Báo cáo khoa học: Inhibition of aryl acid adenylation domains involved in bacterial siderophore synthesis ppt

Inhibition of aryl acid adenylation domains involved in
bacterial siderophore synthesis
Marcus Miethke
1
, Philippe Bisseret
2
, Carsten L. Beckering
1
, David Vignard
2
, Jacques Eustache
2
and Mohamed A. Marahiel
1
1 Philipps-Universita
¨
t Marburg, Fachbereich Chemie ⁄ Biochemie, Marburg, Germany
2 Laboratoire de Chimie Organique et Bioorganique associe
´
au CNRS, Ecole Nationale Supe
´
rieure de Chimie de Mulhouse, Mulhouse,
France
Iron is an essential cofactor for many cellular proces-
ses such as electron transport, synthesis of amino
acids, nucleosides and DNA. However, microorgan-
isms that colonize habitats in an aerobic environment
or in host organisms have to deal with severe iron limi-
tation. Many bacteria have developed high affinity iron
chelators known as siderophores that serve as powerful
tools to extract iron from extracellular sources [1,2].

Due to the fact that these iron chelators are important
virulence factors of various pathogenic bacteria, the
therapeutic potential of siderophore biosynthesis inhi-
bition is obvious [3]. Different classes of siderophores
are known, which contain either aryl, hydroxamoyl or
carboxyl moieties that provide the iron coordinating
ligands. They are generated by different biosynthetic
pathways. Synthesis of hydroxamate and carboxylate
siderophores commonly depends on a diverse spec-
trum of enzymatic activities, including, for example,
Keywords
aryl acid adenylation domain; enzyme
inhibition; iron limitation; pathogenicity;
siderophore
Correspondence
J. Eustache, Laboratoire de Chimie
Organique et Bioorganique (CNRS UMR
7015), Ecole Nationale Supe
´
rieure de
Chimie de Mulhouse, 3 rue Alfred Werner,
F-68093 Mulhouse-Cedex, France
Fax: +33 3 89 33 6860
Tel: +33 3 89 33 6858
E-mail:
M. A. Marahiel, Philipps-Universita
¨
t
Marburg, Fachbereich Chemie ⁄ Biochemie,
Hans-Meerwein-Strasse, D-35032 Marburg,

Germany
Fax: +49 06421 2822191
Tel: +49 06421 2825722
E-mail:
(Received 5 October 2005, accepted
24 November 2005)
doi:10.1111/j.1742-4658.2005.05077.x
Aryl acid adenylation domains are the initial enzymes for aryl-capping of
catecholic siderophores in a plethora of microorganisms. In order to over-
come the problem of iron acquisition in host organisms, siderophore bio-
synthesis is decisive for virulence development in numerous important
human and animal pathogens. Recently, it was shown that growth of
Mycobacterium tuberculosis and Yersinia pestis can be inhibited in an iron-
dependent manner using the arylic acyl adenylate analogue 5¢-O-[N-(sali-
cyl)-sulfamoyl] adenosine that acts on the salicylate activating domains,
MbtA and YbtE [Ferreras JA, Ryu JS, Di Lello F, Tan DS, Quadri LEN
(2005) Nat Chem Biol 1, 29–32]. The present study explores the behaviour
of the 2,3-dihydroxybenzoate activating domain DhbE (bacillibactin syn-
thesis) and compares it to that of YbtE (yersiniabactin synthesis) upon
enzymatic inhibition using a set of newly synthesized aryl sulfamoyl adeno-
sine derivatives. The obtained results underline the highly specific mode of
inhibition for both aryl acid activating domains in accordance with their
natively accepted aryl moiety. These findings are discussed regarding the
structure–function based aspect of aryl substrate binding to the DhbE and
YbtE active sites.
Abbreviations
A, adenylation; AMN, 5¢-O-[N-(aryl)-hydroxamoyl] adenosine; AMS, 5¢-O-[N-(aryl)-sulfamoyl] adenosine; DEAD, diethyl azodicarboxylate;
DHB, 2,3-dihydroxybenzoate; HRMS, high resolution mass spectrometry; NRPS, nonribosomal peptide synthetase; PKS, polyketide
synthase; PP
i

, inorganic pyrophosphate; SAL, salicylate; TFA, trifluoroacetic acid; THF, tetrahydrofluran.
FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 409
monooxygenases, decarboxylases, aminotransferases
and aldolases [4]. In contrast, all aryl-capped sideroph-
ores known so far are mainly assembled by nonribo-
somal peptide synthetases (NRPSs), where polyketide
synthases (PKSs) are sometimes additionally involved
[5,6]. Either 2,3-dihydroxybenzoate (DHB) or salicylate
(SAL) serve as initial substrates for nonribosomal syn-
thesis depending on enzymatic equipment of the micro-
organisms to convert the chorismic acid that is used as
a precursor for both substrates. Both DHB and SAL
containing siderophores represent virulence factors of
important human pathogens (Fig. 1A). Aryl substrate
activation is catalyzed by aryl acid adenylation (A)
domains, forming acyl adenylate intermediates
(Fig. 1B). According to the organism-specific usage of
DHB or SAL as an aryl cap, two types of aryl acid A
domains are known, showing different substrate speci-
ficities for both compounds. So far, aryl acid A
domains are characterized as lone-standing enzymatic
domains within the modular NRPS ⁄ PKS ⁄ NRPS–PKS
A
B
C
Fig. 1. Structures and synthesis of aryl-
capped siderophores. (A) DHB- and SAL-
capped siderophores of human pathogens;
aryl moieties are shown in red; R
1

in
Mycobactin T stands for a variable N-acyl
chain. (B) General reaction catalyzed by aryl
acid A domains; the aryl acid can be either
DHB or SAL. (C) Modular organization of
siderophore biosynthesis exemplified for the
bacillibactin nonribosomal peptide synthe-
tase (NRPS) and the yersiniabactin
NRPS ⁄ nonribosomal peptide polyketide
hybrid synthetase (NRPS-PKS). The lone
standing aryl acid A domains DhbE and YbtE
are shown in red. Abbreviation of domains:
A, adenylation; ACP, acyl carrier protein;
ArCP, aryl carrier protein; AT, acyltrans-
ferase; C, condensation; Cy, cyclization ⁄ con-
densation; ICL, isochorismate lyase; KR,
a-ketoreductase; KS, ketoacyl synthase; MT,
methyltransferase; PCP, peptidyl carrier pro-
tein; TE, thioesterase.
Inhibition of aryl acid adenylation domains M. Miethke et al.
410 FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS
clusters (Fig. 1C). The crystal structure of Bacillus sub-
tilis DhbE activating DHB for bacillibactin synthesis
has been solved, and the cocrystallization with the
DHB acyl adenylate revealed the binding nature of the
activated aryl substrate [7]. This was the first step
towards the development of acyl adenylate analogues
as potential inhibitors of aryl acid A domains.
Recently, the first analogue of this kind, a 5¢-O-[N-(sal-
icyl)-sulfamoyl] adenosine (SAL-AMS), was found to

be a potent inhibitor of several SAL A domains and
was shown to inhibit growth of SAL-capped sidero-
phore producing Mycobacterium tuberculosis and Yer-
sinia pestis in iron-depleted medium [8].
In this context it is remarkable that the class of
aryl-capped siderophores is widely distributed among
pathogenic bacteria. For example, DHB-capped sider-
ophores are produced by Vibrio spp., Bacillus spp. or
enteric bacteria like Escherichia coli or Salmonella spp.
[9–12]. Therefore, information on how a DHB A
domain behaves upon inhibition with the known SAL-
AMS or novel synthetic analogues would be desirable.
This study shows the inhibition of the DHB activating
domain DhbE with SAL-AMS and new derivatives of
this inhibitor. We show that more hydrophobic ana-
logues with expected higher membrane penetrating
potential act as good DhbE inhibitors. Furthermore,
we present a quantitative estimation of the preferential
inhibition of DhbE and the SAL A domain, YbtE, by
two analogues corresponding to the native products of
catalysis. The relationship between substrate specificity
and inhibition effectiveness is discussed based on
DhbE structural information, by comparing models of
the aryl acid binding pockets of the two domains.
Results and Discussion
Inhibition of DhbE with SAL-AMS and a set of
new modified inhibitors
In order to study inhibition of a DHB activating A
domain, Bacillus subtilis DhbE, whose structure is
known, was chosen as target enzyme. Six synthetic acyl

analogues, among them SAL-AMS and five new ana-
logues representing either 5¢-O-[N-(aryl)-sulfamoyl]
adenosine (AMS) or 5¢-O-[N-(aryl)-hydroxamoyl] adeno-
sine (AMN) derivatives were screened for inhibitory
effects (Fig. 2). Enzyme activity with and without
inhibitors was analyzed by adenosine triphosphate ⁄
inorganic pyrophosphate (ATP ⁄ PP
i
) exchange. At sat-
urating concentrations of both DHB and ATP and an
inhibitor : DHB ratio of 1 : 20, the four AMS com-
pounds exhibited strong inhibition ranging from 86 to
> 99% loss of DhbE activity (Table 1). DHB-AMS
and SAL-AMS had the greatest inhibitory effects with
A
B
C
F
E
D
Fig. 2. Synthetic acyl adenylate analogues for aryl acid A domain inhibition. (A) 5¢-O-[N-(benzoyl)-sulfamoyl] adenosine (BEN-AMS). (B)
5¢-O-[N-(o-fluorobenzoyl)-sulfamoyl] adenosine (F-BEN-AMS). (C) 5¢-O-[N-(salicyl)-sulfamoyl] adenosine (SAL-AMS), reported by [8]. (D) 5¢-O-
[N-(2,3-dihydroxybenzoyl)-sulfamoyl] adenosine (DHB-AMS). (E) 5¢-O-[N-(benzoyl)-hydroxamoyl] adenosine (BEN-AMN). (F) 5¢-O-[N-(o-fluoro-
benzoyl)-hydroxamoyl] adenosine (F-BEN-AMN). Colour code: Aryl moieties (red), sulfamoyl moieties (blue), hydroxamoyl moieties (green),
adenosyl moieties (black).
M. Miethke et al. Inhibition of aryl acid adenylation domains
FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 411
an inhibition strength of more than 99% under the
chosen conditions. 5¢-O-[N-(o-fluorobenzoyl)-sulfa-
moyl]adenosine (F-BEN-AMS) and 5¢-O-[N-(benzoyl)-

sulfamoyl]adenosine (BEN-AMS) with more
hydrophobic aryl moieties showed about 90% of
DhbE inhibition, indicating that these compounds are
promising candidates for in vivo studies due to their
expected higher membrane penetrating abilities. In
contrast, the AMN derivatives tested here showed inhi-
bition only to a negligible extent and were not studied
further. Compared with the a-phosphate in the native
acyl adenylate, the shortened hydroxamoyl linker of
the AMN compounds may impair fitting of the aryl
and ⁄ or adenosine moiety to the active site, resulting in
weak inhibitory properties. In contrast, the sulfamoyl
linker of the AMS inhibitors shows almost no struc-
tural difference to the native linker, enabling both the
aryl and adenosine moieties to bind properly to their
cognate pockets as shown in the model for the DHB-
AMS in Fig. 3D.
Determination of inhibition constants for
SAL-AMS and DHB-AMS with DhbE and YbtE
The DHB-AMS and SAL-AMS were selected for
detailed inhibition studies. Target enzymes were
Table 1. Results of inhibition studies. Acyl adenylate analogues
(Fig. 2) were tested for DhbE inhibition at a concentration of
12.5 l
M. ATP and DHB concentrations were at saturating levels of
2m
M and 250 lM, respectively. ATP ⁄ PP
i
exchange reactions were
carried out for 5 min at 37 °C.

Acyl analogue DhbE inhibition (%)
BEN-AMS 86.4
F-BEN-AMS 91.1
SAL-AMS 99.6
DHB-AMS 99.8
BEN-AMN 1.8
F-BEN-AMN 0.1
Fig. 3. Schematic presentation of aryl acid A domain binding pockets. (A) DhbE binding pocket with DHB substrate based on crystal struc-
ture data [7]. (B) Suggested model of the YbtE binding pocket with SAL substrate based on sequence alignments. Colour code of depicted
amino acid residues: Unpolar (blue), polar (yellow), basic (green). DHB and SAL are shown in grey; carboxy groups (dotted) and hydroxy
groups (striped) are indicated. (C) Alignment of DhbE and YbtE amino acids forming the aryl substrate binding pockets. The differing amino
acids are indicated by asterisks. The amino acids with the highest proposed impact for the aryl substrate specificity are shown in red. (D)
Model of the DHB-AMS inhibitor bound to the DhbE active site. Polar interactions are indicated by dashed lines.
Inhibition of aryl acid adenylation domains M. Miethke et al.
412 FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS
B. subtilis DhbE and the SAL activating domain YbtE
of Y. pestis. At first, specificities of the enzymes for
either DHB or SAL were confirmed by ATP ⁄ PP
i
exchange. Michaelis–Menten constants (K
m
) were
K
m
(DHB) ¼ 1.3 ± 0.1 lm and K
m
(SAL) ¼ 81.8 ±
10.6 lm for DhbE and K
m
(DHB) ¼ 325.1 ± 10.5 lm

and K
m
(SAL) ¼ 3.5 ± 0.5 lm for YbtE. These values
were comparable with the corresponding K
m
values
determined previously [9,13]. Then, inhibition con-
stants (K
i
) were determined by ATP ⁄ PP
i
exchange.
ATP was used at saturating concentrations, the DHB
and SAL substrates were used at three concentrations
close to the corresponding K
m
values while the concen-
trations of the inhibitors were varied. The observed
strength of inhibition was inversely proportional to the
aryl substrate concentration, indicating a substrate
dependent mode of inhibition in this kinetic range.
The resulting inhibition constants ranged from 29 to
106 nm revealing effective inhibition of both A
domains with DHB-AMS and SAL-AMS (Table 2).
Furthermore, the K
i
values showed significant differ-
ences depending on the hydroxylation pattern of the
inhibitor aryl moiety and the aryl substrate specificity
of the A domain. DhbE was inhibited 1.25-fold better

with DHB-AMS than with SAL-AMS and YbtE 1.8-
fold better with SAL-AMS than with DHB-AMS. The
observed differences clearly indicate preferential inhibi-
tion by the analogue containing the natively accepted
aryl moiety of the A domain.
Comparison of DhbE and YbtE aryl acid binding
pockets with respect to preferential A domain
inhibition
Comparing the K
i
and K
m
values, the observed prefer-
ential inhibition of DhbE and YbtE by DHB- and
SAL-based inhibitors did not reflect the larger differ-
ences of the aryl substrate specificities. Indeed, the K
m
values for both DHB and SAL differed by approxi-
mately 60-and 90-fold for DhbE and YbtE, respect-
ively. The domain specificities for the aryl substrates
might be explained by comparison of the amino acid
residues that are critical for substrate recognition
based on the DhbE crystal structure. Sequence align-
ments of several DHB and SAL activating domains
showed significant differences between the amino acid
residues forming the aryl acid binding pockets accord-
ing to the nonribosomal code of A domain specificity
[7,14]. Therefore, a model of a putative binding pocket
valid for SAL activating domains can be proposed and
is exemplified here for YbtE in comparison with the

DHB binding pocket of DhbE (Fig. 3A–C). In agree-
ment with previous analyses [7,8], amino acid residues
Ser240 and Val337 of DhbE and the corresponding
residues Cys227 and Leu324 of YbtE, located at the
bottom of the aryl acid binding pockets, seem to be
involved in key interactions with the mono- or dihy-
droxylated aryl substrates. Especially, the hydrophobic
residues Val (which is conserved in DHB A domains)
or Leu (which is conserved in SAL A domains, but
can be replaced by Ile, e.g. in PchD of Pseudomonas
aeruginosa) with different C-chain length in close prox-
imity to the hydroxylation-variable C
3
position of
the aromatic substrate ring might contribute to the
observed substrate specificities of both domain types.
However, considering the whole A domain, this effect
seems to be overshadowed by the strong contribution
of the adenylate moiety to overall binding, which prob-
ably explains the smaller differences of the K
i
values
for DHB-AMS and SAL-AMS compared to the differ-
ences of the K
m
values for DHB and SAL. This, again,
is in agreement with former observations for aminoacyl
tRNA synthetases revealing higher dissociation con-
stants for the substrates than for the aminoacyl adeny-
lates [15]. Additionally, the linker between aryl and

adenylate moieties might also contribute to effective
binding of the reaction intermediates and their syn-
thetic analogues. The a-phosphate of the native DHB
acyl adenylate as well as the sulfamoyl linker of the
DHB-AMS are in very close proximity to the His234
residue of DhbE ([7] and Fig. 3D), indicating ionic
or at least polar interactions between linker and A
domain. In summary, the model-based structural–func-
tional relationship of aryl acid A domain inhibition
supports the use of acyl adenylate analogues as effect-
ive inhibitors and is in accordance with the experimen-
tal data, indicating that AMS derivatives with high
structural resemblance to the native intermediates can
be favoured over derivatives with, for example, a shor-
tened molecular linker such as AMN compounds.
Conclusion
The development of nonhydrolyzable analogues for
inhibition of acyl adenylate forming enzymes like
aminoacyl tRNA synthetases and NRPS A domains
Table 2. Results of inhibition studies. Inhibition constants (K
i
) were
determined with DHB-AMS and SAL-AMS using DhbE and YbtE
(each 300 n
M) as target enzymes. ATP ⁄ PP
i
exchange reactions
were carried out for 30 s at 37 °C.
Acyl analogue
Inhibition constants (nM)

DhbE YbtE
SAL-AMS 106 29
DHB-AMS 85 54
M. Miethke et al. Inhibition of aryl acid adenylation domains
FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 413
emerged in the last years [16–18]. A novel contribution
to pathogenicity control was the development of the
SAL-AMS compound for aryl acid A domain inhibi-
tion of SAL-capped siderophore producing pathogens
[8]. Inhibition of aryl acid A domains bears the
advantage that higher vertebrates, including humans,
do not possess such class of enzymes, making cross
reactions unlikely. Now, another type of aryl acid A
domain, activating DHB for siderophore synthesis, has
been inhibited for the first time with SAL-AMS and
new AMS derivatives. Testing the SAL-AMS and the
herein presented DHB-AMS with both a DHB and a
SAL activating domain resulted in preferential inhibi-
tion with the analogue containing the aryl moiety of
the native reaction intermediate, indicating that DHB-
AMS might be more suitable for inhibition of DHB-
capped siderophore producing bacteria. The presented
in vitro results and the discussed model for substrate
specificity will lead to a deeper understanding of aryl
acid A domain inhibition mechanisms and may help to
extend the possibilities of pathogen inhibition based on
iron limitation. We now intend to confirm the effect-
iveness of the new AMS compounds by growth inhi-
bition (in vivo studies) using a broad range of
microorganisms producing aryl-capped siderophores.

We will also study in detail the relationship between
inhibitor structure and uptake with the aim of scaling-
down in vivo effective doses to levels enabling potential
therapeutic applications.
Experimental procedures
Synthesis of acyl adenylate analogues
Description of chemical synthesis strategies
Salicyl-derived 5¢-O-sulfamoyladenosine derivatives 8–10
were synthesized from 2¢,3¢-O-isopropylidene-5¢-sulfamoyl-
adenosine 1, which was prepared as already reported
(Scheme 1) [19]
.
For the key coupling reaction between 1
and benzoic acid derivatives, we initially tested, without
success, the coupling conditions previously used for the pre-
paration of amino acid derived sulfamates [20,21]. In par-
ticular, the condensation between 1 and benzoic acid using
carbonyldiimidazole as a coupling agent in the presence of
DBU was not satisfactory. The desired sulfamate 5 was
formed in low yield under these conditions together with
unidentified products of similar polarity. (While this manu-
script was in preparation, the synthesis of the sulfamoyl
derivative 10 was reported [8]. This study showed that
coupling worked well when starting from 2¢,3¢-O-silylated-
5¢-O-sulfamoyl-adenosine instead of the isopropylidene
derivative 1.) Finally, we found that the couplings with 1
could be realized, albeit in modest yields, using the succin-
imidyl-derivatives 2–4 [22–24] and cesium carbonate as a
base. Using other bases like DBU or Et
3

N yielded complex
mixtures containing only traces of the desired coupling
product. Deprotection of the isopropylidene was best real-
ized using 50% aqueous trifluoroacetic acid (TFA) and the
final compounds were purified by thin-layer chroma-
tography (TLC). The hydrolysis step was effective to obtain
Scheme 1. Preparation of salicyl-5¢-O-sulfamoyladenosine derivatives 8–10. Reagents and conditions: (a) Cs
2
CO
3
(2 equiv), room tempera-
ture, 20 h (5, 16%; 6, 31%; 7, 23%); (b) TFA : H
2
O(1:1,v⁄ v), room temperature, 4 h (8, 16%; 9, 63%; 10, 92%).
Inhibition of aryl acid adenylation domains M. Miethke et al.
414 FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS
9 and 10 but, however, more problematic in the case of the
nonsubstituted benzoyl compound 8, which appeared to be
more sensitive to acidic conditions and could be obtained
only in low yield.
Extension of this procedure for preparation of the
dihydroxybenzoyl derivative 13 starting from the succini-
mide derivative of dihydroxybenzoic acid was unsuccessful.
The desired coupling could be achieved, however, using the
benzylidene protected equivalent 11. Both the ispopropylid-
ene and benzylidene groups were removed by TFA treat-
ment as shown above to afford 13 in fair yield (Scheme 2).
We also prepared the hydroxamoyl derivatives 18 and 19
by Mitsunobu coupling of the O-protected N-hydroxy-
amides 14 and 15 (Scheme 3). This was best realized using

polymer-supported triphenylphosphine [25].
Preparation of 5¢-O-sulfamoyladenosine derivatives
8–10 and 13: general procedure
To a solution of 2¢,3¢-O-isopropyliden-5¢-O -sulfamoyladeno-
sine 1 in CH
2
Cl
2
: DMF (7 : 3, v ⁄ v) were added the succin-
imide derivative (2–4 and 11, 1.1 equiv) followed by cesium
Scheme 2. Preparation of 2,3-dihydroxybenzoyl-5¢-O-sulfamoyladenosine 13. Reagents and conditions: (a) Cs
2
CO
3
(2 equiv), room tempera-
ture, 20 h, 30%; (b) TFA : H
2
O (1 : 1, v ⁄ v), room temperature, 4 h, 40%.
Scheme 3. Preparation of hydroxamoyl derivatives 18 and 19. Reagents and conditions: (a) polymer-supported triphenylphosphine (4 equiv),
Diethyl azocarboxylate (DEAD) (4 equiv), room temperature, tetrahydrofluran (THF), 1 h, 80%; (b) TFA : H
2
O(2:1,v⁄ v), room temperature,
3 h, 70%.
M. Miethke et al. Inhibition of aryl acid adenylation domains
FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 415
carbonate (2 equiv). After stirring for 20 h at room tem-
perature, methanol was added and the mixture was concen-
trated under reduced pressure. The residue was purified on
a SiO
2

column [EtOAc : MeOH (9 : 1)] to afford the pro-
tected salicyl derivatives 5–7 and 12 as white solids in the
following yields: 5, 16%; 6, 31%; 7, 23%; 12, 30%.
The protected sulfamates (5–7 and 12) were then stirred
for 4 h at room temperature in a mixture of TFA : H
2
O
(1 : 1). After addition of MeOH and toluene ( 1 : 1), the
reaction medium was concentrated under reduced pressure
and purified by TLC using CHCl
3
: MeOH (2 : 1) as eluent
to afford the free salicyl derivatives 8–10 and 13 in the fol-
lowing yields: 8, 16%; 9, 63%; 10, 92%; 13, 40%.
Preparation of hydroxamoyl derivatives 18 and 19:
general procedure
A 40% solution of diethyl azodicarboxylate (DEAD) in
toluene (4 equiv) was added dropwise at room temperature
to polymer-supported triphenylphosphine (4 equiv), 2¢,3¢-O-
isopropyliden-adenosine (1 equiv) and 14 or 15 (1.2 equiv)
in suspension in tetrahydrofluran (THF). After 1 h of stir-
ring at room temperature, the polymer was filtered-off and
washed with CHCl
3
. The filtrate was concentrated under
reduced pressure and the residue was purified by TLC using
EtOAc : MeOH (9 : 1) as an eluent, yielding the protected
hydroxamoyl derivative 16 or 17 (80% yield), that were
then dissolved into a solution of TFA : H
2

O (2 : 1). After
3 h of stirring at room temperature, MeOH and toluene
(1 : 1) were added and the medium was concentrated under
reduced pressure yielding, after TLC purification [EtO-
Ac : MeOH (9 : 1)], the deprotected derivative 18 or 19 in
70% yield.
Analytical chemistry
Analytical TLC was performed on Merck 60F 254 silica gel
plates (Merck KGaA, Darmstadt, Germany) (spots visual-
ized with UV light at 254 nm and at 366 nm after aspersion
of a 0.1% ethanolic solution of berberine chlorhydrate
and ⁄ or with a vanillin-MeOH ⁄ H
2
SO
4
solution followed by
heating). Column chromatography was carried out on MN
Kieselgel 60 (30–270 mesh) (Macherey-Nagel GmbH & Co.
KG, Du
¨
ren, Germany). NMR spectra were recorded using
a Bruker AVANCE 400 spectrometer (
1
H, 400 MHz;
13
C,
100.69 MHz) (Bruker BioSpin GmbH, Rheinstein, Ger-
many) in CDCl
3
or CD

3
OD as solvent. High resolution MS
were performed under electrospray.
5:R
f
¼ 0.34 (EtOAc : MeOH: 4 : 1, v ⁄ v); [a]
25
D
¼ )33
(c ¼ 0.25; MeOH)
1
H NMR (CD
3
OD, 300 K): d ¼ 8.47 (1H, s), 8.12 (1H,
s), 7.96 (2H, d, J ¼ 7.8 Hz), 7.40 (2H, t, J ¼ 7.5 Hz), 7.31
(1H, t, J ¼ 7.5 Hz), 6.19 (1H, d, J ¼ 3.3 Hz), 5.35 (1H, dd,
J ¼ 3.3 & 6 Hz), 5.13 (1H, dd, J ¼ 2 & 6 Hz), 4.57 (1H,
m), 4.27 (2H, d, J ¼ 3.8 Hz), 1.59 (3H, s), 1.35 (3H, s).
13
C NMR (CD
3
OD, 300 K): d ¼ 175.2, 157.3, 153.9,
150.5, 141.4, 138.9, 132.1, 129.9, 129.5, 128.8, 120.1, 115.2,
91.9, 85.8, 85.7, 83.4, 69.7, 27.5, 25.4.
High resolution mass spectrometry (HRMS) calculated
for C
20
H
23
N

6
O
7
S (MH
+
): 491.1349; found: 491.1350.
6:R
f
¼ 0.37 (EtOAc : MeOH, 8 : 2, v ⁄ v); [a]
25
D
¼ )37
(c ¼ 0.3; MeOH)
1
H NMR (CD
3
OD, 300 K): d ¼ 8.51 (1H, s), 8.18 (1H,
s), 7.70 (1H, t, J ¼ 7.5 Hz), 7.39 (1H, m) 7.14 (1H, t, J ¼
7.5 Hz), 7.05 (1H, dd, J ¼ 8.6; 10.5 Hz), 6.24 (1H, d,
3.3 Hz), 5.39 (1H, dd, J ¼ 3.3; 6 Hz), 5.19 (1H, dd, J ¼ 2;
6 Hz), 4.60 (1H, m), 4.33 (2H, d, J ¼ 3.5 Hz), 1.60 (3H, s),
1.38 (3H, s).
13
C NMR (CD
3
OD, 300 K): d ¼ 173.6, 161.9 (d, J ¼
257 Hz), 157.3, 153.9, 150.5, 141.4, 134.5, 132.7 (d, J ¼
8.4 Hz), 131.8, 124.7, 117.1 (d, J ¼ 13.4 Hz), 115.2, 91.9,
85.8, 85.6, 83.7, 69.9, 27.5, 25.5.
HRMS calculated for C

20
H
22
N
6
O
7
FS (MH
+
): 509.1255;
found: 509.1243.
7:R
f
¼ 0.52 (EtOAc : MeOH, 4 : 1, v ⁄ v)
1
H NMR (CD
3
OD, 300 K): d ¼ 8.47 (1H, s), 8.11 (1H, s),
7.90 (1H, dd, J ¼ 7.8; 1.8 Hz), 7.28 (1H, dt, J ¼ 1.8;
6.8 Hz), 6.74 (2H, m), 6.23 (1H, d, J ¼ 3.1 Hz), 5.38 (1H,
dd, J ¼ 3.1 & 6 Hz), 5.13 (1H, dd, J ¼ 2.3 & 6 Hz), 4.57
(1H, m), 4.33 (2H, d, J ¼ 3.8 Hz), 1.55 (3H, s), 1.31 (3H, s).
13
C NMR (CD
3
OD, 300 K): d ¼ 175.3, 162.0, 157.2,
153.9, 150.3, 141.5, 134.4, 131.4, 120.3, 120.2, 119.3, 117.9,
115.3, 91.9, 85.7, 85.6, 83.2, 69.9, 27.4, 25.4.
HRMS calculated for C
20

H
23
N
6
O
8
S (MH
+
): 509.1298;
found: 509.1286.
8:R
f
¼ 0.40 (CHCl
3
: MeOH, 2 : 1, v⁄ v); [a]
25
D
¼ )18
(c ¼ 0.5; MeOH)
1
H NMR (CD
3
OD): d ¼ 8.55 (1H, s), 8.17 (H, s), 8.02
(2H, d, J ¼ 7.1 Hz), 7.43 (1H, t, J ¼ 7.1 Hz), 7.35 (2H, t,
J ¼ 7.1 Hz), 6.10 (1H, d, J ¼ 6.0 Hz), 4.72 (1H, t, J ¼
6.0 Hz), 4.38 (4H, m).
13
C NMR (CD
3
OD): d ¼ 175.3, 157.2, 153.8, 150.9,

141.2, 138.9, 132.1, 129.9, 128.8, 120.1, 92.5, 89.2, 76.1,
72.4, 69.2.
HRMS calculated for C
17
H
19
N
6
O
7
S (MH
+
): 451.1036;
found: 451.1029.
9:R
f
¼ 0.41 (CHCl
3
: MeOH, 2 : 1, v⁄ v); [a]
25
D
¼ )13
(c ¼ 0.5; MeOH)
1
H NMR (CD
3
OD): d ¼ 8.53 (1H, s), 8.19 (1H, s), 7.73
(1H, t, J ¼ 7.6 Hz), 7.38 (1H, m), 7.13 (1H, t, J ¼ 7.6 Hz),
7.07 (1H, dd, J ¼ 8.3; 10,6 Hz), 6.10 (1H, d, J ¼ 5.7 Hz),
4.73 (1H, t, J ¼ 5.7 Hz), 4.43 (4H, m).

13
C NMR (CD
3
OD): d ¼ 174.5, 163.8, 162.9 (d, J ¼
250 Hz), 158.1, 154.7, 151.7, 141.9, 133.8 (d, J ¼ 8.4 Hz),
132.8, 129.2, 121.0, 118.0 (d, J ¼ 29.7 Hz), 90.3, 85.4, 76.8,
73.2, 70.4.
HRMS calculated for C
17
H
18
N
6
O
7
FS (MH
+
): 469.0942;
found: 469.0952.
10:R
f
¼ 0.44 (CHCl
3
: MeOH, 2 : 1, v ⁄ v); [a]
25
D
¼ )14
(c ¼ 0.3; MeOH)
Inhibition of aryl acid adenylation domains M. Miethke et al.
416 FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS

1
H NMR (CD
3
OD): d ¼ 8.49 (1H, s), 8.13 (1H, s), 7.90
(1H, d, J ¼ 8 Hz), 7.23 (1H, t, J ¼ 8 Hz), 6.74 (2H, m),
6.05 (1H, d, J ¼ 5.8 Hz), 4.68 (1H, t, J ¼ 5.3 Hz), 4.68
(4H, m).
13
C NMR (CD
3
OD): d ¼ 175.1, 162.1, 157.2, 153.8,
150.9, 141.1, 134.3, 131.4, 120.6, 120.1, 119.2, 117.8, 89.1,
84.6, 76.1, 72.4, 69.5, 69.0.
HRMS calculated for C
17
H
19
N
6
O
8
S (MH
+
): 467.0985;
found: 467.0994.
12 (mixture of diastereomers): R
f
¼ 0.27 (EtO-
Ac : MeOH, 4 : 1, v ⁄ v)
1

H NMR (CD
3
OD, 300 K): d ¼ 8.4 (1H, s), 8.05 (1H,
m), 6.7–7.5 (9H, m), 6.08 (1H, broad s), 5.18 (1H, broad s),
5.02 (1H, broad s), 4.42 (1H, broad s), 4.21 (2H, broad s),
1.47 (3H, s), 1.19 (3H, s).
13
C NMR (CD
3
OD, 300 K): although the purity of the
corresponding final deprotected compound 13 was at the
end excellent, 12 itself was difficult to purify and did not
give a good
13
C NMR.
13:R
f
¼ 0.21 (CHCl
3
: MeOH, 2 : 1, v ⁄ v)
1
H NMR (CD
3
OD): d ¼ 8.43 (1H, s), 8.06 (1H, s), 7.33
(1H, d, J ¼ 8.0 Hz), 6.74 (1H, d, J ¼ 8.0 Hz), 6.51 (1H, t,
J ¼ 8.0 Hz), 5.98 (1H, d, J ¼ 5.8 Hz), 4.61 (1H, t, J ¼
5.8 Hz), 4.3 (4H, m).
13
C NMR (CD
3

OD): d ¼ 175.2, 164.3, 157.3, 153.8,
150.6, 146.8, 141.1, 121.9, 120.9, 119.3, 118.5, 89.1, 84.7,
76.1, 72.5, 69.5.
16:R
f
¼ 0.46 (EtOAc : MeOH, 9 : 1, v ⁄ v); [a]
25
D
¼ )20
(c ¼ 0.5; CHCl
3
)
1
H NMR (CDCl
3
): d ¼ 8.29 (1H, s), 8.00 (1H, s), 7.55
(2H, d, J ¼ 7.5 Hz), 7.30 (5H, m), 6.85 (2H, d, J ¼
8.6 Hz), 6.14 (1H, d, J ¼ 2.5 Hz), 5.62 (1H, broad s), 5.29
(1H, dd, J ¼ 2.5 & 6.3 Hz), 5.12 (1H, dd, J ¼ 2.8 &
6.3 Hz), 5.02 (2H, s), 4.52 (1H, m), 4.43 (1H, dd, J ¼ 3.8 &
11.0 Hz), 4.32 (1H, dd, J ¼ 5.0 & 11.0 Hz), 3.79 (3H, s),
1.61 (3H, s), 1.34 (3H, s).
13
C NMR (CDCl
3
): d ¼ 159.4, 155.6, 153.9, 153.0, 149.4,
139.5, 130.3, 130.2, 130.0, 129.3, 128.3, 126.9, 120.0, 114.4,
113.7, 90.6, 85.6, 84.2, 81.4, 76.5, 71.0, 55.2, 27.1, 25.2.
17:R
f

¼ 0.45 (EtOAc : MeOH, 9 : 1, v ⁄ v)
1
H NMR (CDCl
3
): d ¼ 8.23 (1H, s), 8.08 (1H, s), 7.30
(7H, m), 7.02 (2H, dd, J ¼ 7.6 & 10.3 Hz), 6.15 (1H, d,
J ¼ 2.8 Hz), 5.84 (1H, broad s), 5.20 (1H, dd, J ¼ 2.8 Hz
& 6.3 Hz), 5.09 (2H, s), 5.06 (1H, m), 4.51 (1H, m), 4.28
(1H, dd, J ¼ 3.3 Hz & 11.3 Hz), 4.18 (1H, dd, J ¼ 4.6 Hz
& 11.3 Hz), 1.61 (3H, s), 1.34 (3H, s).
13
C NMR (CDCl
3
): d ¼ 161.5, 158.9, 155.2, 152.8, 150.6
(d, J ¼ 234 Hz), 139.6, 137.3, 132.2, 131.2, 128, 4, 128.3,
127.9, 124.3, 119.9, 116.0, 114.4, 90.7, 85.3, 84.6, 81.4, 70.6,
27.2, 25.3.
HRMS calculated for C
27
H
28
N
6
O
5
F (MH
+
): 535.2105;
found: 535.2083.
18:R

f
¼ 0.30 (EtOAc : MeOH, 9 : 1, v ⁄ v); [a]
25
D
¼ )17
(c ¼ 0.2; MeOH)
1
H NMR (CD
3
OD): d ¼ 8.20 (1H, s), 8.10 (1H, s), 7.96
(2H, d, J ¼ 7.1 Hz), 7.60 (1H, t, J ¼ 7.7 Hz), 7.46 (2H, t,
J ¼ 7.7 Hz), 6.02 (1H, d, J ¼ 4.3 Hz), 4.71 (1H, dd, J ¼
3.5 & 12.1 Hz), 4.58 (2H, m), 4.37 (1H, dd, J ¼ 4.8 &
8.6 Hz).
13
C NMR (CDCl
3
): d ¼ 167.7, 157.3, 153.9, 150.5, 141.4,
134.4, 131.0, 130.6, 129.6, 90.7, 83.5, 74.9, 71.8, 64.9.
19:R
f
¼ 0.30 (AcOEt : MeOH, 9 : 1, v ⁄ v); [a]
25
D
¼ )42
(c ¼ 0.3; MeOH)
1
H NMR (CDCl
3
): d ¼ 8,19 (1H, s), 8,09 (1H, s), 7.89

(1H, t, J ¼ 7.3 Hz), 7.63 (1H, m), 7.23 (2H, m), 6.03 (1H,
d, J ¼ 4.3 Hz), 4.82 (1H, m, H-2), 4.72 (1H, dd, J ¼ 3.2 &
12 Hz), 4.58 (2H, m), 4.38 (1H, m)
13
C NMR (CDCl
3
): d ¼ 165.4, 163.2 (d, J ¼ 259 Hz),
157.3, 153.9, 153.5, 150.5, 141.1, 136.3, 136.2, 133.2, 125.4,
120.5, 118.1, 117.9, 90.5, 83.5, 75.1, 71.7, 65.2.
Overproduction and purification of proteins
The His6-tagged fusions of B. subtilis DhbE and Y. pestis
YbtE were produced and purified according to described
procedures [9,13]. Proteins were stocked with and without
10% glycerol at )20 °C without observed differences in
activity.
ATP-pyrophosphate exchange assays and
determination of kinetic constants
The ATP ⁄ PP
i
exchange reaction [26] was used to determine
substrate specificity of DhbE and YbtE for DHB and SAL
and the quality of inhibition for the A domain inhibitors.
For all assays, the enzyme concentration was 300 nm and
ATP concentration was at a saturating level of 2 mm. All
reactions were performed at 37 °C. To confirm the aryl
substrate specificities, DHB and SAL concentrations were
varied from 0 to 250 lm for testing DhbE and 0–500 lm
for testing YbtE. ATP ⁄ PP
i
exchange reactions were carried

out for 30 s for each substrate concentration. K
m
values
were determined by using the nonlinear fit modeling option
of Microcal
tm
origin
tm
5.0 software (Microcal Software
Inc., Northampton, MA, USA). The inhibitory effect of the
six acyl adenylate analogues on DhbE was tested using
12.5 lm of inhibitor and 250 lm of DHB. The ATP ⁄ PP
i
exchange reactions were stopped after 5 min. To deter-
mine the K
i
values for DhbE with both DHB-AMS and
SAL-AMS, the DHB concentration was set around the K
m
value at concentrations of either 0.5, 1 or 2 lm, while the
concentration of the inhibitors was varied from 0 to 50 nm.
In the case of YbtE, the SAL concentration was set at 2.25,
4.5 or 9 lm and the concentration of the inhibitors was
varied from 0 to 25 nm. The ATP ⁄ PP
i
exchange reactions
were carried out for 30 s. The inhibition constants were cal-
culated using the Dixon plot method.
M. Miethke et al. Inhibition of aryl acid adenylation domains
FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 417

Acknowledgements
We are indebted to Prof. Dr Christopher T. Walsh,
Harvard Medical School, Boston, Massachusetts, for
the kind supply of the YbtE overexpression strain. We
would like to thank furthermore Prof. Dr Lars-Oliver
Essen for in silico analyses, Dr Uwe Linne for analytical
measurements and Oliver Klotz for practical support.
The work in Germany was supported by grants from the
EC (‘Bacterial stress management relevant to infectious
disease and pharmaceuticals’, Bacell Health, LSHG-
CT-2004-503468), the Deutsche Forschungsgemeinsc-
haft and Fonds der Chemischen Industrie, the work in
France by the Ministe
`
re de la Recherche and the Centre
National de la Recherche Scientifique (CNRS).
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