Design and synthesis of new hydroxyethylamines as inhibitors of d-alanyl-d-lactate ligase (VanA) and d-alanyl-d-alanine ligase (DdlB)

Article abstract of DOI:10.1016/j.bmcl.2009.01.034

The Van enzymes are ATP-dependant ligases responsible for resistance to vancomycin in Staphylococcus aureus and Enteroccoccus species. The de novo molecular design programme SPROUT was used in conjunction with the X-ray crystal structure of Enterococcus faecium d-alanyl-d-lactate ligase (VanA) to design new putative inhibitors based on a hydroxyethylamine template. The two best ranked structures were selected and efficient syntheses developed. The inhibitory activities of these molecules were determined on E. faecium VanA, and due to structural similarity and a common reaction mechanism, also on d-Ala-d-Ala ligase (DdlB) from Escherichia coli. The phosphate group attached to the hydroxyl moiety of the hydroxyethylamine isostere within these systems is essential for their inhibitory activity against both VanA and DdlB.

Full text of DOI:10.1016/j.bmcl.2009.01.034

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1376–1379
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of new hydroxyethylamines as inhibitors of
D-alanyl-D-lactate ligase (VanA) and D-alanyl-D-alanine ligase (DdlB)
a
a
a
b
b
c
c
ˇ
Matej Sova , Gašper Cade zˇ , Samo Turk , Vita Majce , Slovenko Polanc , Sarah Batson , Adrian J. Lloyd ,
c
d
a,
*
David I. Roper , Colin W. G. Fishwick , Stanislav Gobec
Faculty of Pharmacy, University of Ljubljana, Ašker cˇ eva 7, 1000 Ljubljana, Slovenia
Faculty of Chemistry, University of Ljubljana, Ašker cˇ eva 5, 1000 Ljubljana, Slovenia
Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK
a
b
c
d
School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The Van enzymes are ATP-dependant ligases responsible for resistance to vancomycin in Staphylococcus
aureus and Enteroccoccus species. The de novo molecular design programme SPROUT was used in conjunc-
tion with the X-ray crystal structure of Enterococcus faecium D-alanyl-D-lactate ligase (VanA) to design
new putative inhibitors based on a hydroxyethylamine template. The two best ranked structures were
selected and efficient syntheses developed. The inhibitory activities of these molecules were determined
Received 28 November 2008
Revised 9 January 2009
Accepted 14 January 2009
Available online 19 January 2009
on E. faecium VanA, and due to structural similarity and a common reaction mechanism, also on D-Ala-D-
Keywords:
Ala ligase (DdlB) from Escherichia coli. The phosphate group attached to the hydroxyl moiety of the
hydroxyethylamine isostere within these systems is essential for their inhibitory activity against both
VanA and DdlB.
ATP-dependant ligases
De novo molecular design
Enzyme inhibitors
Antibacterial agents
Ó 2009 Elsevier Ltd. All rights reserved.
Staphylococcus aureus and Enteroccoccus species are Gram-posi-
of
triphosphate (ATP), forming ADP and
noacyl-phosphate is subsequently attacked by the hydroxyl group
of -lactate, which eliminates inorganic phosphate and yields the
product
To date, only a few inhibitors of the Van enzymes have been
D
-alanine is activated by phosphorylation by adenosine
1
tive bacteria and the major causes of nosocomial infections. The
D
-alanyl phosphate. This ami-
glycopeptide antibiotic vancomycin2 is used clinically as the last
line of defence for the treatment of these life-threatening infec-
D
3
11,12
tions. However, due to the recent emergence of vancomycin-resis-
D-Ala-D-Lac (Fig. 1).
1,4
tant S. aureus and vancomycin-resistant enterococci there is an
urgent need for the development of novel antibacterial agents
and agents that can overcome the resistance to vancomycin.
Gram-positive clinical isolates resistant to vancomycin are
characterized by a subtle change in the structure of the peptidogly-
1
3,14
described.
analogues have been reported to be reversible inhibitors of VanA.
Inhibitors of the Van enzymes could be used in combination with
Phosphinate and phosphonate transition-state
13
can termini, as a result of the action of
Van enzymes). These enzymes replace the
D
-alanyl-
D-X ligases (the
D
-Ala- -Ala termini with
D
O-
ATP
ADP
OH
O
O-
either
described according to the particular vancomycin-resistance phe-
notype. Replacement of -alanine by -lactate eliminates a key
D
-Ala-
D
-Lac or
D
-Ala-
D
-Ser. VanA-G enzymes have been
H N
H
N
P
5
D
D
O
O
O
D-Ala
amide NH hydrogen bond interaction with vancomycin, leading
to a thousand-fold decrease in binding affinity.6 The depsipeptide
,7
D-Lac
D-Ala
D
-Ala-D-Lac is synthesized by the ATP-dependent depsipeptide
8
,9
ligase VanA.
VanA DdlB
VanA is a 38.5-kDa protein that can use both D-lactate and D-Ala
O
O
as substrates, with a concomitant pH-dependent switch between
ester and peptide bond formation.10 The catalytic mechanism is
H
N
O
H N
O-
H N
O-
similar to that of D-Ala-D-Ala ligase (DdlB). Initially, the carboxyl
O
D-Ala-D-Lac
O
D-Ala-D-Ala
*
Corresponding author. Tel: +386 1 4769585; fax: +386 1 4258031.
E-mail address: gobecs@ffa.uni-lj.si (S. Gobec).
Figure 1. The mechanisms of the reaction catalyzed by VanA and DdlB.
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.034

M. Sova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1376–1379
1377
O-
OH
vancomycin to eliminate the formation of the
D
-Ala-
D
-Lac termini,
O
O
O-
OH
and thus to prevent the drop in binding affinity to vancomycin.
Therefore, the aim of the present study was to design and synthe-
size novel small-molecule inhibitors of VanA, and to evaluate their
enzyme-inhibitory activities. Ddl activity is replaced by VanA in
vancomycin-resistant Gram-positive pathogens.15 However, Ddl
P
P
O
O
H
N
+
H N
O-
N
O
O-
O
is an essential enzyme that is found in all bacterial pathogens,
-cycloserine,1 a rarely
5
and is a drug target for antibiotics such as
D
Figure 3. The structure of the high-energy transition state intermediate of the
reaction catalyzed by VanA (left), and the phosphorylated HEA molecular template
used for the present design of inhibitors (right).
used drug except in all but highly resistant tuberculosis infections,
because of neurological side-effects.16 Targeting Ddl could thus
provide considerable therapeutic benefit. Therefore, due to the
structural similarity and common reaction mechanisms of the
Van and Ddl enzymes,12 the VanA inhibitors designed here were
number of additional potential H-bonding sites within the putative
binding cavity. Thus, Arg290, Gly311, Ser316 and Arg317 were
chosen due to their close proximities to His244. In addition,
Ser177, Ser178 and Asn307 were also selected as H-bond donors/
acceptors. Furthermore, examination with SPROUT also revealed
the presence of a small hydrophobic region (not in direct contact
with the phosphorylated phosphinate inhibitor; yellow in Fig. 2)
bordered by the phenyl ring of Phe241 and the isobutyl side chain
of Ile243.
also evaluated for inhibitory activities against Escherichia coli DdlB,
to determine if, additionally, these molecules have broader poten-
tial efficacies against not only Gram-positive, but also Gram-nega-
tive pathogens.
To design a new class of Van and Ddl inhibitors, we applied
17
computer-aided design using the SPROUT software to a crystal
1
8
structure of VanA. SPROUT, a powerful suite of software for de
novo ligand design has previously been successfully used for the
1
9
20
Hydroxyethylamines (HEAs) have been described as analogues
of tetrahedral high-energy reaction intermediates of protein-
development of novel inhibitors of MurD
and DdlB
from
21
E. coli, of the human NK2 receptor, of Plasmodium dihydroorotate
2
5,26
2
2,23
24
ases,
inhibitors.
of HEAs, we wanted to use the HEA template as a basis for the
and have often been used for the design of potent enzyme
dehydrogenase,
crystallographic structure of the
of Enterococcus faecium complexed with ADP and a phosphorylated
and of the bacterial RNA polymerase. The
2
7,28
Taking advantage of our experience in the synthesis
D
-alanyl- -lactate ligase (VanA)
D
2
9,30
1
8
design of VanA inhibitors.
To mimic the high-energy interme-
phosphinate inhibitor has been resolved to 2.5 Å resolution,
diate of the reaction catalyzed by VanA, we decided to use the
phosphorylated HEA scaffold (Fig. 3).
where the inhibitor makes direct contacts with essentially all of
the active-site residues (Fig. 2).18
In the next step, the selected phosphorylated HEA template was
used to gradually build a small-molecule inhibitor using SPROUT.
During this analysis, a substituted benzene ring was selected to
bind in the hydrophobic region close to Phe241 and Ile243. A sul-
fonamide group, which has H-bond donor/acceptor properties, was
introduced to link the benzene ring and phosphorylated HEA tem-
plate, which was predicted by SPROUT to make contacts with
His244, Gly311 and Arg317. Additionally, the design required the
presence of another ring attached to the amino group of our HEA
template. Application of SPROUT suggested a tetrahydrofuryl ring
may be suitable due to the possible hydrogen bonding interaction
with Ser177. However, for reasons of synthetic availability, we
decided to use a morpholine ring, which still retains the oxygen
as the H-bond acceptor.
SPROUT analysis of the VanA active site revealed a large number
of residues that could have bonding interactions with a potential
inhibitor. By using the SPROUT strategy, we wanted to design a
small inhibitor with a reasonably high affinity for VanA. Therefore,
only a small number of the active site residues were selected. In
particular, due to the involvement of the imidazole ring within
1
8
His244 in dictating the preference of VanA for lactate as a ligand,
His244 was chosen for ligand contacting. Within the high level
vancomycin resistance phenotypes found in a variety of different
enterococcal strains (and also Vancomycin resistant S. aureus) the
D
-Ala-D-Lac ligases all have a His244 and surrounding residues.
There is general conservation of active site residues particularly
with respect to the ATP site and high affinity 1st subsite for ala-
nine. Additionally, SPROUT analysis revealed the presence of a
The two most promising de novo designed small molecules, for
which the predicted binding affinities according to SPROUT would
be in the micromolar range or better (Table 1, compounds 7a and
7
b), were selected and their predicted orientations and interac-
tions within the VanA active site were further analyzed. Analysis
of inhibitor 7a (Fig. 4) revealed that residues His244 and Gly311
Table 1
VanA and DdlB inhibitory activities of compounds 6a, b and 7a, b
1
R
O
S
N
H
N
O
O
P
O
O
O
R
OH
.
2
Compound
R1
R2
% Inhibiton at 500 l
M a
VanA
NI
83 (224 ± 19
65
DdlB
6a
7a
6b
7b
MeO
MeO
F
Ph
H
Ph
H
NI
l
M)
M)
83 (110 ± 28
NI
77 (136 ± 12
l
M)
M)
Figure 2. The bonding interactions between the phosphorylated phosphinate
inhibitor and VanA based on the crystal structure 1e4e.pdb. The hydrophobic
region, which does not interact with the phosphorylated phosphinate inhibitor, is
presented in yellow.
F
75 (290 ± 23
l
l
a
NI = no inhibition observed; values of IC50 ± standard deviation are given in
parentheses.

1
378
M. Sova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1376–1379
H
N
H
N
O
O
O
O
Cl
S
i
S
ii
S
O
O
O
R1
R1
R1
1a-b
2a-b
iv
3a-b
Ph
O
O
P
Ph
OH
O
H
N
H
O
N
O
O
iii
S
S
O
N
O
O
N
O
R1
R1
4
a-b
5a-b
HO
HO
O
OH
P
Ph
P
O
O
H
N
O
H
N
O
v
O
O
S
1
a: R = OCH3
S
1
O
N
+
O
N
b: R = F
R1
R1
O
O
6a-b
7a-b
Figure 4. SPROUT designed inhibitor 7a (blue) docked to the active site of VanA.
Scheme 1. Reagents and conditions: (i) allylamine, CH
ii) mCPBA, CH Cl , rt, 48 h, 73–76%; (iii) morpholine, Ca(OTf)
20 °C, 4 min, 84–88%; (iv) diphenylphosphoryl chloride, DMAP, CH
h, 75–81%; (v) H , PtO , glacial acetic acid, 4 days, then separation by preparative
CN/H O/MeOH (3/1/1) as mobile phase.
2
Cl
2
, 0 °C to rt, 1 h, 88–91%;
, dioxane, MW,
Cl , 0 °C to rt,
ADP is magenta and Mg2 is green.
+
(
1
1
2
2
2
2
2
2
2
thin-layer chromatography using CH
3
2
Due to the very similar topologies of the active sites of VanA and
DdlB, and in light of reported activity of the phosphinate inhibitor
1
2
for both VanA and DdlB, we tested compounds 4a and b, 6a and b
and 7a and b for their inhibitory activities against both VanA from
E. faecium and DdlB from E. coli. The residual activities and IC50
values were determined using a pyruvate kinase/lactate dehydroge-
3
2
nase-coupled assay. The results are presented as percentages of
inhibition of VanA and DdlB in the presence of the inhibitors (Table
1), and for the most active compounds 7a and 7b, also as IC50 values.
Interestingly, compounds lacking the phosphate group (4a and 4b)
showed no inhibitory activities against both VanA and DdlB even at
1
0 mM. The monophenyl phosphate esters 6a and 6b showed no or
only moderate inhibitory activities (% inhibition = 65 at 500 M for
b). Since only the phosphorylated HEA derivatives 7a and 7b inhib-
l
6
ited VanA and DdlB, it can be concluded that the presence of a phos-
phate group is essential for inhibition of both of these enzymes.
Compound 7a, which has a methoxy group on the para-position of
the aromatic ring, is a slightly better inhibitor of both VanA and DdlB
Figure 5. SPROUT designed inhibitor 7b (blue) docked to the active site of VanA.
ADP is magenta and Mg2 is green.
+
(
IC50 values of 224 and 110 lM, respectively). This may be due to the
are important in making hydrogen bonds with the phosphate
group of the designed inhibitor. Additionally, a substituted ben-
zene ring interacts with the hydrophobic region within the cavity.
The morpholine ring oxygen makes a hydrogen bond with the side
chain of Ser177. On the other hand, for inhibitor 7b, the predicted
interactions are with His244, Gly311 and Arg317 (involving
H-bonds with phosphate groups), Asn 307 (H-bond with the mor-
pholine moiety) and the hydrophobic region located near Phe241
ability of the methoxy group to accept hydrogen bond.
During the determination of the IC50 values for these inhibitors,
we noted that 7a and 7b exert hyperbolic inhibition on DdlB,
whilst exerting sigmoidal inhibition on VanA (Fig. 6). This sug-
gested that for VanA, more than one binding event accounts for
the inhibitory kinetics of 7a and 7b, and that this second interac-
tion was absent with DdlB. A simple explanation might be that
7a and 7b can occupy only a single DdlB D-alanine binding site,
(
(
designed to interact with the aromatic ring within the inhibitor)
Fig. 5).
hence giving rise to the hyperbolic inhibition kinetics. In contrast,
the relaxed specificity of the VanA site in binding the second acid
Following molecular design, efficient synthetic approaches to
substrate (D-Ala or D-lac) might allow additional interactions that
compounds 7a and 7b were developed and optimized to obtain
these molecules in high yields (Scheme 1).3 The synthesis started
from benzenesulfonyl chlorides 1a and 1b, which were coupled
with allylamine to give 2a and 2b, respectively. Epoxides 3a and
are not seen with DdlB and 7a or 7b. In this context, it is relevant
to note that an X-ray crystallographic analysis of S. aureus Ddl
1
33
revealed that an inhibitor of this enzyme (3-chloro-2,2-dimethyl-
N-[4(trifluoro-methyl)phenyl]propanamide) binds at a position
adjacent to the active site. It may be that a related situation per-
tains here with VanA, where inhibitors 7a and 7b may bind to
the site occupied by one of the substrates, and in addition, also
to an otherwise cryptic binding site that is not present in E. coli
DdlB, hence giving rise to the sigmoid VanA inhibition kinetics.
In summary, we have designed and synthesized novel small-
molecule inhibitors of VanA and DdlB. with IC50 values in the
micromolar range. The results show that a phosphate attached to
the hydroxyl group of an hydroxyethylamine moiety is essential
3
b were then prepared by oxidation with meta-chloroperbenzoic
acid and subsequently opened with morpholine in the presence
3
0
2
of calcium trifluoromethanesulphonate (Ca(OTf) ) as a catalyst,
to give the HEA derivatives 4a and 4b. The diphenylphosphate
group was introduced using diphenylphosphoryl chloride, with
DMAP as a catalyst/ base. Finally, deprotection of phenyl esters
5
a and 5b was performed using hydrogenation in the presence of
PtO . The compounds 6a and b and 7a and b were isolated follow-
ing preparative thin-layer chromatography.
2

M. Sova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1376–1379
1379
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1. Spectral data for compounds 6–7: 1-(4-methoxyphenylsulfonamido)-3-
morpholinopropan-2-yl phenyl hydrogen phosphate (6a): Yield 35%; white
crystals (hygroscopic), mp 96–100 °C; IR (KBr):
mmax 3447, 2361, 2343, 1719,
1
5
639, 1597, 1492, 1406, 1264, 1158, 1094, 1059, 1028, 927, 782, 640, 612, and
À1
1
64 cm
;
H NMR (D
2
O, NaOD) d (ppm) 2.39–2.46 (m, 5H, CH
N), 2.76 (dd, 1H, J = 8.5 and 12.5 Hz,
), 3.05 (dd, 1H, J = 3.8 and 12.5 Hz, NHCH ), 3.62 (t, 4H, J = 4.5 Hz,
-morpholine), 3.77 (s, 3H, CH ), 4.28–4.35 (m, 1H, CH), 6.93–7.01 (m,
a 2 2
N(CH ) -
morpholine), 2.55–2.61 (m, 1H, CH
NHCH
O(CH
3
b
a
b
)
2 2
4
7
H, Ar), 7.28 (t, 1H, J = 7.3 Hz, Ar), 7.28 (dd, 2H, J = 7.8 and 7.8 Hz, Ar), 7.58–
.63 (m, 2H, Ar), NH and OH not observed, exchanged. 31P NMR (D
O, NaOD) d
PSNa (M+Na ): 509.1123. Found:
2
Figure 6. Dose dependence of inhibition of VanA and DdlB by compounds 7a and
b. Data were fitted with Prism 4.0 by non-linear regression to the equation given
above, and are means ± standard error from assays carried out in triplicate for 7a
and duplicate for 7b.
+
(
5
ppm) À4.21. ESI HRMS Calcd for C20
H
27
N
2
O
8
7
3
4
R
09.1138. HPLC: t = 15.321 min (95.7%).
1
-(4-Fluorophenylsulfonamido)-3-morpholinopropan-2-yl
phenyl
hydrogen
phosphate (6b): Yield 47%; white crystals, mp 125–129 °C; IR (KBr):
m
max
À1
1
3
446, 1565, 1414, 1265, 1155, 1096, 896, 840, 760, and 552 cm
(D O, NaOD) d (ppm) 2.36–2.42 (m, 4H, N(CH -morpholine), 2.51–2.56 (m,
H, CH N), 2.73 (dd, 1H, J = 8.7 and 12.3 Hz, NHCH ), 2.99 (dd, 1H, J = 3.2 and
2.3 Hz NHCH ), 3.58 (t, 4H, J = 4.3 Hz, O(CH -morpholine), 4.24–4.31 (m, 1H,
; H NMR
for inhibitory activity against both VanA and DdlB. The designed
compounds represent an important starting point for further opti-
mization and modifications, to improve these inhibitory activities
against VanA and DdlB. These types of inhibitors have the potential
to be developed into drugs that would reverse bacterial resistance
to vancomycin. Furthermore, the potency of these compounds
against both VanA and DdlB suggest that it will be possible to
develop broad-spectrum antimicrobials that target both Gram-
negative and Gram-positive infections.
2
2 2
)
2
1
2
a
b
2 2
)
CH), 6.98 (d, 2H, J = 7.8 Hz, Ar), 7.04–7.14 (m, 3H, Ar), 7.28 (dd, 2H, J = 7.5 and
7.5 Hz, Ar), 7.60–7.65 (m, 2H, Ar), NH and OH not observed, exchanged. 3
1
P
À
NMR (D
73.0948. Found: 473.0934. HPLC: t
-(4-Methoxyphenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate
(7a): Yield 30%; white crystals (hygroscopic), mp 119–124 °C; IR (KBr):
2
O, NaOD) d (ppm) À4.24. ESI HRMS Calcd for C19
H 42 3FN
2 7
O PS (M-H ):
4
1
R
= 15.68 min (95.1%).3
m
max
3
1
2
433, 2360, 2341, 1711, 1640, 1564, 1501, 1412, 1303, 1262, 1158, 1094, 1058,
À1
1
021, 803, 668, 640, 611, and 561 cm
.15 (m, 2H, CH N), 2.24–2.34 (m, 4H, N(CH
), 2.98 (dd, 1H, J = 3.8 and 12.3 Hz, NHCH
-morpholine), 3.76 (s, 3H, CH ), 4.00–4.05 (m, 1H, CH), 6.97 (d, 2H,
J = 8.6 Hz, Ar), 7.61 (d, 2H, J = 8.6 Hz, Ar), NH and 2OH not observed, exchanged.
;
H NMR (D
-morpholine), 2.66 (dd, 1H, J = 7.8
), 3.52–3.55 (m, 4H,
2
O, NaOD) d (ppm) 2.08–
2
2 2
)
and 12.3 Hz, NHCH
O(CH
a
b
)
2 2
3
Acknowledgments
31
À
P NMR (D
409.0835. Found: 409.0822. HPLC: tR = 8.947 min (97.6%).3
1-(4-Fluorophenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate
7b): Yield 40%; white crystals, mp 130–135 °C; IR (KBr): max 3431, 2361, 2342,
717, 1640, 1593, 1496, 1406, 1327, 1294, 1272, 1240, 1157, 1091, 1030, 982,
2
22 2 8
O, NaOD) d (ppm) 3.95. ESI HRMS Calcd for C1 44 H N O PS (M-H ):
This work was supported by the European Union FP6 Integrated
Project EUR-INTAFAR (Project No. LSHM-CT-2004-512138) and the
Ministry of Higher Education, Science and Technology of the
Republic of Slovenia. The authors thank Dr. Chris Berrie for critical
reading of the manuscript.
(
m
1
8
À1
1
42, 775, 668, 640, 612, and 550 cm
N(CH -morpholine), 2.74 (dd, 1H, J = 8.2 and 12.0 Hz, NHCH
dd, 1H, J = 4.1 and 12.0 Hz, NHCH ), 3.63 (t, 4H, J = 3.9 Hz, O(CH -morpholine),
.09–4.14(m, 1H, CH), 7.21(dd, 2H, J = 8.8and 8.8 Hz, Ar), 7.74 (dd, 2H, J = 5.7 and
;
H NMR (D
2
O, NaOD) d (ppm) 2.21-2.48
(m, 6H, CH
(
2
2
)
2
a
), 3.07
b
2 2
)
4
8
3
References and notes
31
.0 Hz, Ar), NH and 2OH not observed, exchanged. P NMR (D
.95. ESI HRMS Calcd for C13
2
O, NaOD) d (ppm)
À
2
H419FN O
7
PS (M-H ): 397.0635. Found: 397.0640.
1
.
Sievert, D. M.; Rudrik, J. T.; Patel, J. B.; McDonald, L. C.; Wilkins, M. J.; Hageman,
J. C. Clin. Infect. Dis. 2008, 46, 668.
HPLC: t
R
= 8.920 min (97.9%).3
32. Pyruvate kinase/lactate dehydrogenase coupled assay (PK/LDH pathway) used
ADP for the formation of pyruvate from phosphoenolpyruvate (PEP) by the
action of PK. LDH then catalyses the reduction of pyruvate to lactate by NADH,
so by using this assay, ligase activity can be measured spectrophotometrically
by following a decrease in absorbance of NADH at 340 nm, concommitant with
2.
3.
4.
5.
Barna, J. C. J.; Williams, D. H. Annu. Rev. Microbiol. 1984, 38, 339.
Schafer, M.; Schneider, T. R.; Sheldrick, G. M. Structure 1996, 4, 1509.
Murray, B. E. N. Engl. J. Med. 2000, 342, 710.
Barreteau, H.; Kova cˇ , A.; Boniface, A.; Sova, M.; Gobec, S.; Blanot, D. FEMS
Microbiol. Rev. 2008, 32, 168.
+
its oxidation to NAD . For DdlB, all of the assays were carried out in a final
6
7
8
.
.
.
Walsh, C. T.; Fisher, S. L.; Park, I.-S.; Prahalad, M.; Wu, Z. Chem. Biol. 1996, 3, 21.
Arthur, M.; Courvalin, P. Antimicrob. Agents Chemother. 1993, 37, 1563.
Bugg, T. D. H.; Wright, G. D.; Dutka-Malen, S.; Arthur, M.; Courvalin, P.; Walsh,
C. T. Biochemistry 1991, 30, 10408.
volume of 200
KCl, pH 7.5, 10% (v/v) DMSO with or without the compound, 2 mM PEP, 0.2 mM
ATP, 1.972 units/mL PK, 2.46 units/mL LDH, 150 M NADH and 4.3 mM -Ala.
Assays were followed spectrophotometrically at 37 °C, at 340 nm, and the
reaction was initiated by the addition of DdlB (1.2 g/mL), which gave an
2
lL and initially contained 100 mM Hepes, 10 mM MgCl , 10 mM
l
D
9
.
Bugg, T. D.; Dutka-Malen, S.; Arthur, M.; Courvalin, P.; Walsh, C. T. Biochemistry
l
1
991, 30, 2017.
initial slope of À0.210 ± 0.032 (n = 6, uninhibited reaction). For VanA, the assays
1
1
0. Park, I.-S.; Lin, C.-H.; Walsh, C. T. Biochemistry 1996, 35, 10464.
1. Healy, V. L.; Mullins, L. S.; Li, X.; Hall, S. E.; Raushel, F. M.; Walsh, C. T. Chem.
Biol. 2000, 7, 505.
were carried out in the same manner as for DdlB above, except that the
concentrations of the substrates in this case were 8 mM
D-Ala, 1.78 mM
D-Lac,
and the reaction was initiated by the addition of VanA (37.5
l
g/mL), which gave
1
2. Healy, V. L.; Lessard, I. A. D.; Roper, D. I.; Knox, J. R.; Walsh, C. T. Chem. Biol.
an initial slope of À0.215 ± 0.019 (n = 6, uninhibited reaction).
2
000, 7, R109.
33. Liu, S.; Chang, J. S.; Herberg, J. T.; Horng, M.-M.; Tomich, P. K.; Lin, A. H.;
Marotti, K. R. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15178.
1
3. Ellsworth, B. A.; Tom, N. J.; Barlett, P. A. Chem. Biol. 1996, 3, 37.
4. Pratt, S. D.; Xuei, X.; Mackinnon, A. C.; Nilius, A. M.; Hensey-Rudloff, D. M.;
Zhong, P.; Katz, L. J. Biomol. Screen. 1997, 2, 241.
1
34. HPLC analyses were performed on an Agilent Technologies HP 1100 instrument
with G1365B UV-vis detector (254 nm), using
a Luna C18 column
1
5. Bugg, T. D. H. In Comprehensive Natural Products Chemistry; Pinto, B. M., Ed.;
Elsevier: Oxford, 1999; Vol. 3, pp. 241–294.
(4.6 Â 250 mm) at a flow rate of 1 mL/min. The eluant was a mixture of 0.1%
TFA in water (A) and acetonitrile (B). The gradient was from 10% B to 80% B in
30 min (for compounds 6a and 6b) or 5% B to 95% B in 15 min (for compounds
7a and 7b).
1
6. Marshall, E. Science 2008, 321, 364.
1
7. Gillett, V. J.; Newell, W.; Mata, P.; Myatt, G.; Sike, S.; Zsoldos, Z.; Johnson, A. P. J.
Chem. Inf. Comput. Sci. 1994, 34, 207.