Aryl substrate activation is catalyzed by aryl acid adenylation A domains, forming acyl adenylate intermediates Fig.. This was the first step towards the development of acyl adenylate ana
Trang 1bacterial siderophore synthesis
Marcus Miethke1, Philippe Bisseret2, Carsten L Beckering1, David Vignard2, Jacques Eustache2 and Mohamed A Marahiel1
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: jacques.eustache@uha.fr
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: marahiel@chemie.uni-marburg.de
(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 tuberculosisand 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 synsyn-thesis) 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.
Trang 2monooxygenases, 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.
Trang 3clusters (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 pestisin 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⁄ PPi) 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
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) [N-(o-fluorobenzoyl)-sulfamoyl] adenosine (F-BEN-AMS) (C) [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).
Trang 4an 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
2 m M and 250 l M , respectively ATP ⁄ PP i exchange reactions were
carried out for 5 min at 37 C.
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.
Trang 5B subtilisDhbE and the SAL activating domain YbtE
of Y pestis At first, specificities of the enzymes for
either DHB or SAL were confirmed by ATP⁄ PPi
exchange Michaelis–Menten constants (Km) were
Km(DHB) ¼ 1.3 ± 0.1 lm and Km(SAL) ¼ 81.8 ±
10.6 lm for DhbE and Km(DHB) ¼ 325.1 ± 10.5 lm
and Km(SAL) ¼ 3.5 ± 0.5 lm for YbtE These values
were comparable with the corresponding Km values
determined previously [9,13] Then, inhibition
con-stants (Ki) were determined by ATP⁄ PPi exchange
ATP was used at saturating concentrations, the DHB
and SAL substrates were used at three concentrations
close to the corresponding Kmvalues 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 Ki 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 Ki and Kmvalues, 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 Km
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 C3 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 Ki values for DHB-AMS and SAL-AMS compared to the differ-ences of the Kmvalues 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 (Ki) 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)
Trang 6emerged 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
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 Et3N 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 CO3(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%).
Trang 79 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 benzylidispopropylid-ene 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 CH2Cl2: 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 CO3(2 equiv), room tempera-ture, 20 h, 30%; (b) TFA : H2O (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 : H2O (2 : 1, v ⁄ v), room temperature,
3 h, 70%.
Trang 8carbonate (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 SiO2 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 : H2O
(1 : 1) After addition of MeOH and toluene ( 1 : 1), the
reaction medium was concentrated under reduced pressure
and purified by TLC using CHCl3: 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 CHCl3 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 : H2O (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 ⁄ H2SO4solution 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 (1H, 400 MHz; 13C,
100.69 MHz) (Bruker BioSpin GmbH, Rheinstein,
Ger-many) in CDCl3or CD3OD as solvent High resolution MS
were performed under electrospray
5: Rf¼ 0.34 (EtOAc : MeOH: 4 : 1, v ⁄ v); [a]25
(c¼ 0.25; MeOH)
1
H NMR (CD3OD, 300 K): d¼ 8.47 (1H, s), 8.12 (1H,
s), 7.96 (2H, d, J¼ 7.8 Hz), 7.40 (2H, t, J ¼ 7.5 Hz), 7.31
(1H, t, J¼ 7.5 Hz), 6.19 (1H, d, J ¼ 3.3 Hz), 5.35 (1H, dd,
J¼ 3.3 & 6 Hz), 5.13 (1H, dd, J ¼ 2 & 6 Hz), 4.57 (1H,
m), 4.27 (2H, d, J¼ 3.8 Hz), 1.59 (3H, s), 1.35 (3H, s)
13C NMR (CD3OD, 300 K): d¼ 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 C20H23N6O7S (MH+): 491.1349; found: 491.1350 6: Rf¼ 0.37 (EtOAc : MeOH, 8 : 2, v ⁄ v); [a]25
(c¼ 0.3; MeOH)
1
H NMR (CD3OD, 300 K): d¼ 8.51 (1H, s), 8.18 (1H, s), 7.70 (1H, t, J¼ 7.5 Hz), 7.39 (1H, m) 7.14 (1H, t, J ¼ 7.5 Hz), 7.05 (1H, dd, J¼ 8.6; 10.5 Hz), 6.24 (1H, d, 3.3 Hz), 5.39 (1H, dd, J¼ 3.3; 6 Hz), 5.19 (1H, dd, J ¼ 2;
6 Hz), 4.60 (1H, m), 4.33 (2H, d, J¼ 3.5 Hz), 1.60 (3H, s), 1.38 (3H, s)
13
C NMR (CD3OD, 300 K): d¼ 173.6, 161.9 (d, J ¼
257 Hz), 157.3, 153.9, 150.5, 141.4, 134.5, 132.7 (d, J¼ 8.4 Hz), 131.8, 124.7, 117.1 (d, J¼ 13.4 Hz), 115.2, 91.9, 85.8, 85.6, 83.7, 69.9, 27.5, 25.5
HRMS calculated for C20H22N6O7FS (MH+): 509.1255; found: 509.1243
7: Rf¼ 0.52 (EtOAc : MeOH, 4 : 1, v ⁄ v)
1
H NMR (CD3OD, 300 K): d¼ 8.47 (1H, s), 8.11 (1H, s), 7.90 (1H, dd, J¼ 7.8; 1.8 Hz), 7.28 (1H, dt, J ¼ 1.8; 6.8 Hz), 6.74 (2H, m), 6.23 (1H, d, J¼ 3.1 Hz), 5.38 (1H,
dd, J¼ 3.1 & 6 Hz), 5.13 (1H, dd, J ¼ 2.3 & 6 Hz), 4.57 (1H, m), 4.33 (2H, d, J¼ 3.8 Hz), 1.55 (3H, s), 1.31 (3H, s)
13
C NMR (CD3OD, 300 K): d¼ 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 C20H23N6O8S (MH+): 509.1298; found: 509.1286
8: Rf¼ 0.40 (CHCl3: MeOH, 2 : 1, v⁄ v); [a]25
(c¼ 0.5; MeOH)
1H NMR (CD3OD): d¼ 8.55 (1H, s), 8.17 (H, s), 8.02 (2H, d, J¼ 7.1 Hz), 7.43 (1H, t, J ¼ 7.1 Hz), 7.35 (2H, t,
J¼ 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 (CD3OD): d¼ 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 C17H19N6O7S (MH+): 451.1036; found: 451.1029
9: Rf¼ 0.41 (CHCl3: MeOH, 2 : 1, v⁄ v); [a]25
(c¼ 0.5; MeOH)
1H NMR (CD3OD): d¼ 8.53 (1H, s), 8.19 (1H, s), 7.73 (1H, t, J¼ 7.6 Hz), 7.38 (1H, m), 7.13 (1H, t, J ¼ 7.6 Hz), 7.07 (1H, dd, J¼ 8.3; 10,6 Hz), 6.10 (1H, d, J ¼ 5.7 Hz), 4.73 (1H, t, J¼ 5.7 Hz), 4.43 (4H, m)
13
C NMR (CD3OD): d¼ 174.5, 163.8, 162.9 (d, J ¼
250 Hz), 158.1, 154.7, 151.7, 141.9, 133.8 (d, J¼ 8.4 Hz), 132.8, 129.2, 121.0, 118.0 (d, J¼ 29.7 Hz), 90.3, 85.4, 76.8, 73.2, 70.4
HRMS calculated for C17H18N6O7FS (MH+): 469.0942; found: 469.0952
10: Rf¼ 0.44 (CHCl3: MeOH, 2 : 1, v⁄ v); [a]25
(c¼ 0.3; MeOH)
Trang 91H NMR (CD3OD): d¼ 8.49 (1H, s), 8.13 (1H, s), 7.90
(1H, d, J¼ 8 Hz), 7.23 (1H, t, J ¼ 8 Hz), 6.74 (2H, m),
6.05 (1H, d, J¼ 5.8 Hz), 4.68 (1H, t, J ¼ 5.3 Hz), 4.68
(4H, m)
13
C NMR (CD3OD): d¼ 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 C17H19N6O8S (MH+): 467.0985;
found: 467.0994
12 (mixture of diastereomers): Rf¼ 0.27
(EtO-Ac : MeOH, 4 : 1, v⁄ v)
1H NMR (CD3OD, 300 K): d¼ 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 (CD3OD, 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 good13C NMR
13: Rf¼ 0.21 (CHCl3: MeOH, 2 : 1, v⁄ v)
1
H NMR (CD3OD): d¼ 8.43 (1H, s), 8.06 (1H, s), 7.33
(1H, d, J¼ 8.0 Hz), 6.74 (1H, d, J ¼ 8.0 Hz), 6.51 (1H, t,
J¼ 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 (CD3OD): d¼ 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: Rf¼ 0.46 (EtOAc : MeOH, 9 : 1, v ⁄ v); [a]25
(c¼ 0.5; CHCl3)
1
H NMR (CDCl3): d¼ 8.29 (1H, s), 8.00 (1H, s), 7.55
(2H, d, J¼ 7.5 Hz), 7.30 (5H, m), 6.85 (2H, d, J ¼
8.6 Hz), 6.14 (1H, d, J¼ 2.5 Hz), 5.62 (1H, broad s), 5.29
(1H, dd, J¼ 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, J¼ 3.8 &
11.0 Hz), 4.32 (1H, dd, J¼ 5.0 & 11.0 Hz), 3.79 (3H, s),
1.61 (3H, s), 1.34 (3H, s)
13
C NMR (CDCl3): d¼ 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: Rf¼ 0.45 (EtOAc : MeOH, 9 : 1, v ⁄ v)
1
H NMR (CDCl3): d¼ 8.23 (1H, s), 8.08 (1H, s), 7.30
(7H, m), 7.02 (2H, dd, J¼ 7.6 & 10.3 Hz), 6.15 (1H, d,
J¼ 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, J¼ 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 (CDCl3): d¼ 161.5, 158.9, 155.2, 152.8, 150.6
(d, J¼ 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 C27H28N6O5F (MH+): 535.2105;
found: 535.2083
18: Rf¼ 0.30 (EtOAc : MeOH, 9 : 1, v ⁄ v); [a]25D¼)17
(c¼ 0.2; MeOH)
1H NMR (CD3OD): d¼ 8.20 (1H, s), 8.10 (1H, s), 7.96 (2H, d, J¼ 7.1 Hz), 7.60 (1H, t, J ¼ 7.7 Hz), 7.46 (2H, t,
J¼ 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, J¼ 4.8 & 8.6 Hz)
13C NMR (CDCl3): d¼ 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: Rf¼ 0.30 (AcOEt : MeOH, 9 : 1, v ⁄ v); [a]25
(c¼ 0.3; MeOH)
1
H NMR (CDCl3): d¼ 8,19 (1H, s), 8,09 (1H, s), 7.89 (1H, t, J¼ 7.3 Hz), 7.63 (1H, m), 7.23 (2H, m), 6.03 (1H,
d, J¼ 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 (CDCl3): d¼ 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⁄ PPiexchange 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 37C 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⁄ PPiexchange reactions were carried out for 30 s for each substrate concentration Km values were determined by using the nonlinear fit modeling option
of Microcaltm
origintm 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⁄ PPi
exchange reactions were stopped after 5 min To deter-mine the Ki values for DhbE with both DHB-AMS and SAL-AMS, the DHB concentration was set around the Km
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⁄ PPiexchange reactions were carried out for 30 s The inhibition constants were cal-culated using the Dixon plot method
Trang 10We 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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