Unsymmetric aryl-alkyl disulfide growth inhibitors of methicillin-resistant Staphylococcus aureus and Bacillus anthracis

Article abstract of DOI:10.1016/j.bmc.2008.05.032

This study describes the antibacterial properties of synthetically produced mixed aryl-alkyl disulfide compounds as a means to control the growth of Staphylococcus aureus and Bacillus anthracis. Some of these compounds exerted strong in vitro bioactivity. Our results indicate that among the 12 different aryl substituents examined, nitrophenyl derivatives provide the strongest antibiotic activities. This may be the result of electronic activation of the arylthio moiety as a leaving group for nucleophilic attack on the disulfide bond. Small alkyl residues on the other sulfur provide the best activity as well, which for different bacteria appears to be somewhat dependent on the nature of the alkyl moiety. The mechanism of action of these lipophilic disulfides is likely similar to that of previously reported N-thiolated β-lactams, which have been shown to produce alkyl-CoA disulfides through a thiol-disulfide exchange within the cytoplasm, ultimately inhibiting type II fatty acid synthesis. However, the mixed alkyl-CoA disulfides themselves show no antibacterial activity, presumably due to the inability of the highly polar compounds to cross the bacterial cell membrane. These structurally simple disulfides have been found to inhibit β-ketoacyl-acyl carrier protein synthase III, or FabH, a key enzyme in type II fatty acid biosynthesis, and thus may serve as new leads to the development of effective antibacterials for MRSA and anthrax infections.

Full text of DOI:10.1016/j.bmc.2008.05.032

Bioorganic & Medicinal Chemistry 16 (2008) 6501–6508
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Unsymmetric aryl–alkyl disulfide growth inhibitors of methicillin-resistant
Staphylococcus aureus and Bacillus anthracis
b
a
a
a
Edward Turos a, , Kevin D. Revell , Praveen Ramaraju , Danielle A. Gergeres , Kerriann Greenhalgh ,
Ashley Young a, Nalini Sathyanarayan b, Sonja Dickey c, Daniel Lim c, Mamoun M. Alhamadsheh d,
Kevin Reynolds d
*
a Center for Molecular Diversity in Drug Design, Discovery, and Delivery, Department of Chemistry, 4202 East Fowler Avenue, CHE 205, University of South Florida, Tampa,
FL 33620, USA
b Department of Chemistry, Murray State University, 456 Blackburn, Murray, KY 42071, USA
c Department of Biology, 4202 East Fowler Avenue, IDRB 404, University of South Florida, Tampa, FL 33620, USA
d Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 February 2008
Revised 8 May 2008
Accepted 14 May 2008
Available online 17 May 2008
This study describes the antibacterial properties of synthetically produced mixed aryl–alkyl disulfide
compounds as a means to control the growth of Staphylococcus aureus and Bacillus anthracis. Some of
these compounds exerted strong in vitro bioactivity. Our results indicate that among the 12 different aryl
substituents examined, nitrophenyl derivatives provide the strongest antibiotic activities. This may be
the result of electronic activation of the arylthio moiety as a leaving group for nucleophilic attack on
the disulfide bond. Small alkyl residues on the other sulfur provide the best activity as well, which for
different bacteria appears to be somewhat dependent on the nature of the alkyl moiety. The mechanism
of action of these lipophilic disulfides is likely similar to that of previously reported N-thiolated b-lac-
tams, which have been shown to produce alkyl-CoA disulfides through a thiol-disulfide exchange within
the cytoplasm, ultimately inhibiting type II fatty acid synthesis. However, the mixed alkyl-CoA disulfides
themselves show no antibacterial activity, presumably due to the inability of the highly polar compounds
to cross the bacterial cell membrane. These structurally simple disulfides have been found to inhibit
b-ketoacyl-acyl carrier protein synthase III, or FabH, a key enzyme in type II fatty acid biosynthesis,
and thus may serve as new leads to the development of effective antibacterials for MRSA and anthrax
infections.
Keywords:
Disulfides
MRSA
Bacillus anthracis
Lipid biosynthesis
FabH protein
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
O
O
SR
R'
SR
N
O
N
The design of new antimicrobial agents has reached unprece-
dented urgency as drug resistance in certain medically relevant
bacteria continues to climb. Our laboratory has recently been
investigating the microbiological properties of N-alkylthio b-lac-
tams1–9 and N-alkylthio-2-oxazolidinones10 (Fig. 1) as potential
antibiotics for treatment of infections caused by methicillin-resis-
tant Staphylococcus aureus (MRSA) and Bacillus anthracis (the caus-
ative agent of anthrax).
These compounds exert selective bacteriostatic properties
against Staphylococcus and Bacillus microbes over most other com-
mon bacterial genera, and demonstrate unique structure–activity
profiles never seen before for b-lactam and 2-oxazolidinone antibi-
otics. One of the structural requirements of these compounds is
that the residues on the heterocyclic ring and sulfur side chain
R''
R''
-thiolated oxazolidinones
Figure 1. Antibacterially active sulfenylating agents.
R'
N
-thiolated β-lactams
N
must be highly lipophilic in order to bestow the most potent anti-
bacterial activities. Most recently our studies have determined that
N-methylthio b-lactams block type II fatty acid biosynthesis in
S. aureus through the initial transfer of the N-alkylthio moiety from
the ring nitrogen onto a cellular target.9 We have since identified
this target as being coenzyme A (CoASH), which is present in large
quantities in the cytoplasm and comprises the redox buffer system
of these bacteria. It is postulated that sulfenylation of coenzyme A
produces an alkyl-CoA disulfide (CoASSR) responsible for inhibiting
lipid biosynthesis (Scheme 1).
* Corresponding author. Tel.: +1 813 974 7312; fax: +1 813 974 1733.
E-mail address: eturos@cas.usf.edu (E. Turos).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.032

6502
E. Turos et al. / Bioorg. Med. Chem. 16 (2008) 6501–6508
nase, and reversibly inhibits acetyl-CoA synthetases.34,35 This cer-
O
SR
R'
O
N
NH
R'
CoASH
tainly suggests similar modes of action in causing intracellular
sulfenylation of their biological targets. A survey of the patent lit-
erature also uncovered two recent US patent applications describ-
ing the effects of mixed alkyl disulfides as inhibitors of redox
buffers in mammalian cells as a means to restore normal cellular
function, a feature attributed to their high reactivity toward
thioredoxin.36,37
CoASSR
+
R''
R''
Scheme 1. Sulfenylation of coenzyme A by N-thiolated b-lactams.
Recent work in the Reynolds laboratory has demonstrated that
these mixed alkyl-CoA disulfides, CoASSR, covalently modify the
fatty acid biosynthesis protein, FabH, and that the inhibitory effects
are highly dependent on the nature of the alkyl side chain R.11 In
our initial investigations, we examined the possibility that the
CoASSR disulfides could directly inhibit microbial growth if fed
to bacteria growing in culture media. However, incubation of S.
aureus cells with CoASSMe does not lead to growth inhibition, as
judged by both Kirby–Bauer well diffusion studies and minimum
inhibitory concentration assays in broth. Although this seemed
surprising at first, given that this compound is likely responsible
for bioactivity in the cell, this suggests that the multiply charged
CoA moiety may not easily traverse the cell membrane to be able
to exert an inhibitory effect within the cytoplasm.12 This led to
the realization that the N-thiolated b-lactam, and presumably the
N-thiolated oxazolidinone as well, may have the requisite cell per-
meability and sulfenylation capabilities for antibacterial activity,
and could generate the CoASSR adduct intracellularly. Since it
was apparent that the lactam or the oxazolidinone rings did not
have any special structural features needed for activity, other than
being relatively lipophilic and able to serve as a leaving group dur-
ing nucleophilic attack on sulfur, we thought that we could utilize
lipophilic disulfides in place of the N-thiolated b-lactams as a
means to deliver electrophilic sulfur species more effectively into
the bacterial cell. In fact, nature has long used disulfides and trisul-
fides as natural sulfenylation agents, as evidenced by the large
number of naturally occurring compounds that have anti-infective
properties.13 Highly notable examples include diallyl disulfide,
diallyl trisulfide, ajoene, S-allylmercaptocysteine, and allicin, found
only in freshly crushed garlic (Fig. 2).14–17 The biological targets
and mode of action of most of these compounds are not precisely
known, but in the case of allicin, there are distinct similarities in
the antibiotic properties to the N-thiolated b-lactams (and N-thio-
lated oxazolidinones). In addition to exhibiting activity against a
wide range of bacteria,18–21 allicin possesses antifungal,22–24 anti-
viral,25,26 antiparasitic,27,28 and anticancer properties. Both the lac-
tams and allicin strongly inhibit fatty acid synthesis,29–32 but also
partially inhibit protein and nucleic acid biosynthesis.33 Both com-
pounds exert their strongest antibacterial activities against mi-
crobes such as S. aureus and B. anthracis that express unusually
low levels of glutathione and relatively high levels of coenzyme
A. Allicin reacts with sulfhydryl residues of various proteins such
as RNA polymerase, thioredoxin reductase, and alcohol dehydroge-
2. Results and discussion
To initiate these investigations, a selection of unsymmetrical
aryl methyl disulfides was synthesized from commercially avail-
able arylthiols as shown in Scheme 2. We selected a group of elec-
tronically diverse arylthiols as coupling partners in this reaction to
obtain aryl methyl disulfides 1–8 for evaluation. The synthesis was
done simply by combining a methanolic solution of the arylthiol
(100 ml of 1.0 M) with a slight excess of the alkyl methanethiol-
sulfonate (105 ml of 1.0 M methanolic solution) and stirring under
an inert atmosphere for 1 h. The solutions were then treated with a
small amount (about 0.3 equiv) of cysteine hydrochloride to con-
sume the excess methanethiolsulfonate. The solution was concen-
trated and taken up in dichloromethane (1 ml), filtered to remove
any cysteine products, treated with Amberlyst 21 resin (weakly ba-
sic), then flushed through a small silica plug to remove impurities.
In most instances, this yielded products >90% pure by 1H NMR.
Yields for these reactions were in most cases high and the disul-
fides were used in microbiological screening assays without fur-
ther purification. We carried out initial antibacterial assays on
these eight aryl methyl disulfides against methicillin-susceptible
S. aureus by Kirby–Bauer disk diffusion on agar plates, according
to National Committee for Clinical Laboratory Standards guide-
lines.38 In each case, 20 lg of the test compound in DMSO was ap-
plied to 6-mm wide wells bored directly into the agar, prior to
inoculation and incubation. The average zones of growth inhibition
(from three trials) produced by the compounds after 24 h of incu-
bation at 37 °C are presented in Table 1. The Kirby–Bauer data indi-
cate that most of the disulfides have weak or no antimicrobial
activity, which suggests that the disulfide bond itself is not enough
of a prerequisite to induce antibacterial activity. Of these eight
compounds, the p-nitrophenyl analogue 8 showed the strongest
S-SMe
SH
MeSSO2Me
MeOH
X
X
1-8
SSCH3
SSCH3
SSCH3
H3C
F
CH3
OCH2CH3
F
S
S
S
S
S
3
2
1
diallyl disulfide
diallyl trisulfide
SSCH3
OH
SSCH3
SSCH3
O
S
N
S
AcHN
S
4
5
6
ajoene
NH2
SSCH3
SSCH3
O
S
S
S
S
H2N
O2N
COOH
allicin
S-allylmercaptocysteine
8
7
Figure 2. Naturally occurring disulfide and trisulfide antibacterials from garlic.
Scheme 2. Synthesis of aryl methyl disulfides 1–8.

E. Turos et al. / Bioorg. Med. Chem. 16 (2008) 6501–6508
6503
Table 1
considerably more active than the o-nitro analogues 10. The en-
hanced bioactivity of these nitrophenyl disulfides compared to
other aryl-substituted analogues suggests that nucleophilic scis-
sion of the disulfide linkage is enhanced by strong electron defi-
ciency on the sulfur centers.
Zones of bacterial growth inhibition data from Kirby–Bauer testing of disulfides 1–8
against S. aureus (ATCC 25923) on agar plates
1
2
3
4
5
6
7
8
Diameter of zone (mm)
11
10
9
14
23
20
0
35
These data revealed that increasing the alkyl chain length of the
aryl–alkyl disulfide from methyl to ethyl to propyl to butyl in-
creased bioactivity somewhat, but not as much as introducing
branching in the chain (Table 2). Thus, the isopropyl disulfides
and sec-butyl, 8c and 8d, respectively, were considerably more ac-
tive than the unbranched side-chain analogues. Additionally, all of
the compounds appeared to be somewhat more active against S.
aureus than MRSA, which is opposite to what we observed previ-
ously for the N-thiolated b-lactams.7 We do not believe that this
difference has anything to do with penicillinase production in
MRSA, since repeating the testing of S. aureus in the presence of
penicillinase protein does not cause a reduction in bioactivity.
The most active series of compounds, p-nitrophenyl disulfides
8a–f, was also tested against Escherichia coli (K12 strain, ATCC
23590) (Table 3). With the lowest MICs being around 16 lg/ml,
none of these derivatives were appreciably active against E. coli.
However, it is interesting that the methyl and ethyl disulfides pos-
sess the strongest activity, which drops off dramatically as the al-
kyl chain is elongated or branched. This is opposite to that
observed for S. aureus and B. anthracis.
in vitro activity against S. aureus, with zones sizes comparable to
some of our previously reported N-thiolated b-lactam com-
pounds.3–8
We then expanded on the structure–activity properties of these
compounds by making structural modifications to the most potent
one, aryl disulfide 8. These include changing the location of the
electron-withdrawing nitro group on the phenyl ring, as well as
the length and branching of the S-alkyl substituent. The same pro-
cedure shown in Scheme 1 was used to prepare m-nitrophenyl
disulfides 9 and o-nitrophenyl disulfides 10, employing the corre-
sponding alkylthio sulfonates as sulfenylating agents.
Antimicrobial screening was done by Kirby–Bauer testing as
well as by determining the minimum inhibitory concentration
(MIC) values of compounds 8–10 by agar serial dilution. The data
shown in Table 2 indicate that all three nitrophenyl disulfide sys-
tems were active against S. aureus and B. anthracis, but it appeared
that p-nitrophenyl disulfides 8 and m-nitro isomers 9 were both
Table 2
Zones of inhibition (on agar plates) and MIC values (in broth) for aryl disulfides 8a–f, 9a–d, and 10a–d against S. aureus, MRSA, and B. anthracis
S-SR
S-SR
S-SR
O2N
NO2
NO2
8a-f
9a-d
10a-d
Compound
Alkyl R
Methyl
S. aureus (ATCC 25923)
MRSA (ATCC 43300)
B. anthracis
8a
35 mm
23 mm
54 mm
1 lg/ml
67 mm
0.25 lg/ml
71 mm
0.125 lg/ml
62 mm
0.5 lg/ml
57 mm
1 lg/ml
52 mm
1 lg/ml
43 mm
8 lg/ml
40 mm
8 lg/ml
54 mm
1 lg/ml
41 mm
8 lg/ml
31 mm
16 lg/ml
35 mm
16 lg/ml
27 mm
32 lg/ml
23 mm
32 lg/ml
nt
16 lg/ml
54 mm
1 lg/ml
85 mm
<0.125 lg/ml
68 mm
0.8 lg/ml
43 mm
16 lg/ml
53 mm
1 lg/ml
36 mm
16 lg/ml
45 mm
8 lg/ml
85 mm
<0.125 lg/ml
45 mm
8 lg/ml
37 mm
16 lg/ml
40 mm
16 lg/ml
31 mm
16 lg/ml
24 mm
32 lg/ml
33 mm
0.03 lg/ml
27 mm
32 lg/ml
38 mm
16 lg/ml
75 mm
<0.125 lg/ml
54 mm
1.0 lg/ml
38 mm
16 lg/ml
28 mm
32 lg/ml
15 mm
64 lg/ml
12 mm
64 lg/ml
38 mm
16 lg/ml
22 mm
32 lg/ml
26 mm
32 lg/ml
28 mm
32 lg/ml
20 mm
32 lg/ml
20 mm
32 lg/ml
14 mm
64 lg/ml
22 mm
8b
Ethyl
8c
Isopropyl
sec-Butyl
n-Propyl
n-Butyl
Methyl
Ethyl
8d
8e
8f
9a
9b
9c
Isopropyl
sec-Butyl
Methyl
Ethyl
9d
10a
10b
10c
Isopropyl
sec-Butyl
10d
Penicillin G
Vancomycin
nt
nt
0.25 lg/ml
0.5 lg/ml
nt
The top value for each compound and microbe is the average diameter of growth inhibition (3 trials) produced after 24 h of incubation using 20 lg of test compound (in
DMSO). The lower value is the minimum inhibitory concentration (MIC) of drug (in lg/ml), the lowest concentration of compound in the agar where bacterial growth is
completely inhibited. MIC values were determined by serial dilution in 24-well plates according to NCCLS protocols.38 nt = not tested.

6504
E. Turos et al. / Bioorg. Med. Chem. 16 (2008) 6501–6508
Table 3
illustrating the need for both a disulfide linkage (in the case of
inactive compound 13) and high lipophilicity (in the case of inac-
tive disulfide 14) for antimicrobial activity (Fig. 4).
Comparison of the minimum inhibitory concentration (MIC) values of p-nitrophenyl
alkyl disulfides 8a–f against E. coli in broth
Compound
E. coli (ATCC 23590)
In addition to the in vitro properties, we were interested to
determine if the antibacterial activity of the disulfides could be
due to inhibition of a key enzymatic step in lipid biosynthesis in
bacteria. Given our recent finding that N-thiolated lactams react
with coenzyme A to create a mixed CoA disulfide and inhibit fatty
acid biosynthesis, we decided to investigate whether the disulfides
could directly inhibit b-ketoacyl-acyl carrier protein synthase III, or
FabH, a key enzyme in type II fatty acid biosynthesis. Thus, addi-
tional experiments using compounds 8a–f to determine inhibition
capabilities against purified E. coli FabH verify that the disulfides
are highly active against this enzyme. This was carried out as de-
scribed previously.39 Each compound in this series showed >90%
inhibition of E. coli FabH at a disulfide concentration of 5 lM (Table
4).
For comparison, N-alkylthio b-lactams 15a and 15b were also
tested, and both of these thiolating lactams exhibited inhibitory
activity against purified E. coli FabH (Fig. 5). At 1 lM, methylthio
lactam 15a inhibits FabH catalytic activity by 89%, while sec-butyl-
thio lactam 15b induces a 30% decrease in activity. These assays
were done in the absence of coenzyme A, thus showing that the
compounds directly act on FabH protein.
8a
8b
8c
8d
8e
8f
16 lg/ml
16 lg/ml
96 lg/ml
128 lg/ml
64 lg/ml
96 lg/ml
The need for an aryl moiety directly on one of the sulfurs was
made apparent when we tested p-nitrobenzyl and p-nitropheneth-
yl disulfides, 11 and 12 (Fig. 3). Neither of these compounds
showed antibacterial activity against S. aureus, but had very weak
activities against B. anthracis (MICs of 32 and 64 lg/ml, respec-
tively). Thus, insertion of a methylene or ethylene group between
the aryl ring and sulfur center dramatically reduces antibacterial
activity, probably due to significantly lower reactivity of the disul-
fide toward nucleophilic displacement.
Likewise, the corresponding sulfide 13 and Ellman’s reagent
(14) were both found to be effectively inactive against S. aureus,
These results provide the first evidence that the disulfides and
the N-alkylthio b-lactams can inhibit bacterial growth in the same
manner, directly through FabH inactivation. However, once inside
the bacterial cell, the disulfides and N-alkylthio b-lactams can indi-
rectly inhibit FabH catalysis by readily forming alkyl-CoA mixed
disulfide adducts, which in turn can impede FabH function. Indeed,
Reynolds has previously reported that methyl-CoA mixed disulfide
effectively inhibits E. coli-expressed FabH (IC50 = 57 lM) (Fig. 6).11
Data is not yet available for inhibition studies on FabH from S.
aureus.
SSCH3
SSCH3
O2N
O2N
12
11
Figure 3. p-Nitrobenzyl and p-nitrophenethyl disulfides 11 and 12.
NO2
It is noteworthy that the trends in activity observed for the
nitrophenyl disulfides 8–10 coincide with previous observations
on the relative dimensions of the active site in each of the micro-
bial species studied. The active site of the E. coli FabH has been re-
ported to preferentially bind S-acetyl-CoA over larger or branched
S-acyl-CoA analogues.40 By contrast, the FabH from both Staphylo-
coccus41 and Bacillus42 have larger active-site pockets, which favor
reactions with branched S-acyl-CoA species such as S-isobutyryl-
CoA.43 These trends are followed precisely in the bioactivities of
the disulfide series, providing strong indirect evidence that these
compounds may selectively bind to FabH in a mechanism-based
manner. One possibility is that the aryl–alkyl disulfides may inhibit
FabH by reversibly ‘capping’ the active-site cysteine through a
thiol-disulfide exchange. The reported X-ray crystal structures44,45
of FabH from S. aureus indicate a fairly deep, lipophilic ‘pocket’ into
which S-acyl-CoA or S-acyl-ACP are able to stretch their lipophilic
arm during binding. A proximal cysteine residue is located within
the active site, and it is this available thiol that we speculate may
be sulfenylated by entry of the aryl–alkyl disulfides into the en-
S
SCH3
S
CO2H
O2N
CO2H
NO2
13
14
Figure 4. Methyl p-nitrophenyl sulfide (13) and Ellman’s reagent (14).
Table 4
Percent inhibitiona of FabH derived from E. coli by disulfides 8a–f
Compound
ecFabH (%)
8a
8b
8c
8d
8e
8f
94
98
98.5
96
91
93
a
Measured as the percent decrease in activity of FabH in the presence of 5 lM of
disulfides 8a–f, as compared to the same enzyme in the absence of the disulfide.
NH3
N
N
O
SR
N
N
O
P
O
P
N
O
O
O
O
O
Me
S
O
O
MeO
S
N
N
O
O
H
H
OH
Cl
O
P
OH
O
15a: R = Me
15b: R = -Bu
O
s
Figure 6. Methyl-coenzyme A mixed disulfide.
Figure 5. N-Alkylthio b-lactams (15a and 15b).

E. Turos et al. / Bioorg. Med. Chem. 16 (2008) 6501–6508
6505
function. This suggests that formation of the cysteine-alkyl FabH
disulfide (either via the alkyl-CoA disulfide, the alkyl–aryl disul-
fide, or N-alkylthio b-lactam) is irreversible under buffered aque-
ous conditions, but that addition of a thiol can regenerate the
catalytic form through thiol-disulfide exchange.
O
S
O
S
OH
16
Figure 7. FabH inhibitor disulfide 16.
The finding that these simple alkyl–aryl disulfides are generally
less active against E. coli than S. aureus or B. anthracis may also re-
flect differences in the thiol-redox buffers of these three mi-
crobes.13,47 Staphylococcus and Bacillus utilize
a coenzyme A
zyme. Reynolds has recently reported that the asymmetric disul-
fide 16 inhibits the activity of Mycobacterium tuberculosis and
E. coli FabH enzymes, by capping an active-site cysteine thiol by
sulfenylation, and that this capping destroys catalytic functioning
of the enzyme (Fig. 7). We presume that a similar event could be
occurring intracellularly in the FabH protein of S. aureus in the
presence of the disulfides.46
based thiol-redox buffer, while E. coli uses a glutathione buffer.
Consequently, the effects of the aryl–alkyl disulfides in glutathi-
one-based eukaryotic cells would likely also be dampened, which
is a question we are now exploring.
For the past decade, there had been considerable interest in the
development of FabH inhibitors as potential antibacterial
agents.48,49 The key role of FabH in Type II fatty acid synthesis, as
well as the unique differences between bacterial and mammalian
FAS pathways, makes this protein a desirable target for the selec-
tive inhibition of bacterial growth. We are exploring potential
applications of these disulfide inhibitors of FabH as antibacterial
agents for life-threatening bacterial infections.
3. Conclusions
The aryl–alkyl disulfides examined in this initial study possess
strong in vitro antimicrobial properties against S. aureus (including
MRSA) and B. anthracis, and only weak activity against E. coli. Of
the structural variants included in our study, the nitrophenyl al-
kyl disulfides exhibit the greatest in vitro potency. Perhaps even
more interestingly, it seems that the efficacy of these compounds
may be ‘tuned’ to select for a particular bacterial species: for E.
coli, the methyl aryl disulfides are optimal, while for Staphylococ-
cus and Bacillus, the isopropyl- and sec-butyl aryl disulfides ap-
pear to be more efficacious. The fact that the in vitro
microbiological properties of these compounds, determined from
agar diffusion and agar MIC measurements, coincide with the
FabH inhibition capabilities certainly points to the fact that this
key enzyme is associated with the biological properties of the
compounds. These results also highlight the fact that the N-thio-
lated b-lactams (and presumably N-thiolated oxazolidinones), re-
ported previously as selective bacteriostatic agents for
Staphylococcusand Bacillus, may act similarly by directly inhibiting
FabH protein in MRSA and B. anthracis.
The structural effects on bioactivity of these compounds raise
several key questions regarding the role of the aryl nitro substitu-
ent, whether it can be satisfactorily substituted for other function-
alities (such as water solubilizing groups), and if the aryl ring
substituent in some way aids in specific binding of the molecule
to the FabH active site prior to sulfenylation. Understanding the
ability of alkyl-CoA mixed disulfides to covalently deactivate bac-
terial fatty acid synthesis represents a major advance in the quest
for novel antibacterials. However, due to the demonstrated inabil-
ity of CoA mixed disulfides to traverse the cell membrane, the al-
kyl-CoA disulfides are not directly useful as therapeutics. This
research shows that prodrugs such as the described disulfides
and the previously reported N-alkylthio b-lactams can be used to
produce the CoA mixed disulfides within the bacterial target.
The activity of methyl-CoA disulfide against ecFabH provides
strong evidence that in prokaryotic cells which contain CoA, the al-
kyl–aryl disulfides and N-alkylthio b-lactams may function as
prodrugs to produce alkyl-CoA disulfides, and that these mixed
disulfides can then inhibit the fatty acid cycle through inhibition
of FabH. This mechanism is unique in that it involves a thiol-disul-
fide exchange from the alkyl-CoA disulfide to the active-site cys-
4. Experimental
4.1. General methods
Reagents were purchased from Sigma–Aldrich Chemical Com-
pany or Acros Chemical Company. Methanethiolsulfonates were
purchased from Toronto Research Chemicals. Reagents were used
without further purification. Solvents were obtained from Fischer
Scientific Company. Thin-layer chromatography (TLC) was carried
out using EM Reagent plates with fluorescence indicator (SiO2-
60, F-254). Products were purified by flash chromatography using
J.T. Baker flash chromatography silica gel (40 lm). NMR spectra
were recorded in CDCl3 unless otherwise noted. 13C NMR spectra
were proton broad-band decoupled. Methylene chloride and THF
were distilled prior to use. Prior to the preparation of the disulfides,
methanol was purged of oxygen by bubbling nitrogen an inert gas
through it for several minutes.
4.2. m-Nitrobenzenethiol50
To a solution of m-nitrobenzene disulfide (300 mg, 0.97 mmol)
in anhydrous THF (2 ml) was added solid NaBH4 (140 mg,
3.4 mmol) in small portions. The resulting mixture was stirred at
rt under an inert atmosphere. After 2 h, the reaction mixture was
cooled in an ice bath, then about 5 ml of icewater was added to
the mixture. The resulting mixture was acidified with HCl (1 M),
then extracted with CH2Cl2 (10 ml). The organic layer was then
washed with water (10 ml), then brine (10 ml), dried over MgSO4,
and concentrated in vacuo to give the thiol (248 mg, 83%) as a pale
yellow oil. 1H NMR (CDCl3, 400 MHz): d 8.10 (d, J = 2.0 Hz, 1H);
7.97 (dd, J = 8.0, 2.0 Hz, 1H); 7.54 (d, J = 8.0 Hz, 1H); 7.38 (t,
J = 8.0 Hz, 1H); 3.68 (s, 1H); 13C NMR (CDCl3, 100 MHz): d 134.9,
130.0, 123.9, 120.7.
4.3. o-Nitrobenzenethiol
teine of FabH,
a suggestion that is supported by protein
Prepared exactly as described above, except that the reaction
was allowed to run for 3 h, and the products required chromatog-
raphy (silica with hexanes:CH2Cl2) to give the thiol (160 mg, 61%)
as a pale yellow flocculent solid. Mp: 44–45 °C. 1H NMR (CDCl3,
400 MHz): d 8.24 (d, J = 8.0 Hz, 1H); 7.42 (d, J = 4.0 Hz, 1H); 7.29–
7.24 (m, 1H); 4.00 (s, 1H). 13C NMR (CDCl3, 100 MHz): d 133.9,
132.2, 126.5, 126.0.
crystallographic studies. Reynolds reported an X-ray crystal struc-
ture of E. coli FabH showing the methylthio group from methyl-CoA
disulfide being covalently bound to the active-site cysteine
(
112Cys). While the catalytic activity of the methylthiolated (deac-
tivated) FabH protein could not be regenerated simply by dialysis,
treatment with dithiothreitol (DTT) quickly restores full catalytic

6506
E. Turos et al. / Bioorg. Med. Chem. 16 (2008) 6501–6508
4.4. p-Nitrobenzyl thiol
4.6.6. p-Acetamidophenyl methyl disulfide (6)
To a solution of p-acetamidobenzenethiol (50 mg, 0.3 mmol) in
dry CH2Cl2 (3 ml) was added triethylamine (127 ll, 0.9 mmol, 3
equiv), followed by acetyl chloride (32 ll, 0.45 mmol). The reac-
tion mixture was stirred at rt for 1 h, then it was diluted with
dichloromethane, washed with water, then 5% aq HCl, then 5%
aq NaHCO3, dried over MgSO4, then concentrated in vacuo to give
a yellow oil which solidified on standing. Chromotography (1:1
petroleum ether:ethyl acetate) gave 18 mg (29%) as a light brown
solid. 1H NMR (400 MHz, CDCl3): d 7.47 (s, 4H); 2.41 (s, 3H); 2.16
(s, 3H).
Thioacetic acid (386 ll, 5.40 mmol) was added to a mixture of
4-nitrobenzyl bromide (600 mg, 2.79 mmol) and anhydrous
K2CO3 (380 mg, 2.75 mmol) in acetone (3 ml), and the resulting
mixture was stirred at rt for 2 h under an inert atmosphere. The
mix was then partitioned between CH2Cl2 (10 ml) and water
(10 ml). The organic layer was separated, then washed quickly
with saturated aqueous NaHCO3 (10 ml) and brine (10 ml). The or-
ganic solution was concentrated in vacuo, then taken up in meth-
anol (5 ml). Aqueous H2SO4 (50%, 1 ml) was added, and the
solution was heated to reflux for 3 h, under N2. The solution was
partitioned between ether (30 ml) and water (30 ml). The ether
layer was washed with water (2Â 30 ml), then brine (10 ml). The
solution was dried over Na2SO4, then concentrated in vacuo to give
the thiol (317 mg, 67%) as a yellow solid. Mp: 49–52 °C. 1H NMR
(200 MHz, CDCl3): d 8.19 (d, J = 8.8 Hz, 2H); 7.02 (d, J = 8.6 Hz,
2H); 3.82 (d, J = 7.8 Hz, 2H); 1.84 (t, J = 8.1 Hz, 1H).
4.6.7. p-Aminophenyl methyl disulfide (7)
To a solution of p-aminobenzenethiol (125 mg, 1 mmol) in
methanol (1 ml) was added methylthio methanesulfonate (94 ll,
1 mmol) in a single portion. The reaction mixture was stirred at
rt under N2. After 2 h, the solution was loaded onto an SCX column
and eluted with methanol, followed by 1 M ammonia in methanol.
The basic eluents were concentrated in vacuo to a yellow oil. Chro-
matography (1:1 hexanes/CH2Cl2 with 1% isopropyl amine) yielded
8 (140 mg, 82%) as a pale yellow oil. 1H NMR (250 MHz, CDCl3): d
7.28 (d, J = 8.6 Hz, 2H); 6.54 (d, J = 8.6 Hz, 2H); 3.60 (br s, 2H); 2.34
(s, 3H). Treatment with HCl/ether gave 5 mg of the HCl salt. Biolog-
ical testing was performed on the acid salt. 1H NMR (400 MHz,
d6-DMSO): d 7.46 (br s, 2H); 7.05 (br s, 2H); 2.47 (s, 3H); 2.43
(s, 3H).
4.5. p-Nitrophenethyl thiol
Prepared according to the general procedure described above,
except that the reaction time was 45 min. Isolated 170 mg (69%)
as
a d 8.17 (d,
yellow liquid. 1H NMR (200 MHz, CDCl3):
J = 8.8 Hz, 2H); 7.35 (d, J = 8.8 Hz, 2H); 3.04 (t, J = 7.7 Hz, 2H);
2.84 (dt, J = 5.9 Hz, 7.7 Hz, 2H); 1.38 (t, J = 5.9 Hz, 1H).
4.6. Synthesis of alkyl–aryl disulfides: general procedure
4.6.8. p-Nitrophenyl methyl disulfide (8a)
Isolated 15.3 mg (76%) as a yellow oil. 1H NMR (400 MHz,
CDCl3): d 8.17 (d, J = 8.8 Hz, 2H); 7.63 (d, J = 8.8 Hz, 2H); 2.46 (s,
3H). Resynthesis using 10Â scale yielded 184 mg (92%) as a slightly
oily, bright yellow solid. Mp: 29–32 °C. 13C NMR (100 MHz, CDCl3):
d 126.6, 126.0, 124.7, 124.4, 22.9.
To a 1.0 M solution of arylthiol in MeOH (100 ll) was added a
solution of alkyl methanethiolsulfonate in MeOH (105 ll of a
1.0 M solution, 1.05 equiv). After stirring at rt under an inert atmo-
sphere for 1 h, the solutions were treated with a small amount
(about 0.3 equiv) of cysteine hydrochloride in order to consume
the excess thiosulfonate. The mixture was then concentrated under
reduced pressure. The solid was taken up in CH2Cl2 (1 ml), and the
insoluble material was filtered off. The solution was stirred with
Amberlyst-21 resin (weakly basic), (approx. 0.2 equiv, pre-swelled
in CH2Cl2) for 3 min. The solution was drawn off of the resin,
quickly passed through a small silica plug using dichloromethane,
and concentrated in vacuo to give the desired product.
4.6.9. Ethyl p-nitrophenyl disulfide (8b)
1H NMR (400 MHz, CDCl3): d 8.14 (d, J = 8.8 Hz, 2H); 7.64 (d,
J = 8.8 Hz, 2H); 2.77 (q, J = 7.0 Hz, 2H); 1.31 (t, J = 7.2 Hz, 3H).
4.6.10. Isopropyl p-nitrophenyl disulfide (8c)
1H NMR (400 MHz, CDCl3): d 8.14 (d, J = 8.8 Hz, 2H); 7.64 (d,
J = 8.8 Hz, 2H); 3.10 (septuplet, J = 6.5 Hz, 1H); 1.30 (d, J = 6.8 Hz,
6H).
4.6.1. 3,4-Difluorophenyl methyl disulfide (1)
9.6 mg (50%) as a colorless oil. 1H NMR (250 MHz, CDCl3): d
7.37–7.29 (m, 1H); 7.18–7.00 (m, 2H); 2.34 (s, 3H).
4.6.11. sec-Butyl p-nitrophenyl disulfide (8d)
1H NMR (400 MHz, CDCl3): d 8.14 (d, J = 8.8 Hz, 2H); 7.64 (d,
J = 8.8 Hz, 2H); 2.88–2.83 (m, 1H); 1.73–1.64 (m, 1H); 1.57–1.50
(m, 1H); 1.27 (d, J = 6.8 Hz, 3H); 0.97 (t, J = 7.2 Hz, 3H).
4.6.2. 3,4-Dimethylphenyl methyl disulfide (2)
Isolated 9.7 mg (53%) as a colorless oil. 1H NMR (250 MHz,
CDCl3): d 7.22 (d, J = 8.4 Hz, 1H); 7.18 (s, 1H); 7.02 (d, J = 7.7 Hz,
1H); 2.70 (s, 3H); 2.19 (s, 3H); 2.18 (s, 3H).
4.6.12. p-Nitrophenyl n-propyl disulfide (8e)
1H NMR (400 MHz, CDCl3): d 8.14 (d, J = 8.8 Hz, 2H); 7.63 (d,
J = 8.8 Hz, 2H); 2.73 (t, J = 7.2 Hz, 2H); 1.68 (tq, J = 7.2, 7.6 Hz,
2H); 0.98 (t, J = 7.6 Hz, 3H).
4.6.3. m-Ethoxyphenyl methyl disulfide (3)
Isolated 5.6 mg (28%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 7.21 (t, J = 8.0 Hz, 1H); 7.09–7.05 (m, 2H); 6.74 (dd,
J = 8.0, 1.2 Hz, 1H); 4.04 (q, J = 7.1 Hz, 2H); 2.43 (s, 3H); 1.41 (t,
J = 7.4 Hz, 3H).
4.6.13. n-Butyl p-nitrophenyl disulfide (8f)
1H NMR (400 MHz, CDCl3): d 8.15 (d, J = 8.0 Hz, 2H); d 7.64 (d,
J = 8.8 Hz, 2H); d 2.95 (t, J = 7.4 Hz, 2H); d 1.65–1.61 (m, 2H); d
1.42–1.37 (m, 2H); d 0.88 (t, J = 7.2 Hz, 3H).
4.6.4. o-(Hydroxymethyl)phenyl methyl disulfide (4)
1H NMR (400 MHz, CDCl3): d 7.73 (d, J = 8.8 Hz, 1H); 7.44 (dd,
J = 6.6, 2.2 Hz, 1H); 7.32–7.24 (m, 2H); 4.83 (d, J = 5.2 Hz, 2H);
2.42 (s, 3H); 2.02 (br t, J = 6.0 Hz, 1H).
4.6.14. Methyl o-nitrophenyl disulfide (9a)
Isolated 13.9 mg (69%) as an oily yellow solid. 1H NMR
(400 MHz, CDCl3): d 8.27–8.23 (m, 2H); 7.69 (t, J = 7.4 Hz, 1H);
7.35 (d, J = 7.2 Hz, 1H); 2.41 (s, 3H).
4.6.5. Methyl 2-pyridinyl disulfide (5)
1H NMR indicates that about 10% of material is unreacted thiol.
1H NMR (400 MHz, CDCl3): d 8.46 (d, J = 4.4 Hz, 1H); 7.68–7.61 (m,
2H); 7.07 (t, J = 6.6 Hz, 1H); 2.49 (s, 3H).
4.6.15. Ethyl o-nitrophenyl disulfide (9b)
Isolated 13.2 mg (62%) as an oily yellow solid. 1H NMR
(400 MHz, CDCl3): d 8.28 (d, J = 8.0 Hz, 1H); 8.24 (d, J = 8.0 Hz,

E. Turos et al. / Bioorg. Med. Chem. 16 (2008) 6501–6508
6507
1H); 7.66 (t, J = 7.4 Hz, 1H); 7.33 (t, J = 7.6 Hz, 1H); 2.74 (q,
J = 7.3 Hz, 2H); 1.31 (t, J = 7.2 Hz, 3H).
phate-buffered saline (pH 7.2) and swabbed across fresh TSA
plates.
4.6.16. Isopropyl o-nitrophenyl disulfide (9c)
4.7.2. Antimicrobial testing: Kirby–Bauer method
Chromatographed on silica with 3% dichloromethane in hex-
anes as eluent. Isolated 13.7 mg (60%) as a yellow oil. 1H NMR
(400 MHz, CDCl3): d 8.28 (d, J = 8.8 Hz, 1H); 8.23 (d, J = 8.8 Hz,
1H); 7.64 (t, J = 7.9 Hz, 1H); 7.31 (t, J = 7.6 Hz, 1H); 3.05 (s,
J = 6.8 Hz, 1H); 1.31 (d, J = 6.8 Hz, 6H).
Sterile saline (5 ml) was inoculated with a swab of bacteria, then
the concentration was adjusted to 0.5 McFarland standard. Bacterial
solution was then streaked across a TSA plate to give an even lawn of
bacteria. Sterile pipet tips (1–30 ll) were used to drill 6 mm wells
into the agar plate, then 20 ll of 1 mg/ml drug in DMSO was added
to the well. Plates were inoculated overnight at 37 °C.
4.6.17. Butyl o-nitrophenyl disulfide (9d)
Chromatographed on silica with 3% dichloromethane in hex-
anes as eluent. Isolated 12.9 mg (54%) as a yellow oil. 1H NMR
(400 MHz, CDCl3): d 8.29 (d, J = 8.0 Hz, 1H); 8.22 (d, J = 8.8 Hz,
1H); 7.64 (t, J = 7.8 Hz, 1H); 7.31 (t, J = 8.0 Hz, 1H); 2.86–2.79 (m,
1H); 1.75–1.60 (m, 1H); 1.58–1.50 (m, 1H); 1.28 (d, J = 7.2 Hz,
3H); 0.98 (t, J = 7.8 Hz, 3H).
4.7.3. Agar dilution minimal inhibitory concentration (MIC)
assay
A stock solution of 1 mg/ml of each disulfide was prepared in
DMSO. Freshly prepared Mueller-Hinton agar (1.5 ml) was added
to each well of a 24-well plate, and to each well was then added
a specified amount of the disulfide test solution in order to a give
a final drug concentration of 256 mg/ml down to 0.125 lg/ml.
One well within each series received 0.1 ml of DMSO as a blank.
The contents of each well were thoroughly stirred to evenly dis-
tribute the antimicrobial solution within the agar. Once the agar
completely solidified, 10 ll of Mueller-Hinton broth saline contain-
ing 0.5 McFarland standard of the test bacteria was pipetted on top
of each well and the plates were then incubated for 24 h at 37 °C.
Bacterial growth was assessed by visual observation of growth.
4.6.18. Methyl m-nitrophenyl disulfide (10a)
Isolated 7.6 mg (38%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 8.39 (s, 1H); 8.04 (d, J = 6.4Hz, 1H); 7.79 (d, J = 8.0 Hz,
1H); 7.49 (t, J = 8.0 Hz, 1H); 2.47 (s, 3H).
4.6.19. Ethyl m-nitrophenyl disulfide (10b)
Isolated 7.7 mg (36%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 8.41 (s, 1H); 8.02 (d, J = 7.2 Hz, 1H); 7.80 (d, J = 7.2 Hz,
1H); 7.47 (t, J = 7.2 Hz, 1H); 2.78 (q, J = 7.3 Hz, 2H); 1.32 (t,
J = 7.2 Hz, 3H).
4.7.4. FabH enzyme assay
FabH assays were carried out using a standard coupled trichlo-
roacetic acid precipitation assay which determines the rate of for-
mation of radiolabeled 3-ketoacyl ACP from malonyl ACP and
radiolabeled acetyl-CoA. In this coupled assay Streptomyces glau-
scescens FabD is used to generate the malonyl ACP substrate from
malonyl-CoA and Streptomyces glauscescens ACP. For inhibition
studies, A standard 20 ll reaction mixture of ecFabH (2 pmol of
monomer, 100 nM) and test compound (0.02 nmol, 1 lM for com-
pounds 15a and 15b; 0.1 nmol, 5 lM for compounds 8a–f) in
50 mM sodium phosphate buffer (pH 7.4) was incubated at room
4.6.20. Isopropyl m-nitrophenyl disulfide (10c)
Isolated 17.0 mg (75%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 8.41 (s, 1H); 8.01 (d, J = 8.0 Hz, 1H); 7.80 (d, J = 7.2 Hz,
1H); 7.46 (t, J = 7.2 Hz, 1H); 3.09 (septet, J = 6.8 Hz, 1H); 1.30 (d,
J = 6.8 Hz, 6H).
4.6.21. Butyl m-nitrophenyl disulfide (10d)
Isolated 12.6 mg (52%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 8.42 (s, 1H) 8.00 (d, J = 8.0 Hz, 1H); 7.80 (d, J = 8.0 Hz,
1H); 7.45 (t, J = 8.0 Hz, 1H); 2.88–2.83 (m, 1H); 1.73–1.68 (m, 1H);
1.57–1.50 (m, 1H); 1.28 (d, J = 7.2 Hz, 3H); 0.97 (t, J = 7.2 Hz, 3H).
temperature for 15 min prior to addition to
a solution of
[1-14C]acetyl-CoA (0.8 nmol, 40 lM) and MACP (0.2 nmol,
10 lM). The inhibition was measured using standard FabH as-
say.51 The % inhibition was determined by measuring FabH activ-
ity in the presence of test compound relative to a negative control
that has no inhibitor (100% activity). The assay was run in
duplicate.
4.6.22. Methyl 4-nitrobenzyl disulfide (11)
Prepared according to the general procedure outlined above for
the aryl–alkyl disulfides. Following chromatography (3:1 hexanes/
CH2Cl2), 3.1 mg (5%) was obtained as a pale yellow solid. 1H NMR
(200 MHz, CDCl3): d 8.20 (d, J = 8.7 Hz, 2H); 7.51 (d, J = 8.6 Hz,
2H); 3.94 (s, 2H); 2.15 (s, 3H).
Acknowledgment
We thank the NIH for supporting these studies through research
Grant R01 AI51351.
4.6.23. Methyl 2-(p-nitrophenethyl) disulfide (12)
Obtained 9.7 mg (15%) as a pale yellow oil. 1H NMR (200 MHz,
CDCl3): d 8.17 (d, J = 8.0 Hz, 2H); 7.37 (d, J = 8.0 Hz, 2H); 3.19–
2.85 (m, 4H); 2.43 (s, 3H). MS (EI) m/z: 229 (M+).
References and notes
1. Turos, E.; Konaklieva, M. I.; Ren, R. X.-F.; Shi, H.; Gonzalez, J.; Dickey, S.; Lim, D.
Tetrahedron 2000, 56, 5571.
4.7. General microbiological methods
2. Long, T. E.; Turos, E. Curr. Med. Chem. Anti-Infect. Agents 2002, 1, 251.
3. Turos, E.; Long, T. E.; Konaklieva, M. I.; Coates, C.; Shim, J.-Y.; Dickey, S.; Lim, D.
V.; Cannons, A. Bioorg. Med. Chem. Lett. 2002, 12, 2229.
4. Coates, C.; Long, T. E.; Turos, E.; Dickey, S.; Lim, D. V. Bioorg. Med. Chem. 2003,
11, 193.
5. Long, T. E.; Turos, E.; Konaklieva, M. I.; Blum, A. L.; Amry, A.; Baker, E. A.;
Suwandi, L. S.; McCain, M. D.; Rahman, M. F.; Dickey, S.; Lim, D. V. Bioorg. Med.
Chem. 2003, 11, 1859.
6. Turos, E.; Coates, C.; Shim, J.-Y.; Wang, Y.; Leslie, J. M.; Long, T. E.; Reddy, G. S.
K.; Ortiz, A.; Culbreath, M.; Dickey, S.; Lim, D. V.; Alonso, E.; Gonzalez, J. Bioorg.
Med. Chem. 2005, 13, 6289.
7. Heldreth, B.; Long, T. E.; Jang, S.; Reddy, G. S. K.; Turos, E.; Dickey, S.; Lim, D. V.
Bioorg. Med. Chem. 2006, 14, 3775.
Staphylococcus aureus (ATCC 25923) and MRSA (ATCC 43300)
were purchased from ATCC sources. Bacillus bacteria from Sterne
spore vaccine was purchased from Colorado Serum Co., Denver, CO.
4.7.1. Culture preparation
From a freezer stock in tryptic soy broth (Difco Laboratories, De-
troit, MI) and 20% glycerol, a culture of each microorganism was
transferred with a sterile Dacron swab to TrypticaseÒ Soy Agar
(TSA) plates (Becton-Dickinson Laboratories, Cockeysville, MD),
streaked for isolation, and incubated at 37 °C for 24 h. A 108 stan-
dardized cell count suspension was then made in sterile phos-
8. Turos, E.; Long, T. E.; Heldreth, B.; Leslie, J. M.; Reddy, G. S. K.; Wang, Y.; Coates,
C.; Konaklieva, M.; Dickey, S.; Lim, D. V.; Alonso, E.; Gonzalez, J. Bioorg. Med.
Chem. Lett. 2006, 16, 2084.

6508
E. Turos et al. / Bioorg. Med. Chem. 16 (2008) 6501–6508
9. Revell, K. D.; Heldreth, B.; Long, T. E.; Jang, S.; Turos, E. Bioorg. Med. Chem. 2007,
15, 2453.
10. Mishra, R. K.; Revell, K. D.; Coates, C. M.; Turos, E.; Dickey, S.; Lim, D. V. Bioorg.
Med. Chem. Lett. 2006, 16, 2081.
32. Augusti, K. T.; Mathew, P. T. Experentia 1974, 30, 468.
33. Feldberg, R. S.; Chang, S. C.; Kotik, A. N.; Nadler, M.; Neuwirth, Z.;
Sundstrom, D. C.; Thompson, N. H. Antimicrob. Agents Chemother. 1988,
32, 1763.
11. Wright, H. T.; Reynolds, K. A. Curr. Opin. Microbiol. 2007, 10, 447.
12. Clarke, K. M.; Mercer, A. C.; La Clair, J. J.; Burkart, M. D. J. Am. Chem. Soc. 2005,
237, 11234.
34. Focke, M.; Feld, A.; Lichtenthaler, H. K. FEBS Lett. 1990, 261, 106.
35. Ozolin, O. N.; Uteshev, T. A.; Kim, I. A.; Deev, A. A.; Kamzolova, S. G. Mol. Biol.
(Mosk.) 1990, 24, 1057.
13. Heldreth, B.; Turos, E. Curr. Med. Chem. Anti-Infect. Agents 2005, 4, 295.
14. Cavallito, C.; Bailey, J. H. J. Am. Chem. Soc. 1944, 66, 1944.
15. Ankri, S.; Mirelman, D. Microbes Infect. 1999, 2, 125.
16. Block, E. Sci. Am. 1985, 252, 94.
36. Kirkpatrick, D. L. Asymmetric disulfides and methods of using same, US Patent
Application 20030176512 (September 18, 2003).
37. Kirkpatrick, D. L.; Powis, G. Asymmetric disulfides and methods of using same,
US Patent Application 20040116496 (June 17, 2004).
17. Koch, H. P.; Lawson, L. D. Garlic: The Science and Therapeutic Application of
Allium sativum L.; Williams and Wilkins: Baltimore, 1996. pp 1–233.
18. Uchida, Y.; Takahashi, T.; Sato, N. Jpn. J. Antibiot. 1975, 28, 638.
19. Cellini, L.; Campli, E.; Masulli, M.; Di Bartolomeo, S.; Allocati, N. FEMS Immunol.
Med. Microbiol. 1996, 13, 273.
20. Gonzales-Fandos, E.; Garcia-Lopez, M. L.; Sierrra, M. L.; Otera, A. J. Appl.
Bacteriol. 1994, 77, 549.
21. Gimenez, M. A.; Solanes, R. E.; Gimenez, D. F. Rev. Argent. Microbiol. 1988, 20,
17.
22. Davis, L. E.; Shen, J.; Royer, R. E. Planta Med. 1994, 60, 546.
23. Hughes, B. G.; Lawson, L. D. Photother. Res. 1991, 5, 154.
24. Yamada, Y.; Azuma, K. Antimicrob. Agents Chemother. 1997, 11, 743.
25. Tsai, Y.; Cole, L. L.; Dav Lockwood, S. J.; Simmons, V.; Wild, G. C. Planta Med.
1985, 5, 460.
26. Tatarintsev, A. V.; Vrzhets, P. V.; Ershov, D. E.; Turgiev, A. S.; Karamov, E. V.;
Kornilaeva, G. V.; Makarova, T. V.; Federov, N. A.; Varfolomeev, S. D. Vestn. Ross.
Akad. Med. Nauk. 1992, 11, 6.
38. NCCLS (National Committee for Clinical Laboratory Standards), Methods for
Dilution of Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically, NCCLS Document M7-A4, 1997; Vol. 17.
39. Alhamadsheh, M. M.; Musayev, F.; Komissarov, A. A.; Sachdeva, S.;
Wright, H. T.; Scarsdale, N.; Florova, G.; Reynolds, K. A. Chem. Biol.
2007, 14, 513.
40. Kang, S. L.; Rybak, M. L. Antimicrob. Agents Chemother. 1995, 39, 1505.
41. He, X.; Reynolds, K. A. Antimicrob. Agents Chemother. 2002, 46, 1310.
42. Choi, K.; Heath, R. J.; Rock, C. O. J. Bacteriol. 2000, 182, 365.
43. Choi, K.-H.; Heath, R. J.; Rock, C. O. J. Bacteriol. 2000, 182, 365.
44. Daines, R. A.; Pendrak, I.; Sham, K.; Van Aller, G. S.; Konstantinidis, A. K.;
Lonsdale, J. T.; Janson, C. A.; Qiu, X.; Brandt, M.; Khandekar, S. S.; Silverman, C.;
Head, M. S. J. Med. Chem. 2003, 46, 5.
45. Qui, X.; Choudhry, A. E.; Janson, C. A.; Grooms, M.; Daines, R. A.; Lonsdale, J. T.;
Khandekar, S. S. Protein Sci. 2005, 14, 2087.
46. Sachdeva, S.; Musayev, F. N.; Alhamadsheh, M. M.; Scarsdale, J. N.; Wright, H.
T.; Reynolds, K. A. Chem. Biol. 2008, 15, 402.
27. Mirelman, D.; Monheit, D.; Varon, S. J. Infect. Dis. 1987, 156, 243.
28. Ankri, S.; Miron, T.; Rabinkov, A.; Wilchek, M.; Mirelman, D. Antimicrob. Agents
Chemother. 1997, 10, 2286.
29. Adetumbi, M.; Javor, G. T.; Lau, B. H. Antimicrob. Agents Chemother. 1986, 30,
499.
30. Ghannoum, M. A. J. Gen. Microbiol. 1988, 134, 2917.
31. Neuwirth, Z.; Sundstrom, D. C.; Thompson, N. H. Antimicrob. Agents Chemother.
1988, 32, 1763.
47. Poole, L. B.; Karplus, P. A.; Claiborne, A. Annu. Rev. Pharmacol. Toxicol. 2004, 44,
325.
48. Marrakchi, H.; Zhang, Y.-M.; Rock, C. O. Biochem. Soc. Trans. 2002, 30,
1050.
49. Campbell, J. W.; Cronan, J. E. Annu. Rev. Microbiol. 2001, 55, 305.
50. Cashman, J. R.; Olsen, L. D.; Young, G.; Bern, H. Chem. Res. Toxicol. 1989, 2,
392.
51. Lobo, S.; Florova, G.; Reynolds, K. A. Biochemistry 2001, 40, 11955.