlsY.6
Abbreviations: ACP, acyl carrier protein; PlsY, acyl-phosphate:glycerol-3-phos-
phate acyltransferase; PlsX, acyl-phosphate:ACP transacylase; acyl-PO4, acyl-phos-
phate; glycerol-PO4, sn-glycerol-3-phosphate; SAR, structure–activity relationship.
⇑
Corresponding author. Tel.: +1 901 595 6617; fax: +1 901 595 5715.
E-mail address: richard.lee@stjude.org (R.E. Lee).
These authors contributed equally to the work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.029

4986
P. T. Cherian et al. / Bioorg. Med. Chem. 20 (2012) 4985–4994
Figure 1. Substrate based inhibitors of PlsY. Palmitoyl-phosphate is a PlsY substrate. Non-hydrolyzable substrate mimics of PlsY were generated by bioisosteric replacement
of the phosphate head group. The acyl-sulfamate class was expanded upon in this study.
Scheme 1. Overall synthetic scheme for preparing acyl-sulfamates. (i) HCOOH, neat (ii) R1OH, DMA (iii) R2COCl, DMAP, NEt3, DCM:DMF.
These chemical classes included acyl-phosphonates, acyl-phospho-
ramides, reverse amide phosphonates, acyl-sulfamates and
acyl-sulfamides (Fig. 1). The most active inhibitors from each series
generally contained a long aliphatic chain that mimicked the
acyl-PO4 substrate of PlsY. These PlsY inhibitors displayed weak
to moderate antimicrobial activity against a representative panel
of Gram-positive bacteria, with the exception of Bacillus anthracis,
for which potent antibacterial activity was observed.
This study expands the SAR and mechanism of action within the
acyl-sulfamate class (Fig. 1), the most potent of the bioisosteric
PlsY inhibitors.6 The acyl-sulfamate scaffold was modified at the
R1 (head) and R2 (tail) positions (Scheme 1) to determine the effect
of substitutions on the potency against PlsY and antimicrobial
activity. Biochemical analysis of acyl-sulfamate treated cells con-
firmed that these molecules target the PlsY pathway for phospho-
lipid synthesis, although these amphipathic molecules can also
exhibit off-target activities, which need to be carefully monitored
to gain a clear understanding of antibacterial structure–activity
relationships.
than the smaller furan (3), cyclopentane (2) and methyl (1) ana-
logs. The potency was also influenced by the hydrophobicity of
the R1 substituent. The more hydrophobic cyclohexane analog 4
was six-fold more potent than the 4-oxygen containing pyran ana-
log 5 and at least twenty-fold more potent than the 4-NH contain-
ing piperidine analog 6. The considerable loss of potency of 6
suggested that a free amine was not tolerated at this site due to
a site specific interaction and a potentially strong desolvation
penalty of this cation. This was further corroborated by the signif-
icant restoration of potency upon protection of the 4-NH of piper-
idine (6) with a Boc-group (7). Replacing the saturated and
comparatively flexible cyclohexane with an aromatic phenyl group
(8) led to a minor loss in potency; nevertheless, the availability of
various substituted phenols made it the better option for further
exploring this site. A 4-Cl substituent on the phenyl ring (9) proved
to be somewhat better than the unsubstituted 8. The 3, 4-dichloro
analog 11 and the 3-chloro analog 10 were less potent than 9 sug-
gesting that the 4-position on the phenyl was the preferred site for
further exploration. We replaced the 4-Cl with substituents of
increasing size including methyl (12), hydroxymethyl (13) methyl
propionate (14), ethyl acetate (15) and 4-(4-NO2) biphenyl (18).
Despite the substantial increase in size at the 4-position, the po-
tency was only two-fold or less compared to 9. The only 4-substit-
utent from our series that did show a significant loss of potency
was the 4-ethylamine analog 16. This loss was attributed to the
presence of the free amine also found in 16 rather than the size be-
cause potency was restored in the Boc-protected analog 17. This
trend was similar to the preceding amine containing pair (6 and
7). We also synthesized bicyclic analogs 19–21 and found that
the benzodioxole (19) and quinoline (20) derivatives had potencies
similar to most of the 4-substituted phenols while the benzofura-
nyl 3-sulfamate (21) was four-fold less potent.
2. Results and discussion
2.1. Chemistry
The acyl-sulfamates were synthesized as described previously.6
Briefly, chlorosulfonyl isocyanate was treated with neat formic
acid to generate sulfamoyl chloride that was reacted with various
alcohols to synthesize a series of sulfamates introducing the R1
substitution. Acylation of the sulfamates with selected acyl chlo-
rides provided the desired acylsulfamates introducing the R2 acyl
tail (Scheme 1).
The SAR at R2 acyl tail position (Scheme 1) was explored by syn-
thesizing derivatives 22–28 that incorporated a phenyl or 4-OCH3
phenyl at R1 head (Table 2). The myristoyl (C14) derivative 22 was
slightly better than the C16 derivative 13 while further decreasing
the chain length to C11 in 23 led to a threefold decrease in potency.
Interestingly, a terminal phenyl on the C11 aliphatic chain (24) at
R2 was well-tolerated and the compound was equipotent to the
palmitoyl analog 8. Further decreasing the chain length between
2.2. SAR of acyl-sulfamates against S. aureus PlsY
The SAR at the acyl-PO4 R1 head site (Scheme 1) was explored
by the synthesis of compounds 1–21 (Table 1) that maintained a
palmitoyl (C16) chain at the R2 tail position to mimic the natural
substrate. The potency of these acyl-sulfamates as PlsY inhibitors
was influenced by the size of the R1 substituent with the larger
six membered and bicyclic analogs being generally more potent

P. T. Cherian et al. / Bioorg. Med. Chem. 20 (2012) 4985–4994
4987
Table 1
SAR at the R1 position
MICb
(lg/ml)
a
IC50
(l
M)
SA-PIsY
SA
SP
EF
BS
BA
No.
Medianc
Ranged
1
2
CH3
80
>200
>200
>200
>200
>200
>200
>200
>200
>100
>100
>100
>100
>100
3
45
25
>200
>200
>200
1.56
0.195–6.25
4
5
6
7
8
5
>200
>200
>200
>200
>200
100
>200
>200
>200
>200
50
>200
>200
>200
>200
50
6.25
3.13
>100
3.13
3.13
0.78–12.5
0.39–6.25
>100
30
>100
15
8
>200
>200
>200
>200
0.78–3.13
0.78–6.25
9
6
>200
>200
100
>200
100
>200
100
6.25
3.13
1.56–12.5
0.78–6.25
10
14
>200
11
9
>200
>200
100
100
12.5
6.25–12.5
12
13
12
8
>200
12.5
>200
12.5
200
200
6.25
3.13
0.78–12.5
0.78–3.13
3.12
3.12
14
11
25
0.78
>200
>200
3.13
0.39–12.5
15
16
10
6.25
1.56
>200
>200
>200
>200
3.13
0.78–3.13
>100
>100
>200
>200
>100
17
18
19
22
11
12
>200
100
3.13
0.78
3.13
>200
>200
>200
>200
>200
>200
1.56
1.56
>100
0.78–3.13
1.56–3.13
>100
100
20
9
>200
1.56
200
200
3.13
0.78–3.13
(continued on next page)

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P. T. Cherian et al. / Bioorg. Med. Chem. 20 (2012) 4985–4994
Table 1 (continued)
IC50
(l
M)
MICb
(lg/ml)
a
SA-PIsY
SA
25
SP
EF
BS
BA
No.
21
Medianc
3.13
Ranged
41
3.13
100
100
1.56–12.5
a
IC50 against S. aureus PlsY.
b
MIC: Whole-cell minimum inhibitory concentration of the following species: SA, S. aureus ATCC 29213; SP, S. pneumoniae R6; EF, E. faecalis ATCC 33186; BS, B. subtilis
ATCC 23857.
c
Median: The median MIC value against 22 virulent strains of B. anthracis (BA).
Range: The highest and lowest MIC values recorded against the 22 B. anthracis strains.
d
Table 2
SAR at the R2 position
MICb
BS
(lg/ml)
a
No.
IC50
SA-PIsY
(
l
M)
SA
SP
EF
BA
Medianc
3.13
Ranged
13
8
12.5
12.5
12.5
3.12
0.78–3.13
22
23
24
6
>200
12.5
6.25
>200
200
6.25
1.6
>200
6.25
0.78
12.5
3.13
0.39–1.56
6.25–25
3.13
27
8
200
12.5
100–200
>200
25
26
25
76
12.5
50
100
50
100
100
6.25
12.5
1.56–6.25
12.5
>200
27
28
100
100
25
50
50
50
>200
>200
100
100
>100
>100
100
25–100
a
IC50 against S. aureus PlsY.
b
MIC: Whole-cell minimum inhibitory concentration of the following species: SA, S. aureus ATCC 29213; SP, S. pneumoniae R6; EF, E. faecalis ATCC 33186; BS, B. subtilis
ATCC 23857.
c
Median: The median MIC value against 22 virulent strains of B. anthracis (BA).
Range: The highest and lowest MIC values recorded against the 22 B. anthracis strains.
d
the terminal phenyl and the head group to C8 (25) led to a three-
fold decrease in potency while the shorter C7 (26) and C6 (27) ana-
logs and the cinnamic acid analog (28) were at least ten-fold less
potent than 24. Thus, for the compound series 24–28, the potency
was directly related to the alkyl chain length at R2.
modified at the R1 position (1–21) and found to be inactive against
S. aureus PlsY (1, 2, 6 and 16) were, in general, inactive against our
panel of Gram-positive bacteria (Table 1). In contrast to their S.
aureus PlsY inhibitory activity, most of the palmitoyl analogs were
inactive against S. aureus in the whole cell assay (Table 1) indicat-
ing that additional factors including permeability may be influenc-
ing the whole cell activity of these compounds. Exceptions in this
case were compounds 15 and 13, which had low MIC of 6.25 and
2.3. Antimicrobial activity
The synthesized compounds were screened against a represen-
tative panel of Gram-positive bacteria including 22 virulent strains
of B. anthracis (Tables 1 and 2). Palmitoyl (C16) analogs that were
12.5 lg/ml, respectively against S. aureus. Interestingly, most of
the palmitoyl analogs were highly active against E. faecalis and B.
anthracis; however, the MIC values of these compounds were

P. T. Cherian et al. / Bioorg. Med. Chem. 20 (2012) 4985–4994
4989
considerably lower than the corresponding PlsY IC50 suggesting
that this class of molecules can affect bacterial growth by a non-
PlsY mechanism (Table 1). Most of these analogs were generally
inactive against S. pneumoniae and B. subtilis except for 13 which
displayed good activity against both (Table 1).
For compounds incorporating a 4-OCH3 phenyl at the R1 posi-
tion, shortening the chain length at R2 from C16 (13) to C14 (22)
led to significant loss of activity against S. pneumoniae, S. aureus
and B. subtilis but caused a slight improvement in activity against
E. feacalis and B. anthracis (Table 2). Further decreasing the chain
length to C11 (23) led to significant restoration of the S. aureus
and B. subtilis activity, further improvement in the E. faecalis activ-
ity and a modest decreasing in the B. anthracis activity. Thus 23 dis-
played a broad antimicrobial profile with good activity against
multiple species in our panel of Gram-positive bacteria.
For compounds 24–27 that incorporated a phenyl group at the
R1 position and a terminal phenyl group on the R2 acyl chain,
decreasing the chain length between the head group and the ter-
minal phenyl at R2 led to a proportional decrease in the S. aureus
inhibitory activity, in similar fashion to their PlsY inhibitory activ-
ity (Table 2). The MIC values of 24–27 against S. aureus are compa-
rable to their PlsY IC50. A similar trend of increasing MIC with
decreasing chain length in 24–27 is also observed for B. anthracis
(Table 2). Compounds 24–27 were generally inactive against S.
pneumoniae and B. subtilis. The cinnamic acid analog 28 showed
moderate to weak activity against S. aureus and E. faecalis but
was generally inactive against the other species (Table 2).
2.4. Target validation
Whether the antibacterial effects of the acyl-sulfamates were
due to PlsY inhibition was addressed in a series of metabolic label-
ing and structural studies. The phenyl (8-phenyloctanoyl) sulfa-
mate 25 was selected for this purpose based on its reasonable
PlsY inhibition, good antimicrobial activity against S. aureus and
B. anthracis and comparatively lower molecular weight. Two genet-
ic systems provided the template for assessing the physiological
changes that occur after PlsY inhibition. One is in Bacillus subtilis
where the plsY gene was silenced7 and the second is the gpsA mu-
tant of S. aureus that conditionally deprives the cells of the glyc-
erol-PO4 for PlsY.8,9 Lipid synthesis inhibitors do not cause cell
lysis or immediate growth cessation. Rather, bacteria continue to
grow for a few generations until the imbalance between mem-
brane lipid and protein synthesis becomes intolerable and the cells
cease proliferation.10 S. aureus cell growth did not immediately
cease following the addition of 25, but slowed down over approx-
Figure 2. Effect of 25 on the growth and lipid synthesis in S. aureus strain RN4220.
(A) Growth curves of S. aureus strain RN4220 treated with 0 (d), 6.25 (s), 12.5 (j),
imately 2 h before stopping completely when treated with 25 lM
or greater of 25 (Fig. 2A). This gradual stop to S. aureus cell growth
was reminiscent of the gpsA mutants deprived of glycerol and PlsY
knockdown cells.8,9 Metabolic labeling in the presence and absence
of 50 lM 25 was used to assess its effect on the four major path-
ways of macromolecular biosynthesis (Fig. 2B). Compound 25
25 (h), 50 (N), or 100
pathways was measured in strain RN4220 treated with 50
untreated cells. Metabolic labeling with [14C]acetate was used for lipid biosynthesis,
l
M (4) of 25. (B) The effect of 25 on the major biosynthetic
l
M 25 relative to
a
3H-labeled amino acid mixture was used to measure protein biosynthesis,
[3H]thymidine incorporation measured DNA biosynthesis, and [3H]uracil measured
stable RNA biosynthesis. (C) The rates of lipid biosynthesis in strain RN4220 treated
selectively inhibited lipid biosynthesis compared to the other ma-
with 0, 12.5, 25, 50, or 100 lM of 25 were measured by the incorporation of
[
14C]acetate into the lipid fraction. [14C]Acetate incorporation in the absence of drug
jor macromolecules. Accordingly, 25 caused
a concentration
dependent decrease in [14C] acetate incorporation into fatty acids
and membrane phospholipids (Fig. 2C). In the Bacillus subtilis PlsY
conditional gene knockout model, knocking out PlsY causes
accumulation of intracellular free fatty acids.7 An increase in
14C-labeled free fatty acids was also observed in 25 treated cells
(not shown), consistent with PlsY inhibition as the in vivo mode
of action for 25 against S. aureus.
was 194,000 cpm per 5 ꢀ 109 cells.
was inhibited, then FabF has a competitive advantage and the acyl-
ACP end-product undergo additional rounds of elongation leading
to an increase in the average chain length in the membrane phos-
pholipids. The major fatty acids in control (DMSO-treated) strain
RN4220 were 15:0 and 17:0 with only small amounts of 19:0
and 20:0 chain lengths (Table 3). Treatment with increasing
amounts of 25 led to a progressive increase in the chain length of
fatty acids produced by the cells. For example, 21-carbon fatty
acids were not detected in untreated strain RN4220, but comprised
Another metabolic defect associated with a PlsY inhibitor was
an increase in the average chain-length of fatty acids in treated
cells. The chain-length of membrane fatty acids was determined
by the competition between the elongation condensing enzyme
(FabF) and the glycerol-PO4 acyltransferase.11 If the acyltransferase

4990
P. T. Cherian et al. / Bioorg. Med. Chem. 20 (2012) 4985–4994
Table 3
Effect of compound 25 on fatty acid composition
Fatty Acida
DMSO
Compound 25
25
lM
50 lM
15:0b
16:0
17:0
18:0
19:0
20:0
21:0
43%c
5%
25%
11%
9%
37%
5%
10%
9%
18%
9%
12%
32%
6%
8%
12%
14%
13%
14%
6%
n.d.d
a
S. aureus strain RN4220 was treated with the indicated concentration of 25 for
2.5 h and the fatty acid composition analyzed as described in the experimental
section.
b
Fatty acid nomenclature X:Y denotes that the fatty acid has X number of total
carbons and Y number of double bonds.
c
Percent values are weight percent from the average of duplicate measures,
rounded to the nearest percent.
d
Not detected, means <1%.
14% of the total fatty acids in S. aureus treated with 50 lM 25. The
appearance of these longer chain lengths correlated with a de-
crease in the 15-carbon fatty acids. Phosphatidylglycerol (PtdGro)
was the major membrane phospholipid in S. aureus, and mass spec-
trometry was used to determine how the distribution of PtdGro
molecular species was impacted by treatment with 25. In
untreated cells, the major molecular species was the 32-carbon
PtdGro consisting of 17:0 in the 1-position and 15:0 in the 2-posi-
tion (Fig. 3A).10 In cells treated with 25
molecular species contained 34 carbons, and in cells treated with
50 M 25, 35-carbon PtdGro was the most abundant species
(Fig. 3B). A 36-carbon molecular species was a major component
in cells treated with 50 M 25, but was barely detected in un-
lM 25, the principle
l
l
treated cells (Fig. 3C). An increase in the average chain-length of
fatty acids in membrane phospholipids was a hallmark feature that
points to the acyl-sulfamates targeting PlsY in vivo.
A blockade at the glycerol-PO4 acyltransferase step should also
lead to the accumulation of long chain acyl-ACP, which continues
to be produced by the fatty acid biosynthetic pathway, but fails
to be utilized due to the block in phospholipid synthesis.12 There-
fore, we examined the composition of the ACP pool in S. aureus
strain RN4220 treated with various concentrations of 25 (Fig. 4).
Acyl-ACPs can be separated by urea gel electrophoresis based on
the hydrophobicity of the attached acyl chain13, and were detected
by western blotting using anti-ACP antibody.14 The ACP pool in un-
treated cells was primarily non-esterified ACP. Treatment with 25
led to a significant increase in the level of long-chain acyl-ACP in
the cells, indicating a block in the utilization of the end products
of fatty acid synthesis as acyltransferase substrates. These mea-
surements of the intracellular ACP pool composition strongly sup-
port the conclusion that PlsY was a target for 25 in S. aureus.
Figure 3. Effect of 25 on the molecular species distribution in the major membrane
phospholipid of S. aureus, phosphatidylglyerol (PtdGro). S. aureus strain RN4220
was grown in Luria-Bertini broth and treated with DMSO as a control (A), or either
25 lM (B) or (C) 50 lM 25 for 2.5 h. The distribution of PtdGro molecular species
was determined by mass spectrometry as described under the Experimental
Section. The numbers above the peaks correspond to the total number of carbons in
the two acyl chains of the PtdGro molecular species at that mass.
2.5. Mode of action in B. anthracis
There are three PlsY homologs in B. anthracis. BaPlsY1 and BaP-
lsY2 have reasonable homology to the SaPlsY (ꢁ43–45% identity).
that either the PlsY of this organism was more sensitive to the
acyl-sulfamates than the S. aureus enzyme, or that the acyl-
sulfamates also have an off-target effect on B. anthracis growth.
The first possibility was tested by cloning and expressing the
B. anthracis PlsY1, and determining the IC50 for the prototypical
acyl-sulfamate, 25 (Fig. 5A). The 2-fold increase in the potency of
25 against B. anthracis PlsY1 was about the same as the difference
between the MIC for S. aureus and the average MIC for the wild-
type B. anthracis strain panel (Table 2). However, the effect of 25
on the growth of B. anthracis Sterne was also not characteristic of
lipid synthesis inhibitors (Fig. 5B). Compound 25 addition caused
BaPlsY3 has
a much lower homology (28% identity). We
reconstituted the enzymatic activity of all three BaPlsY. BaPlsY3
has no detected acyltransferase activity. BaPlsY2 has lower
acyltransferase activity, while BaPlsY1 has the most acyltransfer-
ase activity. Based on bioinformatics predication and enzyme
activity from reconstitution, we believe that BaPlsY1 is the major
PlsY in Bacillus anthracis, and therefore choose that for compound
activity testing. Many PlsY inhibitors had more potent antibacterial
activity against B. anthracis compared to other Gram-positive bac-
teria (Tables 1 and 2). These lower MICs in B. anthracis suggested

P. T. Cherian et al. / Bioorg. Med. Chem. 20 (2012) 4985–4994
4991
sulfamates may have off-target effects on bacterial membrane
function in some bacteria.
3. Conclusions
As part of our SAR study, a series of acyl-sulfamates with mod-
ifications at the R1 head and R2 acyl tail positions were synthesized
and evaluated as S. aureus PlsY inhibitors and for Gram-positive
antibacterial activities. At the R1 position, a wide range of substit-
uents were tolerated (1–21) with the exception of substituents
containing a free amine (6, 16) that led to significant loss of PlsY
activity. At R2, the acyl chain could be shortened from C16 to
C11 with minimal loss of PlsY activity (13 vs 22, 23) and com-
Figure 4. The effect of 25 on the intracellular acyl-ACP pool. S. aureus strain RN4220
was treated with the indicated concentrations of 25 for 2.5 h, cell extracts were
prepared and the proteins fractionated on 15% polyacrylamide gels containing 2 M
urea to separate the ACP species based on the length of the attached fatty acid
chain. ACP species were detected using anti-ACP antibody to visualize the different
acylated ACP species.
pounds incorporating
a terminal phenyl on the acyl chain
(24–27) displayed a direct correlation between chain length and
PlsY inhibitory activity. The acyl-sulfamates, typified by 25, pos-
sessed antibacterial activity through the inhibition of PlsY in vivo.
Compound 25 caused a slow inhibition of S. aureus growth that was
characteristic of other lipid biosynthesis inhibitors. Compound 25
selectively inhibited lipid synthesis compared to other major
branches of macromolecular biosynthesis, caused an increase in
the average chain-length of membrane phospholipid fatty acids
and triggered the accumulation of long-chain acyl-ACP end-prod-
ucts of fatty acid synthesis. All of these metabolic effects are con-
sistent with PlsY inhibition as the ultimate cellular target. A
potential drawback to this series of compounds was revealed by
the analysis of the acyl-sulfamate series against B. anthracis Sterne.
In this system, the amphipathic acyl-sulfamate possessed signifi-
cant off-target effects on B. anthracis metabolism and caused the
immediate cessation of bacterial growth that is not observed in S.
aureus. Nonetheless, the analysis of the acyl-sulfamate class fur-
ther advances PlsY as a druggable target for the development of
novel antibacterial therapeutics. Future development of the acyl-
sulfamate compound series will include finding suitable molecular
replacements for the long aliphatic chain at position R2 (Scheme 1)
that will retain PlsY activity and have improved drug-like proper-
ties and less off-target activity.
4. Experimental section
4.1. General synthetic methods
All reagents and solvents were purchased from commercial
sources. All reactions were performed under inert atmosphere.
The final reaction mixtures were purified on Isolera Flash Purifica-
tion System using high performance SNAP columns from Biotage.
The solvent system used for all purifications was hexane/ethyl ace-
tate (5–100% gradient). The purity and mass of the synthesized
compounds were determined on a Waters ACQUITY UPLC-PDA-
ELSD-MS system using a C18 reverse phase column and 0.1%
formic acid/water—0.1% formic acid/acetonitrile binary solvent
system. All synthesized compounds were at least 95% pure. The
structures of the synthesized compounds were confirmed by 1H
NMR which was recorded on a 400 Mhz Varian AVANCE 400-FT
NMR.
Figure 5. Effect of 25 on B. anthracis PlsY1 enzyme activity and growth. (A) IC50 for
25 using either S. aureus PlsY (d) of B. anthracis PlsY1 (s) as the enzyme source. (B)
Growth of B. anthracis Sterne in the absence (d, DMSO-treated control) or presence
(s) of 5 lM 25.
4.2. General procedure for the synthesis of palmitoylsulfamates
(1–21)
an abrupt cessation of cell growth that was more indicative of a
membrane disruptive agent as opposed to specific inhibition of
membrane biogenesis. Furthermore, the acyl-sulfamates inhibited
protein, DNA and RNA synthesis in B. anthracis Sterne (not shown)
compared to their selective effect on lipid synthesis observed in S.
aureus. An important caveat to these experiments was that we can-
not determine if the sensitivity of B. anthracis Sterne vaccine strain
is due to the loss of the pXO2 plasmid that produces a protective
capsule, which may allow the acyl-sulfamates to target the cell
membrane. Nonetheless, these data suggest that the acyl-
Formic acid (98%, 2 equiv) was added drop-wise to chlorosulfo-
nyl isocyanate (2 equiv) at 0 °C. The mixture was allowed to rise to
room temperature and stirred until the gas evolution stopped
(ꢁ2 h). The desired alcohol (1 equiv) in dimethylacetamide
(DMA) was added drop-wise to the resulting sulfamoyl chloride
at 0 °C. The mixture was stirred at 0 °C for 10 min and then at room
temperature for 3 h. The mixture was then poured into cold brine

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P. T. Cherian et al. / Bioorg. Med. Chem. 20 (2012) 4985–4994
and extracted with EtOAc. The combined extracts were washed
with brine, dried over Na2SO4 and concentrated in vacuo to provide
the desired sulfamate ester which was used directly in the next
step.
To a mixture of the obtained sulfamate ester (1 equiv), trieth-
ylamine (3 equiv) and N,N-dimethylpyridin-4-amine [DMAP]
(0.1 equiv) in anhydrous DCM:DMF (1:1) at 0 °C were added pal-
mitoyl chloride (1 equiv). The mixture was stirred at 0 °C for
10 min and then at room temperature overnight. The reaction
mixture was diluted with DCM and washed with 2.5% HCl, water
and brine, dried over Na2SO4 and concentrated in vacuo. The
crude mixture was separated by column chromatography as de-
scribed above.
4.3.8. Phenyl palmitoylsulfamate (8)
Synthesized with phenol. Yield = 70%. 1H NMR (400 MHz,
CDCl3) d 7.87 (br s, 1H), 7.47–7.27 (m, 5H), 2.40 (t, J = 7.5 Hz,
2H), 1.75–1.46 (m, 2H), 1.26 (br s, 24H), 0.88 (t, J = 6.3 Hz, 3H).
MS-ESI m/z = 410.73 [MꢂH]ꢂ.
4.3.9. 4-chlorophenyl palmitoylsulfamate (9)
Synthesized with 4-chlorophenol. Yield = 63%. 1H NMR
(400 MHz, CDCl3) d 7.84 (br s, 1H), 7.38 (d, 8.3 Hz, 2H), 7.25 (d,
J = 9.1 Hz, 2H), 2.40 (t, J = 7.4 Hz, 2H), 1.64 (q, J = 7.3 Hz, 2H), 1.26
(br s, 24H), 0.88 (t, J = 6.7 Hz, 3H). MS-ESI m/z = 444.55 [MꢂH]ꢂ.
4.3.10. 3-chlorophenyl palmitoylsulfamate (10)
Synthesized with 3-chlorophenol. Yield = 33%. 1H NMR
(400 MHz, CDCl3) d 7.40–7.30 (m, 3H), 7.21 (dt, J = 6.8, 2.3 Hz,
1H), 2.39 (t, J = 7.4 Hz, 2H), 1.79–1.50 (m, 2H), 1.25 (br s, 24H),
0.88 (t, J = 6.7 Hz, 3H). MS-ESI m/z = 444.20 [MꢂH]ꢂ.
4.3.1. Methyl palmitoylsulfamate (1)
Synthesized with methanol. Yield = 17%. 1H NMR (400 MHz,
CDCl3) d 7.95 (br s, 1H), 4.06 (s, 3H), 2.39 (t, J = 7.5 Hz, 2H), 1.66
(p, J = 7.5 Hz, 2H), 1.25 (br s, 24H), 0.88 (t, J = 6.8 Hz, 3H). MS-ESI
m/z = 348.22 [MꢂH]ꢂ
4.3.11. 3,4-dichlorophenyl palmitoylsulfamate (11)
Synthesized with 3,4-dichlorophenol. Yield = 32%. 1H NMR
(400 MHz, CDCl3) d 7.51 (d, J = 7.5 Hz, 1H), 7.44 (d, J = 7.4 Hz, 1H),
7.20 (d, J = 7.18 Hz, 1H), 2.39 (t, J = 7.4 Hz, 1H), 1.78–1.46 (m,
1H), 1.25 (br s, 24H), 0.88 (t, 6.5 Hz, 3H). MS-ESI m/z = 478.16
[MꢂH]ꢂ.
4.3.2. Cyclopentyl palmitoylsulfamate (2)
Synthesized with cyclopentanol. Yield = 9%. 1H NMR (400 MHz,
CDCl3) d 8.00 (br s, 1H), 5.38 (m, 1H), 2.41 (t, J = 7.3 Hz, 2H), 2.10–
1.75 (m, 6H), 1.74–1.58 (m, 4H), 1.27 (br s, 24H), 0.90 (t, J = 5.9 Hz,
3H). MS-ESI m/z = 402.48 [MꢂH]ꢂ.
4.3.12. p-Tolyl palmitoylsulfamate (12)
Synthesized with p-cresol. Yield = 65%. 1H NMR (400 MHz,
CDCl3) d 7.18 (q, J = 8.7 Hz, 4H), 2.50–2. 23 (m, 3H), 1.69–1.57
(m, 2H), 1.26 (br s, 24H), 0.88 (t, J = 6.8 Hz, 3H). MS-ESI m/
z = 424.29 [MꢂH]ꢂ.
4.3.3 Tetrahydrofuran-3-yl palmitoylsulfamate (3)
Synthesized with tetrahydrofuran-3-ol. Yield = 50%. 1H NMR
(400 MHz, CDCl3) d 7.98 (br s, 1H), 5.59 (t, J = 5.0 Hz, 1H), 4.10–
3.79 (m, 5H), 2.36 (t, J = 7.5 Hz, 2H), 2.30–2.16 (m, 2H), 1.74–1.61
(m, 3H), 1.26 (br s, 21H), 0.88 (t, J = 6.8 Hz, 3H). MS-ESI m/
z = 404.31 [MꢂH]ꢂ.
4.3.13. 4-Methoxyphenyl palmitoylsulfamate (13)
Synthesized with 4-methoxyphenol. Yield = 85%. 1H NMR
(400 MHz, CDCl3) d 7.20 (d, J = 9.2 Hz, 2H), 6.90 (d, J = 9.2 Hz, 2H),
3.81 (s, 2H), 2.40 (t, J = 7.5 Hz, 2H), 1.65 (dt, J = 14.6, 7.4 Hz, 2H),
1.26 (br s, 24H), 0.88 (t, J = 6.8 Hz, 3H). MS-ESI m/z = 440.23
[MꢂH]ꢂ.
4.3.4. Cyclohexyl palmitoylsulfamate (4)
Synthesized with cyclohexanol. Yield = 75%. 1H NMR (400 MHz,
CDCl3) d 7.99 (br s, 1H), 4.90 (dt, J = 8.9, 4.8 Hz, 2H), 2.38 (t, J = 6.9,
6.4 Hz, 3H), 2.03–1.93 (m, 2H), 1.83–1.73 (m, 2H), 1.73–1.47 (m,
6H), 1.47–1.15 (m, 24H), 0.88 (t, J = 6.8 Hz, 3H). MS-ESI m/
z = 416.29 [MꢂH]ꢂ.
4.3.14. Methyl 2-(4-(N-palmitoylsulfamoyloxy)phenyl)acetate
(14)
4.3.5. Tetrahydro-2H-pyran-4-yl palmitoylsulfamate (5)
Synthesized with tetrahydro-2H-pyran-4-ol. Yield = 63%. 1H
NMR (400 MHz, CDCl3) d 8.04 (br s, 1H), 5.22–5.13 (m, 1H),
4.02–3.90 (m, 2H), 3.59 (ddd, J = 11.7, 8.5, 3.2 Hz, 2H), 2.38
(t, J = 7.5 Hz, 2H), 213–2.04 (m, 2H), 1.97–1.86 (m, 2H), 1.68
(p, J = 7.3 Hz, 2H), 1.28 (s, 24H), 0.90 (t, J = 6.6 Hz, 3H). MS-ESI m/
z = 418.43 [MꢂH]ꢂ.
Synthesized
with
methyl
2-(4-hydroxyphenol)acetate.
Yield = 41%. 1H NMR (400 MHz, CDCl3) d 7.32 (d, J = 8.7 Hz, 2H),
7.24 (d, J = 8.7 Hz, 2H), 3.71 (s, 3H), 3.64 (s, 2H), 2.39 (t,
J = 7.5 Hz, 2H), 1.65 (dt, J = 14.9, 7.4 Hz, 2H), 1.26 (br s, 24H), 0.88
(t, J = 6.8 Hz, 2H). MS-ESI m/z = 482.32 [MꢂH]ꢂ.
4.3.15. Ethyl 4-(N-palmitoylsulfamoyloxy)benzoate (15)
Synthesized with ethyl 4-hydroxybenzoate. Yield = 51%. 1H
4.3.6. Piperidin-4-yl palmitoylsulfamate (6)
NMR (400 MHz, CDCl3)
d 8.10 (d, J = 8.8 Hz, 2H), 7.36 (d,
Compound (7) was stirred in a 10% solution of Trifluoroacetic
acid in DCM for 30 min and the solvent evaporated to afford (6).
1H NMR (400 MHz, CDCl3) d 5.16–5.04 (m, 1H), 3.34–3.16 (m,
4H), 2.35–2.24 (m, 1H), 2.24–2.06 (m, 3H), 1.62 (p, J = 7.9 Hz,
2H), 1.25 (br s, 24H), 0.88 (t, J = 5.7 Hz, 2H). MS-ESI m/z = 417.42
[MꢂH]ꢂ.
J = 8.8 Hz, 2H), 4.39 (q, J = 7.1 Hz, 2H), 2.40 (t, J = 7.4 Hz, 2H),
1.69–1.60 (m, 2H), 1.40 (t, J = 7.1 Hz, 3H), 1.25 (br s, 24H), 0.88
(t, J = 5.8 Hz, 3H). MS-ESI m/z = 482.42 [MꢂH]ꢂ.
4.3.16. 4-(2-Aminoethyl)phenyl palmitoylsulfamate (16)
Compound (17) was stirred in neat Trifluoroacetic acid (TFA)
for 12 h. The TFA was evaporated and the crude mixture dissolved
in minimal amount of acetone. The product was precipitated by
addition of water and then filtered and dried under vacuum to
provide (16) in 37% yield. 1H NMR (400 MHz, DMSO) d 7.76 (br
s, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.3 Hz, 2H), 3.13–2.92
(m, 2H), 2.89–2.71 (m, 2H), 1.95 (t, J = 7.4 Hz, 3H), 1.48–1.34
(m, 4H), 1.24 (s, 24H), 0.86 (t, J = 6.6 Hz, 3H). MS-ESI m/
z = 453.17 [MꢂH]ꢂ.
4.3.7. tert-butyl 4-(N-palmitoylsulfamoyloxy)piperidine-1-
carboxylate (7)
Synthesized with t-butyl 4-hydroxypiperidine-1-carboxylate.
Yield = 38%. 1H NMR (400 MHz, CDCl3) d 8.49 (br s, 1H), 5.21–
5.06 (m, 1H), 3.72–3.61 (m, 2H), 3.42–3.32 (m, 4.6 Hz, 2H), 2.39
(t, J = 6.5 Hz, 2H), 2.03–1.92 (m, 2H), 1.92–1.80 (m, 2H), 1.75–
1.59 (m, 2H), 1.48 (s, 9H), 1.28 (br s, 24H), 0.90 (t, J = 5.9 Hz, 3H).
MS-ESI m/z = 517.38 [MꢂH]ꢂ.

P. T. Cherian et al. / Bioorg. Med. Chem. 20 (2012) 4985–4994
4993
4.3.17. 4-(2-(tert-Butoxycarbonylamino)ethyl)phenyl
palmitoylsulfamate (17)
4.4.3. Phenyl (11-phenylundecanoyl) sulfamate (24)
Synthesized with 11-phenylundecanoic acid. Yield = 31%. 1H
NMR (400 MHz, CDCl3) d 7.43–7.32 (m, 2H), 7.32–7.20 (m, 5H),
7.19–7.11 (m, 3H), 2.78–2.15 (m, 3H), 1.72–1.39 (m, 5H), 1.17
(dd, J = 14.56, 42.81 Hz, 8H), 0.94–0.65 (m, 2H). MS-ESI m/
z = 416.22 [MꢂH]ꢂ.
Synthesized with t-butyl 4-hydroxyphenethylcarbamate.
Yield = 54%. 1H NMR (400 MHz, DMSO) d 7.30 (d, J = 8.5 Hz, 2H),
7.15 (d, J = 8.5 Hz, 2H), 3.13 (q, J = 6.5 Hz, 2H), 2.71 (t, J = 7.5 Hz,
2H), 2.26 (t, J = 7.2 Hz, 2H), 1.49 (dt, J = 12.5, 5.7 Hz, 2H), 1.24 (br
s, 24H), 0.86 (t, J = 6.5 Hz 3H). MS-ESI m/z = 553.29 [MꢂH]ꢂ.
4.4.4. Phenyl (8-phenyloctanoyl) sulfamate (25)
4.3.18. 40-Nitrobiphenyl-4-yl palmitoylsulfamate (18)
Synthesized with 4’-nitrobiphenyl-4-ol. Yield = 65%. 1H NMR
(400 MHz, CDCl3) d 8.32 (d, J = 8.8 Hz, 2H), 7.68 (dd, J = 17.4,
8.7 Hz, 4H), 7.44 (d, J = 8.7 Hz, 2H), 2.43 (t, J = 7.5 Hz, 2H), 1.86–
1.62 (m, 2H), 1.36–1.16 (m, 24H), 0.88 (t, J = 6.7 Hz, 3H). MS-ESI
m/z = 531.24 [MꢂH]ꢂ.
Synthesized with 8-phenyloctanoic acid. Yield = 40%. 1H NMR
(400 MHz, CDCl3) d 7.42–7.35 (m, 2H), 7.35–7.29 (m, 1H), 7.29–
7.26 (m, 4H), 7.20–7.12 (m, 3H), 2.58 (td, J = 8.0, 3.6 Hz, 2H), 2.34
(td, J = 7.6, 3.0 Hz, 2H), 1.59 (br s, 4H), 1.29 (br s, 6H). MS-ESI m/
z = 374.09 [MꢂH]ꢂ.
4.4.5. Phenyl (7-phenylheptanoyl) sulfamate (26)
4.3.19. Benzo[d][1,3]dioxol-5-yl palmitoylsulfamate (19)
Synthesized with Benzo[d][1,3]dioxol-5-ol. Yield = 62%. 1H NMR
Synthesized with 7-phenylheptanoic acid. Yield = 64%. 1H NMR
(400 MHz, CDCl3) d 7.45–7.37 (m, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.28
(d, J = 5.4 Hz, 3H), 7.17 (t, J = 8.2 Hz, 3H), 2.59 (t, J = 7.7 Hz, 2H),
2.34 (dt, J = 15.2, 7.5 Hz, 2H), 1.62 (q, J = 8.5, 7.9 Hz, 4H), 1.34 (dq,
J = 7.3, 4.4, 3.6 Hz, 4H). MS-ESI m/z = 360.13 [MꢂH]ꢂ.
(400 MHz, CDCl3)
d 6.87–6.65 (m, 3H), 6.02 (s, 2H), 2.41
(t, J = 7.4 Hz, 2H), 1.67–1.62 (m, 2H), 1.25 (br s, 24H), 0.88
(t, J = 5.9 Hz, 3H). MS-ESI m/z = 454.32 [MꢂH]ꢂ.
4.3.20. Quinolin-6-yl palmitoylsulfamate (20)
4.4.6. Phenyl (6-phenylhexanoyl) sulfamate (27)
Synthesized with quinolin-6-ol. Yield = 21%. 1H NMR (400 MHz,
DMSO) d 8.88 (dd, J = 4.2, 1.7 Hz, 1H), 8.38 (dd, J = 8.4, 1.6 Hz, 1H),
8.02 (d, J = 9.1 Hz, 1H), 7.79 (d, J = 2.6 Hz, 1H), 7.61 (dd, J = 9.1,
2.6 Hz, 1H), 7.54 (dd, J = 8.3, 4.2 Hz, 1H), 2.07 (t, J = 7.3 Hz, 2H),
1.40 (p, J = 7.1 Hz, 2H), 1.33–1.06 (m, 24H), 0.86 (t, J = 6.8 Hz,
3H). MS-ESI m/z = 461.30 [MꢂH]ꢂ.
Synthesized with 6-phenylhexanoic acid. Yield = 74%. 1H NMR
(400 MHz, CDCl3) d 7.39 (dt, J = 8.1, 4.1 Hz, 2H), 7.35–7.30 (m,
2H), 7.30–7.26 (m, 3H), 7.16 (t, J = 6.0 Hz, 3H), 2.59 (t, J = 7.5 Hz,
2H), 2.43–2.23 (m, 2H), 1.62 (dq, J = 14.8, 7.5 Hz, 4H), 1.46–1.28
(m, 2H). MS-ESI m/z = 346.11 [MꢂH]ꢂ.
4.4.7. (E)-Phenyl 3-(biphenyl-4-yl)acryloylsulfamate (28)
Synthesized with 4-phenylcinnamic acid. Yield = 11%. 1H NMR
(400 MHz, CDCl3) d 7.87 (d, J = 15.6 Hz, 1H), 7.63–7.60 (m, 6H),
7.46 (q, J = 8.3, 7.8 Hz, 2H), 7.39 (t, J = 6.7 Hz, 3H), 7.37–7.30 (m,
3H), 6.56 (d, J = 15.6 Hz, 1H). MS-ESI m/z = 378.13 [MꢂH]ꢂ.
4.3.21. 3-(Sulfamoyloxy)benzofuran-6-yl palmitoylsulfamate
(21)
Synthesized with 6-hydroxybenzofuran-3(2H)-one. Yield = 2%.
1H NMR (400 MHz, DMSO) d 8.25 (br s, 1H), 8.06 (s, 1H), 7.65 (d,
J = 8.6 Hz, 1H), 7.58 (d, J = 1.7 Hz, 1H), 7.28 (dd, J = 8.6, 1.9 Hz,
1H), 2.20 (t, J = 7.1 Hz, 2H), 1.50–1.41 (m, 2H), 1.31–1.13 (m,
24H), 0.86 (t, J = 6.8 Hz, 3H). MS-ESI m/z = 545.23 [MꢂH]ꢂ.
4.5. Membrane preparation and PlsY assay
PlsY is an intrinsic membrane protein and was expressed and
assayed in
FB23281 (
a
native membrane environment. E. coli strain
4.4. General procedure for the synthesis of 22–28
D
plsY) was used as the host for plasmids expressing
either the S. aureus PlsY (SA1187) or B. anthracis PlsY1
(BAS3399). B. anthracis has 3 PlsY homologs, and PlsY1 was the
most similar to the single prototypical single PlsY proteins ex-
pressed in Gram-positive bacteria. Membranes were purified and
acyltransferase activity was measured as described previously.5
Briefly, the reaction buffer (pH 7.4) contained 100 mM Tris–HCl,
To a solution of the desired carboxylic acid (1 equiv) in anhy-
drous DCM containing 2 drops of DMF was added oxalyl chloride
(1 equiv) at 0 °C. The mixture was stirred at 0 °C for 15 min and
then at room temperature for 30 min. The solution was then added
to a mixture of DMAP (0.1 equiv), triethylamine (3 equiv) and
either 4-methoxyphenyl sulfamate (1 equiv) (22–23) or Phenyl
sulfamate (24–28) in anhydrous DCM/DMF (1:1). After stirring
overnight, the reaction mixture was extracted with 2.5% HCl, H2O
and brine and dried with Na2SO4. The organic layer was evaporated
under vacuum and the crude mixture separated by column
chromatography as described above.
150 mM NaCl, 1 mg/mL BSA, 5 mM Na3VO4, 100
erol-PO4 (8 mCi/mmol), inhibitor (200 M) and 4
branes. 16:0-PO4 (final concentration 100 M) was added to start
the reaction. Reactions were terminated after incubation at 37 °C
for 20 min by aliquotting the reaction mixture (20 L) onto a
l
M [U-14C]glyc-
lg purified mem-
l
l
l
Whatman 3 mm cellulose filter disc. Filter discs were washed in
10%, 5%, and 1% ice-cold trichloroacetic acid (20 min, 20 mL/disk)
to remove unreacted glycerol-PO4 prior to scintillation counting.
4.4.1. 4-Methoxyphenyl tetradecanoylsulfamate (22)
Synthesized with Myristic acid. Yield = 61%. 1H NMR
(400 MHz, CDCl3) d 7.78 (br, 1H), 7.20 (d, J = 9.1 Hz, 2H), 6.89
(d, J = 9.1 Hz, 2H), 3.81 (s, 3H), 2.40 (t, J = 7.5 Hz, 2H), 1.69–
1.59 (m, 2H), 1.26 (br s, 20H), 0.88 (t, J = 6.7 Hz, 3H). MS-ESI
m/z = 412.25 [MꢂH]ꢂ.
4.6. MIC determinations
The MIC of each test compound was determined by the micro-
broth dilution method in Mueller–Hinton (MH) media according to
the Clinical Laboratory Standards Institute (CLSI) document M7A7
for testing of the antibiotic susceptibility of aerobic bacteria. For
growth of S. pneumoniae and S. pyogenes, MH broth was supple-
mented with 5% lysed horse blood from BD Diagnostic Systems
(Loveton Circle, Sparks, MD, USA). All test compounds were dis-
solved in DMSO at a concentration of 10 mg/mL and stored at
ꢂ80 °C. Twofold serial dilutions of test compound were prepared
in MH broth in 96-well plates to give drug concentrations that
4.4.2. 4-Methoxyphenyl undec-10-enoylsulfamate (23)
Synthesized with undec-10-enoic acid. Yield = (62%). 1H NMR
(400 MHz, CDCl3)
d 7.23–7.16 (m, 2H), 6.90 (dd, J = 2.12,
7.01 Hz, 2H), 5.81 (ddt, J = 6.66, 10.24, 16.89 Hz, 1H), 5.08–4.82
(m, 2H), 3.81 (s, 3H), 2.54–2.33 (m, 2H), 2.04 (q, J = 6.74 Hz,
2H), 1.77–1.46 (m, 5H), 1.46–1.11 (m, 12H). MS-ESI m/
z = 367.94 [MꢂH]ꢂ.

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P. T. Cherian et al. / Bioorg. Med. Chem. 20 (2012) 4985–4994
ranged from 400–0.025
l
g/mL. Bacterial inoculums were prepared
4.9. ACP immunoblotting
by streaking at ꢂ80 °C stock bacterial culture onto an MH agar
plate, which was incubated overnight at 37 °C. A single colony is
picked into 10 ml of MH broth and incubated in 37 °C shaking incu-
bator. The culture is grown to mid-log phase (OD600 = 0.5) and then
Cultures of S. aureus RN4220 cells were grown to OD600 = 0.5,
and then split into 50 mL aliquots. Appropriate concentrations of
inhibitors were added to the aliquots and grown at 37 °C for
30 min and the OD600 was collected. Cells were harvested,
extracted and immunoblotted to determine the ACP species as
described previously.10 The amount of supernatant loaded is
adjusted to OD600 such that a similar amount of total protein is
loaded for each drug treated sample.
diluted to OD600 = 0.001. An aliquot of culture (100
lL) was then
added to each well of the 96-well plate to give an
OD600 = 0.0005, which corresponded to about 105 CFU/mL, and fi-
nal antibiotic concentrations that ranged from 200–0.0125 lg/
mL. The 96-well plates were incubated overnight at 37 °C, and
the MIC was recorded as the lowest concentration of drug that
inhibited 90% of visible bacterial growth. The MIC for B. anthracis
was determined as previously described.15,16
4.10. Gas chromatography and mass spectrometry
S. aureus was grown in Luria–Bertani broth at 37 °C with rigor-
4.7. Acetate labeling
ous shaking (225 rpm). Cells (250 mL) were grown to OD600 of 0.5,
aliquoted to 50 mL cultures, and treated with no inhibitor, 25 lM,
[
14C]Acetate incorporation experiments were conducted to
or 50 lM of 25 for 2.5 hours. Cells were washed with PBS and
measure lipid metabolism activity in S. aureus or B. anthracis. For
both bacteria, the culture was grown overnight and the starter cul-
ture was back diluted to an initial OD600 of 0.1 and grown until
OD600 of 0.5. The culture was split into 10 mL aliquots and each ali-
quot was incubated with the appropriate concentration of inhibi-
tors or DMSO (control) for 15 min and the OD600 of the culture
harvested for lipids using the method of Bligh and Dyer.17 Gas
chromatography was performed on S. aureus strain RN4220 as de-
scribed.10 The PtdGro molecular species were also analyzed by
mass spectrometry as described previously.10
Acknowledgements
was collected. [14C]Acetate (10
lCi) was added to each aliquot for
30 min. The cells are harvested by centrifugation and washed 4
times with phosphate-buffered saline. Pelleted cells are then lysed
to harvest for lipids using the method of Bligh and Dyer.17 The
radioactivity from the lipid extraction was counted on a LS6500
Multipurpose Scintillation Counter and normalized to the OD600
of the cell culture. All measurements were made in duplicate and
the averages with standard error were reported. The radioactive li-
pid extract was also separated on Silica Gel G layers developed
with CHCl3/methanol/acetic acid (98:2:1) to separate the different
lipid species. The radioactivity of the plate was counted using the
Bioscan imaging system.
We thank Caroline Pate for her expert technical assistance. This
research was supported by National Institutes of Health Grants
AI079653 and GM034496, Cancer Center (CORE) Support Grant
CA21765 and the American Lebanese Syrian Associated Charities.
The content is solely the responsibility of the authors and does
not necessarily represent the official views of the National Institute
of General Medical Sciences or the National Institutes of Health.
References and notes
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4.8. Pathway Labeling
The method for protein, DNA, and RNA pathway labeling was
similar to acetate labeling experiments. Cells were grown to
OD600 of 0.5 before being split to 10 mL aliquots and incubated
with appropriate inhibitors for 15 min. Then, either a 3H-labled
amino acid mix (10
midine (10
Ci) for DNA pathway metabolism, or [3H]uracil
(10 Ci) for stable RNA synthesis was added to the culture and
grown for 30 min. The labeled cells are then collected via vacuum
filtration through 0.45 m HA filters, and the radioactivity was
l
Ci) for protein pathway metabolism, [3H]thy-
l
l
l
counted and normalized to the OD600 of the culture. The cell den-
sity adjusted activity of the metabolic pathways from cells treated
with inhibitor is divided by the metabolic activity of the untreated
cells to determine the effect of the inhibitor on the metabolic path-
ways. Duplicated measures were made with averages and standard
error were reported.