Syntheses and Iron Binding Affinities of the Bacillus anthracis Siderophore Petrobactin and Sidechain-Modified Analogues

Article abstract of DOI:10.1002/ejoc.201301340

An improved synthesis of petrobactin and analogues has been developed that provides reliable access to siderophores in sufficient quantities for biological studies. The determination of the iron affinity of different petrobactin derivatives showed that functionalization at the central amino group of the spermidine sidechain has only a minor influence on the iron binding properties of the siderophore. Accordingly, such derivatives are promising starting points for the development of biological probes to study bacterial siderophore transport, a possible new target for antibiotics.

Full text of DOI:10.1002/ejoc.201301340

FULL PAPER
DOI: 10.1002/ejoc.201301340
Syntheses and Iron Binding Affinities of the Bacillus anthracis Siderophore
Petrobactin and Sidechain-Modified Analogues
Nikolas Bugdahn[a] and Markus Oberthür*[a,b]
Keywords: Binding affinity / Bioorganic chemistry / Iron / Siderophores / Ligand design
An improved synthesis of petrobactin and analogues has
been developed that provides reliable access to siderophores
in sufficient quantities for biological studies. The determi-
nation of the iron affinity of different petrobactin derivatives
showed that functionalization at the central amino group of
the spermidine sidechain has only a minor influence on the
iron binding properties of the siderophore. Accordingly, such
derivatives are promising starting points for the development
of biological probes to study bacterial siderophore transport,
a possible new target for antibiotics.
however, only a limited number import systems have been
characterized biochemically and structurally.[8,9,12–14]
Introduction
The acquisition of the essential element iron is a difficult
task for all organisms. Despite its widespread abundance in
the earth’s upper crust, the environmental concentration of
FeIII ions is rather low because iron is sequestered in the
form of insoluble oxides and hydroxides.[1] Consequently,
organisms have developed a number of acquisition and
storage strategies to enable iron homeostasis. Whereas hu-
mans simply rely on their diet to supply sufficient quantities
of iron for all essential cellular functions, smaller organisms,
such as bacteria[2,3] and fungi,[4,5] were forced to come up
with more ingenious solutions to this problem. The most
abundant strategy features the secretion of siderophores,
which are polar, low molecular weight molecules with ex-
We have recently developed an identification strategy for
siderophore-binding proteins that is based on the actual sid-
erophore-binding event during affinity chromatography
rather than on sequence homologies.[15,16] For our initial
studies, we selected petrobactin (1, Figure 1), a siderophore
that is produced by different bacteria, most importantly by
the two pathogens Bacillus cereus[17] and Bacillus an-
thracis.[18,19] As a proof of concept, we prepared a biotinyl-
ated petrobactin derivative 2 (Figure 1) in which the biotin
ceptionally high binding affinities for FeIII [6,7]
Upon iron
.
binding, for example by removal from iron storage proteins
within a host, the siderophore-iron complex is internalized
by dedicated import systems,[8,9] and the iron is extracted
from the complex by either reductive or hydrolytic processes
for further use.
Inhibition of the siderophore cycle offers a potentially
new target for antibacterial and antifungal agents, because
siderophore-mediated iron uptake is essential for both the
survival and the virulence of many pathogens.[10] In ad-
dition to blocking their biosynthesis,[11] interfering with sid-
erophore uptake would provide an additional means by
which to deplete the cytoplasmic iron concentration. So far,
[a] Fachbereich Chemie, Philipps-Universität Marburg,
Hans-Meerwein-Straße, 35032 Marburg, Germany
E-mail: oberthuer@chemie.uni-marburg.de
www.uni-marburg.de/fb15
Figure 1. Structure of petrobactins used in this study to determine
the influence of sidechain substitution on iron binding. The func-
tionalized petrobactins 2 and 3 contain a biotin group connected
to a short linker that is attached to the central amino group of the
spermidine sidechain through amidation or alkylation, respectively.
Mono- and di-N4Ј-acetyl petrobactin 4 and 5, respectively, were
prepared as model compounds to assess the general influence of
N-acylation as well as steric effects.
[b] Current address: Deutsches Textilforschungszentrum Nord-
West, Universität Duisburg-Essen, NETZ/DTNW gGmbH,
Carl-Benz-Str. 199, 47057 Duisburg, Germany
E-mail: oberthuer@dtnw.de
www.dtnw.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301340.
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Bacillus anthracis Siderophore Petrobactin and Analogues
group is attached to the central side chain amine through a 2 and 3, Figure 1), or to racemic mixtures when two dif-
γ-butyric acid linker.
ferent achiral sidechains are used, for example the mono-
Immobilization of this derivative on streptavidin-modi- Ac derivative 4 (Figure 1). For our exploratory experiments
fied agarose and subsequent affinity chromatography of regarding iron binding and the isolation of binding proteins
crude cell extracts then led to the identification of the petro-
bactin-binding protein FpiA and the corresponding petro-
bactin uptake system fpiABCD in the model organism Ba-
cillus subtilis.[15,20] Notwithstanding the success of our pro-
tein fishing experiments, information regarding the iron-
binding abilities of such modified petrobactins was still
lacking. Accordingly, we prepared a set of additional deriv-
atives carrying substituents at this position. We then deter-
mined their iron-binding affinity, which for the first time
allowed quantitative assessment of the influence that a side-
chain modification exerts on iron binding. This information
will help the design of functionalized petrobactins for other
possible applications, for example pathogen capture[21,22]
and siderophore-mediated cargo delivery.[23–26]
Results and Discussion
Petrobactin (1) is a citric acid derived siderophore that
carries two spermidine sidechains with 3,4-catecholic end
groups. For our binding protein capture experiments, we
modified the central amino group of one of the sidechains
with a biotin group through a linker (see derivative 2, Fig-
ure 1). The spermidine chains are presumably not directly
involved in the iron complexation process, but provide a
suitable orientation of the primary ligands, i.e., the two cat-
echols and the hydroxyl and carbocylic acid groups of the
citric acid moiety. Nevertheless, it was not clear at the be-
ginning of our studies in which way a modification at this
position might influence the stability of the FeIII complex
either sterically or electronically. This information is, of
course, vital for the design of future petrobactin probes be-
cause iron binding is required for the biological function.
To study possible effects in more detail, we prepared bio-
tinylated petrobactin 3, in which the linker is of similar size,
but attached to the spermidine sidechain through alkylation
instead of amidation of N-4Ј. As a consequence, derivative
3 is a closer analogue of petrobactin (1) than amide 2 be-
cause the sidechain amine retains a similar geometry (tetra-
hedral) and basicity to that in 1. To study possible steric
effects of amides attached to the spermidine sidechains, we
also prepared two N4Ј-acetyl analogues 4 and 5, which carry
acyl groups of minimal size, by an improved synthetic route.
Synthesis of Petrobactins
N-Alkyl-linked biotinyl petrobactin 3 was prepared by
analogy to the procedure already developed for
2
(Scheme 1),[15] which features the opening of a cyclic citric
acid anhydride with the linker containing side chain amine,
followed by attachment of the regular spermidine side
chain. It should be noted that the addition of two different
sidechains to the citric acid core leads to diastereomeric
mixtures when additional stereogenic centers are present
Scheme 1. Synthesis of biotinyl petrobactin 3. Dde-OH = acetyldi-
medone, PPTS = pyridinium para-toluenesulfonate, NHS = N-hy-
within at least one of the side chains (e.g., biotin derivatives droxysuccinimide, DIC = diisopropylcarbodiimide.
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N. Bugdahn, M. Oberthür
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from crude cell extracts, however, this limitation was de-
emed acceptable because it simplified the synthesis dramati-
cally.
The synthesis of 3 started with monoalkylation of the
known spermidine side chain 8[15] by reductive amination
with 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde)
protected β-amino aldehyde 7, which, in turn, was obtained
from commercially available acetal 6 (71%, two steps). The
Dde group[27] is stable under a variety of reaction condi-
tions and can be removed selectively by using hydrazine.
Optimal yields for the reductive amination (66%) were ob-
tained when NaBH(OAc)3 (1.8 equiv.) in anhydrous MeOH
Scheme 2. Problematic imide formation by intramolecular attack
of one of the amide nitrogen atoms during acidic removal of the
tert-butyl ester.
was used as the reducing agent and molecular sieves (3 Å) trobactin derivatives that might also lead to a further im-
and Et3N (Ǟ pH 8) were added to the reaction mixture. proved route to petrobactin (1) itself. After a careful survey
The use of NaBH3CN (2 equiv.), on the other hand, gave of the available literature, we decided to explore the use of
significantly lower yields. Following removal of the Boc corresponding isopropyl derivatives, which are also easily
group [trifluoroacetic acid (TFA)/CH2Cl2], primary amine accessible and were reported to be stable in the presence of
10 was treated with cyclic anhydride 11, which afforded the primary amines. The removal of such citric acid isopropyl
corresponding crude monoacid. Activation of the carbox- esters by treatment with aqueous hydroxide solution was
ylic acid as the N-hydroxysuccinimide (NHS) ester and cou- successfully achieved by the Miller group without imide for-
pling with the regular side chain amine 12[15] then gave the mation in the case of the siderophore schizokinen,[32] but
fully assembled petrobactin derivative 13 carrying the Dde- was never tested in the petrobactin system.
protected linker (42%, two steps).
The practicality of the isopropyl group was first estab-
Removal of the Dde group with hydrazine and subse- lished in the synthesis of mono-N4Ј-acetyl petrobactin de-
quent reaction with biotin NHS ester 14 then led to the rivative 4 (Scheme 3) in a sequence similar to that used for
protected biotinyl petrobactin 15 in good yields (77%, two the preparation of 2. For the generation of an appropriate
steps). Finally, global deprotection was achieved by using N4-acetylated spermidine, petrobactin side chain 8 was first
conditions established previously in a two-step procedure, acetylated (AcCl), followed by removal of the Boc group
which afforded pure biotinyl petrobactin 3 following purifi- with TFA to afford amine 16 in good overall yields (82%,
cation by reverse-phase (RP) HPLC.
two steps). Reaction with the isopropyl-protected cyclic an-
Although this final sequence easily afforded enough ma- hydride 18, obtained from 2-isopropyl citrate 17[32,35]
terial for our studies, the quite low yields (15%) of the de- (Scheme 4) by activation with N,N-dicyclohexylcarbodi-
protection steps left room for further improvement. In ad- imide (DCC),[36] afforded the corresponding monoacid 19
dition to some losses during the RP HPLC purification pro- in good yields (74%). This acid was then coupled with
cess due to the highly polar nature of the product, the re- spermidine 12 as described for the preparation of 2 to af-
moval of the tert-butyl ester was judged to be the most ford protected N4Ј-acetyl petrobactin 20. Gratifyingly, the
problematic step. So far, our synthetic efforts relied on citric isopropyl ester proved to be easily removable by saponifica-
acid building blocks in which the central carboxylic ester tion of 20 with aqueous lithium hydroxide without any for-
was blocked with a tert-butyl group, similar to the two total mation of the imide side product. Removal of the Cbz and
syntheses that had been published previously.[28–30] The ad- Bn groups by hydrogenolysis (45 bar, Pd/C) then afforded
vantages of the central tert-butyl ester in citric acid systems mono-N4Ј-Ac petrobactin 4 in high purity after RP HPLC
are twofold: its ease of preparation[31] and its stability purification (51%, two steps).
against various nucleophiles as a result of steric hindrance.
Based on these encouraging results, we also modified our
A major drawback, however, becomes apparent when the established route to symmetric petrobactins, including 1, by
protecting group has to be removed. Under the acidic con- using isopropyl-protected citric acid building block 17. This
ditions required for deprotection of the tert-butyl ester, in- led to an improved total synthesis of petrobactin (1) itself,
tramolecular attack of one of the amide nitrogen atoms on as well as other symmetric petrobactins such as di-N4Ј-Ac
the central carboxy group is a common side reaction derivative 5 (Scheme 4). After some exploratory studies, we
(Scheme 2).[28,32,33]
found that optimal yields for amide coupling reactions were
This imide formation was observed for different sidero- obtained when 17 was activated by treatment with para-
phores even under neutral conditions,[34] but is greatly fa- nitrophenol and DCC.[32] The active ester was easily iso-
cilitated by protonation of the carbonyl group. Accordingly, lated by acidic and base extractions and filtration through
the removal of the tert-butyl ester requires careful optimiza- a short pad of silica gel. Reaction of this diester with
tion of reaction and work-up conditions. In addition, in our 2.4 equiv. of sidechain amine 12 then delivered the fully
hands, previously successful deprotection protocols for a protected petrobactin 21 in a good overall yield (69%).
specific system were found to be difficult to reproduce from Again, the isopropyl ester was saponified cleanly by treat-
time to time. Therefore, we wanted to develop a different ment with aqueous lithium hydroxide without imide forma-
protecting group strategy for the synthesis of additional pe- tion. Removal of the Cbz and Bn groups by hydrogenolysis
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Bacillus anthracis Siderophore Petrobactin and Analogues
Scheme 4. Synthesis of petrobactin (1) and di-N4Ј-Ac petrobactin
5. PNP = para-nitrophenol.
17 with the N4Ј-acetylated sidechain amine 16 afforded pro-
tected petrobactin 22 even in slightly better yields (77%).
Saponification and hydrogenolysis then led to the depro-
tected product 5 in 62% yield after purification by RP
HPLC.
Determination of the Iron Binding Affinities of Modified
Petrobactins
Previous to our studies, the iron binding affinity of petro-
bactin (1) was determined by using two different ap-
proaches,[38,39] however, no information was available that
showed to what extent structural modifications of the petro-
bactin skeleton altered the stability of the iron complex.
The precise determination of the stability constant for the
formation of a siderophore-iron complex is difficult for
various reasons. Direct measurement of the formation con-
stant is usually not possible because no significant amount
of unbound iron is present at equilibrium. Therefore, the
iron affinity of the siderophore is often determined by using
Scheme 3. Synthesis of mono-N4Ј-Ac petrobactin 4.
(55 bar, Pd/C) then gave petrobactin (1) in high purity after a competition assay against ethylenediaminetetraacetic acid
RP HPLC purification (62%). Our optimized synthesis is a (EDTA) or another ligand for which the formation con-
further improvement of our initial approach using the tert- stant of the iron complex is known.[40] In addition, the de-
butyl derivative and afforded petrobactin (1) in ten steps termination of “standard” pH independent binding con-
and 20.2% overall yield starting from 3,4-dihydroxybenzoic stants that are usually reported requires the knowledge
acid. Because only five chromatographic separations are re- of all acidity constants of dissociable protons within the
quired, the process is amenable to scale-up and offers easy siderophore, because the overall formation constant
access to gram quantities of the siderophore. This is espe- βFe–siderophore is expressed in terms of a fully deprotonated
cially noteworthy because the isolation of petrobactin (1) ligand. For some siderophores, these acidity constants are
in quantities of more than 100 mg by fermentation is not known, but often only estimated values are used, because
practical for many laboratories because of safety issues and factors such as complex aggregation or precipitation and
the low production levels within the producing organ- degradation of the siderophore makes their experimental
isms.[37]
determination difficult. In the case of petrobactin (1), for
Finally, the synthesis of the diacetyl derivative di-N4Ј-Ac example, oxidation of the 3,4-dihydroxybenzamide residue
petrobactin 5 proved that this reaction sequence also pro- at elevated pH prevents an accurate determination of the
ceeds in reliably good yields with other spermidine side- pKa values of the catecholic hydroxyl groups.[41] Accord-
chains. Thus, reaction of the bis-para-nitrophenyl ester of ingly, the group of Butler has shown that only three of the
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429

N. Bugdahn, M. Oberthür
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eight dissociation constants of petrobactin can be accu- complexation, a change of the linker type (acyl vs. alkyl)
rately determined by potentiometric measurements, which, has only a minor influence on the binding of the modified
in turn, made a precise calculation of a pH independent siderophore by purified FpiA [2.4 μm (3) vs. 18 μm (2)]. De-
binding constant using estimated values difficult.[39]
spite these similarities, the solubility of the various iron
To overcome these obstacles, the “pM” scale has been complexes at physiological pH can differ quite substantially.
introduced and is being increasingly used to compare the Compound 3, with the amine linker, was better soluble in
binding affinities of a variety of structurally unrelated sider- pH 7.4 buffer than derivatives that contain an amide
ophores for iron (M = FeIII) or other metals.[42] The pFeIII linker.[43] Therefore, possible probes for biological experi-
value describes the concentration of unbound iron ments have to be chosen carefully to obtain reliable data.
–log[FeIII] in equilibrium with the metal-ligand complex(es)
at fixed concentrations of ligand and metal ([siderophore]
Conclusions
= 10 μm, [FeIII] = 1 μm) and at a physiological pH of 7.4.
The exchange of the previously used citric acid 2-tert-
butyl ester for the corresponding isopropyl derivative 17 has
vastly improved the overall yield and efficiency of our syn-
thetic route to petrobactin (1), a siderophore that is pro-
duced by different bacteria, most notably by two pathogens
B. cereus and B. anthracis. Because the deprotection of the
By definition, the pFeIII value is independent of the nature
and protonation state of the metal-ligand complexes formed
under these conditions and is proportional to the free en-
ergy released by metal ligand binding. For the determi-
nation of the pFeIII values for different petrobactin deriva-
tives, we used a spectrophotometric EDTA competition as-
isopropyl ester is much less prone to imide formation, our
say by following a protocol developed by Abergel et al.[38]
synthesis can reliably provide quantities of 1 that are diffi-
cult to obtain by fermentation. In addition, this flexible
By using this method, we determined a pFeIII value for pe-
trobactin (1) of 23.4Ϯ0.16, which is close to the value ob-
route makes functionalized petrobactin derivatives for
tained by the Raymond group (23.0)[38] under similar condi-
chemical and biological studies easily available. Accord-
tions. The pFeIII values for various sidechain modified ana-
ingly, further analogues, for example, fluorescent derivatives
or photoaffinity probes, should be accessible in a straight-
forward way. Finally, we were able to determine the pre-
logues that were obtained by this method are shown in
Table 1. All acylated analogues (2, 4, and 5) proved to have
a slightly higher binding affinity than petrobactin (1). In
viously unknown influence of structural modifications to
addition, there was no dramatic change even when both
petrobactin on the iron binding affinity of the siderophore.
sidechain amines were acylated, as demonstrated by the
Our data shows that both alkylation and acylation of the
spermidine sidechain has only a minor effect on iron bind-
very similar value obtained for diacetate 5 and monoacetate
4. Finally, derivative 3, in which the biotin residue was at-
ing, which makes this type of petrobactin analogues prom-
tached through an alkyl linkage, has a pFeIII value close to
ising tools for the study of iron homeostasis in pathogenic
bacteria.
that of petrobactin (1). Despite these small differences, all
pFeIII values are within a close range (23.42–24.60), i.e., one
can conclude that a structural modification of petrobactin
at the secondary amine of the spermidine sidechain has no
significant influence on iron binding.
Experimental Section
General Remarks: All reactions were performed under an argon
atmosphere at ambient pressure unless indicated otherwise. All re-
actions leading to unprotected petrobactin and petrobactin ana-
logues were performed under the exclusion of light. Spermidine
sidechain 12 and biotinylated petrobactin 2 were prepared as de-
scribed.[15] Commercial reagents were used as received. All extracts
were dried with MgSO4 and evaporated below 40 °C under reduced
pressure. Thin-layer chromatography (TLC) was performed on alu-
minum plates coated with silica gel 60 F254. Detection was carried
out by fluorescence quenching under UV light (λ = 254 nm) or by
staining with 20% H2SO4 or ninhydrin solution followed by heating
to ca. 300 °C. Flash chromatography was performed on silica gel
60 (0.040–0.063 mm). For chromatography solvents that contained
Table 1. pFeIII values determined for petrobactin (1) and analogues
2–5.
Entry
Siderophore
pFeIII
1
2
3
4
5
1
2
3
4
5
23.42Ϯ0.16
24.60Ϯ0.08
23.55Ϯ0.09
23.90Ϯ0.14
23.84Ϯ0.09
Accordingly, functionalized derivatives of this type are
valid probes for biological experiments. We could already NH4OH, a solution of 25wt.-% (NH4OH/H2O) was used. Prepara-
tive high-performance liquid chromatography was performed by
using a Nucleodur 100–5 C18 ec column (25 mmϫ250 mm) or a
Luna 5 μm C18 column (4.60ϫ250 mm). Chemical shifts (δ) are
given in ppm referring to the solvent residual signal. In many cases,
cis/trans rotational isomers were observed even at elevated tempera-
show that biotinyl derivative 2 can be successfully used as
an immobilized ligand for the isolation of siderophore-
binding proteins through affinity chromatography, which
led to the identification of the siderophore-binding protein
FpiA in B. subtilis. Interestingly, protein retention was
achieved despite the fact that the binding affinity of FpiA
for the modified ligand 2 dropped quite substantially by
three orders of magnitude compared with petrobactin (1)
itself [18 μm (2) vs. 51 nm (1)].[15] With alkylated petrobactin
1
tures. In the H NMR spectroscopic data, peaks for the same H-
atoms corresponding to such rotamers are combined and listed
with a subscript (e.g., 2ϫ scis/trans). The integral given corresponds
to that of the combined peaks. The signals were assigned with the
aid of COSY, HMBC and HMQC spectra. All coupling patterns
3 in hand, we were now able to show that, similar to iron were characterized according to their actual appearance (phenome-
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Bacillus anthracis Siderophore Petrobactin and Analogues
nological data) and not according to the multiplicity that is theore-
tically expected. All coupling constants are JH,H couplings unless
indicated otherwise and are the actual values observed for each
signal and not averaged values. Mass spectra were recorded with a
LTQ FT spectrometer; the resolution was set to 100.000.
120.6, 127.4, 127.5, 127.8, 128.3, 128.4, 136.9, 137.1, 147.6, 150.5,
155.5, 165.4, 172.6, 196.3 ppm. HRMS (ESI): calcd. for
C46H63N4O7 [M + H]+ 783.4691; found 783.4690.
3
N1-[3Ј,4Ј-Bis(benzyloxy)benzoyl]-N4-(N-{1-[(4,4-dimethyl-2,6-dioxo-
cyclo-hexylidene)ethyl]}aminopropyl)spermidine·2TFA (10): A solu-
tion of amine 9 (0.46 g, 0.58 mmol) in CH2Cl2 (12 mL) was stirred
at 0 °C for 30 min. A solution of TFA (1.4 mL) in CH2Cl2 (12 mL)
was added dropwise over 1 h and the mixture was stirred for an
additional 30 min at 0 °C before it was allowed to reach room tem-
perature. The solution was stirred for 12 h at room temperature,
then coevaporated with toluene (4ϫ) and CHCl3 (2ϫ), to afford
amine 10 (di-TFA salt, 438 mg, 83%) as an amorphous solid. Rf =
0.10 (CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz, [D6]DMSO): δ =
0.93 (s, 6 H, 2ϫ CH3Dde), 1.49–1.72 (m, 4 H, 6-H2, 7-H2), 1.84–
2.02 (m, 4 H, 2-H2, 10-H2), 2.26 (s, 4 H, 2ϫ CH2Dde), 2.47 (s, 3 H,
CH3Dde), 2.76–2.88 (m, 2 H, 8-H2), 3.06–3.18 (m, 6 H, 3-H2, 5-H2,
9-H2), 3.31 (q, J1,2 = 5.9 Hz, 2 H, 1-H2), 3.48 (q, J = 6.5 Hz, 2 H,
N-{[1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl]-3-oxopropyl}-
amine (7): To a solution of acetyldimedone (Dde-OH, 0.99 g,
5.5 mmol)[44] and Et3N (0.48 mL, 3.5 mmol) in EtOH (30 mL) was
added freshly activated molecular sieves (3 Å) and the mixture was
stirred at room temperature for 10 min. 3,3-Diethoxy-1-propyl-
amine (6, 1.4 mL, 8.8 mmol) was added in one portion. The mix-
ture was stirred at 25 °C for 16 h, filtered through a pad of Celite,
and concentrated under reduced pressure. The resulting residue was
dissolved in EtOAc, extracted with aq. citric acid (20%), aq. NaOH
(1 m), and brine. The combined organic phases were dried, filtered,
and concentrated to dryness to afford Dde-protected acetal (1.66 g,
97%) as a yellow oil, which was used directly in the next reaction
step.
11-H2), 5.17, 5.20 (2ϫ s, 4 H, 2ϫ CH2Bn), 7.13 (d, J5,6 = 8.5 Hz,
4
1 H, 5-HAr), 7.29–7.47 (m, 11 H, 2ϫ Ph, 6-HAr), 7.57 (d, J2,6
=
=
1.92 Hz, 1 H, 2-HAr), 7.83 (br. s, 3 H, 8-NH3+), 8.53 (t, JNH,2
To a solution of this crude acetal (1.65 g, 5.30 mmol) in acetone/
H2O (4:1, 60 mL), a catalytic amount of pyridinium toluene-4-sulf-
onate was added and the solution was stirred at 50 °C for 36 h.
After the solvents were removed in vacuo, the colorless residue was
dissolved in EtOAc and extracted with brine, aq. NaOH (1 m), and
water. The solution was dried (MgSO4), filtered, and the solvents
were evaporated to dryness. The oily residue was purified by flash
chromatography (CH2Cl2/MeOH, 20:1Ǟ10:1) to give 7 (0.89 g,
71%) as a clear oil. Rf = 0.40 (CH2Cl2/MeOH, 10:1). 1H NMR
(300 MHz, [D6]DMSO): δ = 1.01 (s, 6 H, 2ϫ CH3Dde), 2.35 (s, 4
H, 2ϫ CH2Dde), 2.58 (s, 3 H, CH3Dde), 2.90 (t, J1,2 = 6.6 Hz, 2 H,
2-H2), 3.71 (q, J = 6.3 Hz, 2 H, 1-H2), 9.83 (s, 1 H, 3-H), 13.55
(br. s, 1 H, NHDde) ppm. 13C NMR (75 MHz, [D6]DMSO): δ =
17.7, 28.2, 30.1, 36.3, 43.0, 52.8, 108.1, 173.7, 198.1, 198.2 ppm.
HRMS (ESI): calcd. for C13H19N1O3Na [M + Na]+ 260.1257;
found 260.1260.
5.4 Hz, 1 H, 1-NH), 9.70 (br. s, 1 H, 4-NH+), 13.26 (t, J = 5.3 Hz,
11-NH) ppm. 13C NMR (75 MHz, [D6]DMSO): δ = 17.3, 20.1,
26.3, 26.6, 27.4, 27.8, 29.6, 36.4, 38.2, 40.7, 48.9, 49.0, 51.3, 52.4,
69.9, 70.2, 107.1, 113.2, 113.3, 120.8, 127.4, 127.5, 127.8, 128.3,
128.4, 136.9, 137.1, 147.8, 150.5, 165.9, 173.0, 196.6 ppm.
HRMS (ESI): calcd. for C41H55N4O5 [M + H]+ 683.4167; found
683.4165.
1-(N1Ј-[3ЈЈЈ,4ЈЈЈ-Bis(benzyloxy)benzoyl]-N4Ј-{1-[(4,4-dimethyl-2,6-di-
oxocyclohexylidene)ethyl]aminopropyl}spermidinyl)-3-{N1ЈЈ
-
[3ЈЈЈ,4ЈЈЈ-bis(benzyloxy)benzoyl]-N4ЈЈ-benzyloxycarbonyl-
spermidinyl}-2-tert-butyl Citrate (13): To a solution of citric acid
anhydride 11[36] (0.20 g, 0.86 mmol) and freshly activated molecular
sieves (3 Å) in DMF (2 mL), was added amine 10 (di-TFA salt,
0.36 g, 0.40 mmol) and Et3N (0.46 mL, 3.4 mmol). The mixture
was stirred at room temperature for 36 h, filtered through a pad of
Celite, and the solvents were evaporated to dryness. The oily resi-
due was dissolved in EtOAc, extracted with aq. HCl (1 m, 2ϫ) and
brine, dried, and filtered. The organic layer was concentrated in
vacuo and purified by flash chromatography (SiO2; CH2Cl2/
MeOH/AcOH, 10:1:0.1) to give the monoacid (234 mg) as a clear
oil; Rf = 0.05 (CH2Cl2/MeOH, 10:1).
N1-[3Ј,4Ј-Bis(benzyloxy)benzoyl]-N8-tert-butyloxycarbonyl-N4-(N-
{1-[(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]}aminopropyl)-
spermidine (9): To a solution of aldehyde 7 (270 mg, 1.14 mmol)
and amine 8[15] (0.49 g, 0.88 mmol) in anhydrous MeOH (20 mL)
was added freshly activated molecular sieves (3 Å) and Et3N until
pH 8 was reached. The resulting mixture was stirred for 1 h at room
temperature before NaBH(OAc)3 (335 mg, 1.58 mmol) was added
in one portion. The mixture was stirred for 48 h, then the resulting
slurry was filtered through a pad of Celite and H2O (5 mL) was
added to quench the reaction. The solvents were removed under
reduced pressure and the resulting oil was dissolved in EtOAc and
extracted with satd. aq. NaHCO3 and brine. The organic layer was
dried and filtered, the solvent was removed in vacuo, and the oily
residue was purified by flash chromatography (SiO2; CH2Cl2/
MeOH/NH4OH, 10:1:0Ǟ10:1:0.1) to afford spermidine derivative
To a solution of this acid (0.23 g, 0.26 mmol) in anhydrous THF
(5 mL) was added N-hydroxysuccinimide (45 mg, 0.39 mmol) and
diisopropylcarbodiimide (73 μL, 0.47 mmol). The mixture was
stirred at room temperature for 18 h, then the solvents were evapo-
rated to dryness to afford the crude N-hydroxysuccinimide ester,
which was used for the next step without further purification.
To a mixture of amine 12 (TFA salt, 0.30 g, 0.42 mmol) and Et3N
(0.65 mL, 4.7 mmol) in anhydrous CH2Cl2 (5 mL) was added the
crude N-hydroxysuccinimide ester dissolved in anhydrous dioxane
9 (452 mg, 66 %) as an amorphous solid. Rf = 0.35 (CH2Cl2/
1
MeOH, 10:1). H NMR (600 MHz, [D6]DMSO): δ = 0.92 (s, 6 H, (3 mL). The mixture was stirred at room temperature for 72 h, then
2ϫ CH3Dde), 1.33–1.39 (m, 13 H, 6-H2, 7-H2, CH3Boc), 1.60–1.66 the solvents were evaporated to dryness. The residue was dissolved
(m, 2 H, 2-H2), 1.66–1.73 (m, 2 H, 10-H2), 2.24 (s, 4 H, 2 ϫ in EtOAc and extracted with aq. HCl (1 m), aq. NaOH (1 m), and
CH2Dde), 2.30–2.38 (m, 2 H, 5-H2), 2.38–2.46 (m, 4 H, 3-H2, 9-H2), brine. The organic phase was dried, filtered, and concentrated to
2.47 (s, 3 H, CH3Dde), 2.86–2.91 (m, 2 H, 8-H2), 3.24 (q, J1,2
=
dryness. The obtained residue was purified by flash chromatog-
raphy (SiO2; CH2Cl2/MeOH, 10:1) to give petrobactin derivative 19
(248 mg, 42%, 2 steps) as an amorphous solid. Rf = 0.30 (CH2Cl2/
6.5 Hz, 2 H, 1-H2), 3.43 (q, J = 6.4 Hz, 2 H, 11-H2), 5.16, 5.19 (2ϫ
s, 4 H, 2ϫ CH2Bn), 6.76 (t, JNH,8 = 5.3 Hz, 1 H, 8-NH), 7.11 (d,
J5,6 = 8.5 Hz, 1 H, 5-HAr), 7.30–7.47 (m, 11 H, 2ϫ Ph, 6-HAr),
MeOH, 10:1). H NMR (400 MHz, [D6]DMSO): δ = 0.91 (s, 6 H,
1
4
7.55 (d, J2,6 = 1.98 Hz, 1 H, 2-HAr), 8.24–8.27 (m, 1 H, 1-NH), 2ϫ CH3Dde), 1.30–1.42 (m, 13 H, tBu, 7Ј-H2, 7ЈЈ-H2), 1.43–1.59
13.25 (t, J = 5.2 Hz, 11-NH) ppm. 13C NMR (150 MHz, [D6]-
DMSO): δ = 17.2, 23.7, 26.3, 26.6, 27.4, 27.8, 28.2, 29.6, 37.6, 39.3,
40.7, 50.3, 51.0, 52.4, 52.9, 69.9, 70.2, 77.3, 106.9, 113.2, 113.3,
(m, 4 H, 6Ј-H2, 6ЈЈ-H2), 1.60–1.80 (m, 6 H, 2Ј-H2, 2ЈЈ-H2, 10Ј-H2),
2.23 (s, 4 H, 2ϫ CH2Dde), 2.35–2.48 (m, 2 H, CHaHcitr), 2.46 (s, 3
2
H, CH3Dde), 2.55 (d, J = 14.7 Hz, 2 H, CHHbcitr), 2.95–3.05 (m, 4
Eur. J. Org. Chem. 2014, 426–435
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431

N. Bugdahn, M. Oberthür
FULL PAPER
H, 8Ј-H2, 8ЈЈ-H2), 3.15–3.35 (m, 12 H, 1Ј-H2, 1ЈЈ-H2, 3Ј-H2, 3ЈЈ-H2, The residue was dissolved in methanol (10 mL) and ammonium
5Ј-H2, 5ЈЈ-H2), 3.35–3.50 (m, 4 H, 9Ј-H2, 11Ј-H2), 5.02–5.08 (br. s, formate (82 mg, 1.3 mmol) and Pd/C (30%, 25 mg) were added,
2 H, CH2Cbz), 5.15, 5.19 (2ϫ s, 8 H, 4ϫ CH2Bn), 5.65 (s, 1 H,
and the resulting mixture was heated to reflux for 4 h. The mixture
OHcitr), 7.11 (d, J5,6 = 8.5 Hz, 2 H, 2ϫ 5-HAr), 7.30–7.48 (m, 27 was filtered through a pad of Celite and the solvents were evapo-
H, 2ϫ 6-HAr, 5ϫ Ph), 7.55 (m, 2 H, 2ϫ 2-HAr), 7.90 (m, 2 H, 8Ј- rated to dryness to give an amorphous solid. The residue was puri-
NH, 8ЈЈ-NH), 8.29 (br. s, 2 H,1Ј-NH, 1ЈЈ-NH), 13.24 (t, JNH,11
=
fied by preparative HPLC (column: C18, 4.60 mmϫ250 mm sol-
vent: CH3CN/H2O/0.1% TFA, flow rate: 1.1 mLmin–1, gradient:
5–35% over 35 min) and the appropriate fractions were lyophilized
5.1 Hz, 1 H, 11Ј-NH) ppm. 13C NMR (100 MHz, [D6]DMSO): δ =
17.2, 23.8, 24.2, 26.3, 26.8, 27.4, 27.8, 29.6, 36.9, 38.1, 40.6, 43.2,
44.8, 46.5, 49.3, 50.2, 51.9, 52.3, 69.6, 69.9, 70.2, 73.9, 80.1, 106.9, to give biotinylated petrobactin 3 (7.6 mg, 15%) as an amorphous
1
113.2, 113.3, 120.6, 127.3, 127.4, 127.7, 127.8, 128.1, 128.3, 128.4,
128.5, 136.9, 137.0, 147.7, 150.6, 155.2, 165.5, 169.3, 172.2, 172.7,
196.3 ppm. HRMS (ESI): calcd. for C87H108N7O15 [M + H]+
1490.7898; found 1490.7905.
1-{N1Ј-[3ЈЈЈ,4ЈЈЈ-Bis(benzyloxy)benzoyl]-N4Ј-[N-(biotinyl)aminoprop-
yl]spermidinyl}-3-{N1ЈЈ-[3ЈЈЈ,4ЈЈЈ-bis(benzyloxy)benzoyl]-N4ЈЈ-benzyl-
oxycarbonyl-spermidinyl}-2-tert-butyl Citrate (15): To a solution of
Dde-protected petrobactin 13 (0.24 g, 0.16 mmol) in EtOH (5 mL),
was added hydrazine (0.11 mL, 3.4 mmol) at 0 °C. The mixture was
stirred at room temperature for 16 h, then toluene (10 mL) was
added and the mixture was evaporated to dryness to give the crude
amine as a clear oil.
solid. HPLC: tR = 11.39 min. H NMR (600 MHz, [D6]DMSO): δ
= 1.23–1.35 (m, 2 H, 15Ј-H2), 1.38–1.45 (m, 6 H, 7Ј-H2, 7ЈЈ-H2,
16Ј-H2), 1.45–1.63 (m, 6 H, 6Ј-H2, 6ЈЈ-H2, 14Ј-H2), 1.71–1.77 (m, 2
H, 2ЈЈ-H2), 1.78–1.87 (m, 4 H, 2Ј-H2, 10Ј-H2), 2.05–2.10 (m, 2 H,
13Ј-H2), 2.50–2.53 (m, 2 H, CHaHcitr, 18Ј-Ha), 2.56–2.61 (m, 3 H,
2
CHHbcitr), 2.81 (dd, J = 12.4, J18,19 = 5.1 Hz, 1 H, 18Ј-Hb), 2.87–
2.95 (m, 4 H, 3ЈЈ-H2, 5ЈЈ-H2), 3.03–3.15 (m, 13 H, 3Ј-H2, 5Ј-H2, 8Ј-
H2, 8ЈЈ-H2, 9Ј-H2, 11Ј-H2, 17Ј-H), 3.25–3.30 (m, 4 H, 1Ј-H2, 1ЈЈ-
H2), 4.10–4.15 (m, 1 H, 21Ј-H), 4.28–4.35 (m, 1 H, 19Ј-H), 5.55
(br. s, 1 H, OHcitr), 6.38, 6.40 (2ϫ s, 2 H, 19Ј-NH, 21Ј-NH), 6.76
(d, J5,6 = 7.8 Hz, 2 H, 2ϫ 5-HAr), 7.18 (dd, J5,6 = 8.3, J2,6
=
4
1.5 Hz, 2 H, 2ϫ 6-HAr), 7.27 (br. s, 2 H, 2ϫ 2-HAr), 7.93 (t, J13,NH
= 5.5 Hz, 1 H, 12Ј-NH), 7.99 (t, J8,NH = 5.4 Hz, 2 H, 8Ј-NH, 8ЈЈ-
NH), 8.28–8.32 (m, 3 H, 1Ј-NH, 1ЈЈ-NH, 4ЈЈ-NH), 9.10 (br. s, 2 H,
2ϫ 3-OHAr), 9.19 (br. s, 1 H, 4Ј-NH+), 9.53 (br. s, 2 H, 2ϫ 4-
OHAr), 12.63 (br. s, 1 H, CO2Hcitr) ppm. 13C NMR (125 MHz, [D6]-
DMSO): δ = 20.3, 22.9, 23.8, 25.2, 26.0, 26.1, 26.2, 28.0, 28.2, 35.1,
35.6, 36.1, 36.3, 37.7, 39.5, 43.3, 44.8, 46.5, 49.9, 50.2, 51.6, 55.4,
59.2, 61.1, 73.5, 114.8, 115.1, 118.9, 125.3, 125.4, 144.8, 148.5,
1 6 2 . 7 , 1 6 6 . 6 , 1 6 6 . 7 , 1 6 9 . 5 , 1 6 9 . 6 , 1 7 2 . 6 , 1 7 5 . 0 pp m.
HRMS (MALDI): calcd. for C47H72N9O13S1 [M + H]+ 1002.797;
found 1002.793.
The amine was dissolved in DMF (5 mL), biotin N-hydroxysuc-
cinimide ester (14, 80 mg, 0.23 mmol) and iPr2EtN (80 μL,
0.47 mmol) were added, and the mixture was stirred for 48 h. After
evaporation to dryness, purification of the residue by flash
chromatography (SiO2; CH2Cl2/MeOH/NH4OH, 10:1:0.1) gave
biotinylated petrobactin 15 (202 mg, 77%) as an amorphous solid.
Rf = 0.60 (CH2Cl2/MeOH/NH4OH, 10:1:0.1). HPLC: tR
=
29.91 min (C18, 4.6 mmϫ150 mm, CH3CN/H2O/0.1% TFA, flow
rate: 0.6 mL min–1, gradient: 10–90 % over 45 min). 1H NMR
(600 MHz, [D6]DMSO): δ = 1.26–1.38 (m, 17 H, tBu, 6Ј-H2, 6ЈЈ-
H2, 7Ј-H2, 15Ј-H2), 1.41–1.54 (m, 7 H, 7ЈЈ-H2, 10Ј-H2, 14Ј-H2, 16Ј-
Ha), 1.56–1.66 (m, 3 H, 2Ј-H2, 16Ј-Hb), 1.68–1.78 (m, 2 H, 2ЈЈ-H2),
2.02–2.08 (m, 2 H, 13Ј-H2), 2.30–2.40 (m, 6 H, 3Ј-H2, 5Ј-H2, 9Ј-
H2), 2.45 (d, 2J = 14.6 Hz, 2 H, CHaHcitr), 2.52–2.59 (m, 3 H,
N1-[3Ј,4Ј-Bis(benzyloxy)benzoyl]-N4-acetyl Spermidine·TFA (16): To
a solution of amine 8 (700 mg, 1.25 mmol) and Et3N (0.28 mL,
1.5 mmol) in CH2Cl2 (20 mL) was added AcCl (0.11 mL,
1.5 mmol) at 0 °C and the resulting mixture was stirred at room
temperature for 18 h. The reaction was quenched by the addition
of H2O (50 mL), and the solution was extracted with CH2Cl2 (3ϫ
). The organic layer was dried and evaporated, and the residue was
purified by flash chromatography (SiO2; CH2Cl2/MeOH, 20:1) to
give the N4-acetyl derivative (647 mg, 86%) as an amorphous solid.
citr
CHHb , 18Ј-Ha), 2.79 (dd, 2J = 12.5, J18,19 = 5.2 Hz, 1 H, 18Ј-
Hb), 2.96–3.08 (m, 7 H, 8Ј-H2, 8ЈЈ-H2, 11Ј-H2, 17Ј-H), 3.18–3.27
(m, 8 H, 1Ј-H2, 1ЈЈ-H2, 3ЈЈ-H2, 5ЈЈ-H2), 4.08–4-11 (m, 1 H, 21Ј-H),
4.26–4.28 (m, 1 H, 19Ј-H), 5.02–5.07 (m, 2 H, CH2Cbz), 5.15, 5.18,
5.19 (s, 8 H, 4ϫ CH2Bn), 5.65 (s, 1 H, OHcitr), 6.34 (s, 1 H, 19Ј-
NH), 6.39 (s, 1 H, 21Ј-NH), 7.11 (d, J5,6 = 8.6 Hz, 2 H, 2ϫ 5-HAr),
To a solution of acetyl derivative (647 mg, 1.07 mmol) in CH2Cl2
(20 mL) was added a solution of TFA (3 mL) in CH2Cl2 (30 mL)
dropwise over 1 h at 0 °C. After stirring for 30 min at 0 °C, the
mixture was stirred for 4 h at room temperature. Coevaporation
with toluene (4ϫ) and CHCl3 (2ϫ) then afforded amine 16 (TFA
salt, 633 mg, 96%) as an amorphous solid. Rf = 0.05 (CH2Cl2/
2
7.28–7.47 (m, 27 H, 2ϫ 6-HAr, 5ϫ Ph), 7.56 (d, J2,6 = 2.0 Hz, 2
H, 2ϫ 2-HAr), 7.74 (br. s, 1 H, 11Ј-NH), 7.90 (t, J8,NH = 5.2 Hz, 2
H, 8Ј-NH, 8ЈЈ-NH), 8.30 (br. s, 2 H, 1Ј-NH, 1ЈЈ-NH) ppm. 13C
NMR (100 MHz, [D6]DMSO): δ = 24.0, 25.1, 25.3, 26.3, 26.9, 27.4,
28.0, 28.2, 28.6, 34.9, 35.2, 36.8, 37.7, 38.1, 38.4, 39.5, 43.2, 44.7,
46.4, 51.0, 51.1, 53.4, 55.4, 59.1, 60.9, 65.9, 69.8, 70.2, 73.9, 80.1,
113.2, 113.3, 120.6, 127.3, 127.4, 127.5, 127.6, 127.8, 128.3, 128.4,
136.9, 137.0, 147.6, 150.5, 155.5, 162.6, 165.4, 169.3, 172.2,
174.1 ppm. HRMS (ESI): calcd. for C87H110N9O15S1 [M + H]+
1552.7837; found 1552.7807.
1
MeOH, 10:1). H NMR (300 MHz, [D6]DMSO): δ (cis/trans rota-
mers were observed) = 1.42–1.60 (m, 4 H, 6-H2, 7-H2), 1.65–1.82
(mcis/trans, 2 H, 2-H2), 1.95, 2.00 (2ϫ s, 3 H, CH3Ac), 2.72–2.88 (m,
2 H, 8-H2), 3.16–3.34 (m, 6 H, 1-H2, 3-H2, 5-H2), 5.16, 5.20 (2ϫ
s, each 2 H, CH2Bn), 7.11–7.14 (mcis/trans, 1 H, 5-HAr), 7.32–7.48
(m, 11 H, 2ϫ Ph, 6-HAr), 7.56 (br. s, 1 H, 2-HAr), 7.66–7.80 (br. s,
3 H, 8-NH3+), 8.35, 8.42 (2 ϫ tcis/trans, JNH,1 = 5.4 Hz, 1 H, 1-
NH) ppm. 13C NMR (75 MHz, [D6]DMSO): δ = 21.1, 24.1, 26.6,
28.2, 36.0, 38.4, 42.1, 43.8, 45.8, 47.1, 69.5, 69.8, 112.9, 113.0,
120.2, 127.1, 127.4, 127.5, 127.8, 128.1, 128.9, 136.9, 137.0 ppm,
no C=O signals observed (broad). HRMS (ESI): calcd. for
C30H38N3O4 [M + H]+ 504.2857; found 504.2851.
1-{N1Ј-[3ЈЈЈ,4ЈЈЈ-Bis(hydroxy)benzoyl]-N4Ј-[N-(biotinyl)aminoprop-
yl]spermidinyl}-3-{N1ЈЈ-[3ЈЈЈ,4ЈЈЈ-bis(hydroxy)benzoyl]spermidinyl}
Citrate (3): To a solution of protected biotinylated petrobactin 15
(81 mg, 52 μmol) in AcOH (3 mL) was added a mixture of conc.
HCl (0.5 mL) and AcOH (3 mL) at 15 °C with vigorous stirring.
The mixture was stirred at room temperature for 3 h, then the sol-
vents were removed by coevaporation with toluene (3 ϫ ) and
CHCl3 (3ϫ ), and the obtained crude monoacid was used for the
next step without further purification. HPLC showed complete re-
m ova l o f t h e t e rt - bu t y l e s t e r [ t R = 2 8 . 2 5 m i n ( C 1 8 ,
4.6 mm ϫ 150 mm, CH3 CN/H2 O/0.1 % TFA, flow rate:
0.6 mLmin–1, gradient: 10–90% over 45 min)].
1-{N1Ј-[3ЈЈ,4ЈЈ-Bis(benzyloxy)benzoyl]-N4Ј-acetyl-spermidinyl}-2-iso-
propyl Citrate (19): Preparation of cyclic anhydride 18:[32,35,36] To a
solution of 2-isopropyl citrate (17, 586 mg, 2.50 mmol) in THF
(15 mL) was added freshly activated molecular sieves (3 Å) and
432
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Bacillus anthracis Siderophore Petrobactin and Analogues
DCC (516 mg, 2.50 mmol), and the resulting suspension was stirred
for 1 h at room temperature. The solution was filtered through a
pad of Celite and the solvent was removed under reduced pressure
to afford the cyclic anhydride 18 (460 mg, 82%), which was used
directly in the next reaction step.
127.3, 127.4, 127.5, 127.8, 128.3, 128.4, 136.9, 137.0, 147.7, 150.6,
155.6, 165.3, 165.4, 169.0, 169.2, 169.3, 172.6 ppm. HRMS (ESI):
calcd. for C75H88N6O14Na [M + Na]+ 1319.6251; found 1319.6233.
1-{N1Ј-[3ЈЈЈ,4ЈЈЈ-Bis(hydroxy)benzoyl]-N4Ј-acetyl-spermidinyl}-3-
{N1ЈЈ-[3ЈЈЈ,4ЈЈЈ-bis(hydroxy)benzoyl]spermidinyl} Citrate (4): Pro-
tected petrobactin 20 (0.27 g, 0.21 mmol) was dissolved in a mix-
ture of THF/H2O (15 mL, 2:1) and cooled to 0 °C. After the ad-
dition of LiOH (13 mg, 0.25 mmol), the mixture was stirred for
60 min at this temperature. Strongly acidic ion exchange resin
(Dowex 50WX8-100, 50–100 mesh) was added and the mixture was
stirred for 5 min, filtered, and the solvents evaporated to dryness.
The residue was used for the next step without further purification.
To a solution of the anhydride 18 (460 mg, 2.00 mmol) in DMF
(2 mL) was added molecular sieves (3 Å) and a solution of amine
16 (TFA salt, 882 mg, 1.43 mmol) and Et3N (0.79 mL, 5.72 mmol)
in DMF (5 mL). The resulting mixture was stirred for 36 h at room
temperature, filtered, and the solvents evaporated. The residue was
dissolved in EtOAc and extracted with aq. HCl (1 m). The organic
layer was dried, filtered, and evaporated, and the residue was puri-
fied by flash chromatography (SiO2; CH2Cl2/MeOH/HOAc,
20:1:0.1Ǟ10:1:0.1) to afford monoacid 19 (755 mg, 74%) as a clear
oil. Rf = 0.05 (CH2Cl2/MeOH/HOAc, 20:1:0.1). 1H NMR
(300 MHz, [D6]DMSO): δ (cis/trans rotamers were observed) = 1.15
(d, J = 6.2 Hz, 6 H, 2ϫ CH3iPr), 1.28–1.54 (m, 4 H, 6Ј-H2, 7Ј-H2),
1.62–1.82 (mcis/trans, 2 H, 2Ј-H2), 1.93, 1.99 (2 ϫ scis/trans, 3 H,
CH3Ac), 2.42–2.53, 2.56–2.68, 2.72–2.82 (3ϫ m, 4 H, 2ϫ CH2citr),
2.94–3.12 (m, 2 H, 8Ј-H2), 3.14–3.33 (m, 6 H, 1Ј-H2, 3Ј-H2, 5Ј-H2),
4.87 (sept, J = 6.7 Hz, 1 H, CHiPr), 5.16, 5.19 (2ϫ s, 2 H, CH2Bn),
5.95 (br. s, 1 H, OHcitr), 7.12 (d, J5,6 = 8.5 Hz, 1 H, 5-HAr), 7.30–
7.48 (m, 11 H, 2ϫ Ph, 6-HAr), 7.57 (br. s, 1 H, 2-HAr), 7.98 (q,
The crude acid was dissolved in EtOH/H2O (20 mL, 10:1) and Pd/
C (30%, 80 mg) was added. The mixture was stirred vigorously at
room temperature for 72 h under a H2 atmosphere (35 bar), then
the mixture was filtered through a pad of Celite, concentrated, and
the residue was purified by preparative HPLC (column: C18,
4.60 mmϫ250 mm, solvent: CH3CN/H2O/0.1% TFA, flow rate:
1.10 mLmin–1, gradient: 5Ǟ35% CH3CN over 35 min). Appropri-
ate fractions (tR = 8.0–9.1 min) were lyophilized to give monoacyl-
ated petrobactin 4 (81 mg, 51%, two steps) as an amorphous solid.
1H NMR (500 MHz, [D6]DMSO): δ (cis/trans rotamers were ob-
served) = 1.25–1.59 (m, 8 H, 6Ј-H2, 6ЈЈ-H2, 7Ј-H2, 7ЈЈ-H2), 1.65,
1.75 (2ϫ quint.cis/trans, J = 7.2 Hz, 1 H, 2Ј-H2), 1.80 (quint., J =
7.1 Hz, 2ЈЈ-H2), 1.95, 1.99 (2ϫ scis/trans, 3 H, CH3Ac), 2.48–2.52,
2.53–2.65 (2ϫ m, each 2 H, 2ϫ CH2citr), 2.85–2.95 (m, 4 H, 3Ј-
H2, 5Ј-H2), 3.00–3.10 (m, 4 H, 8Ј-H2, 8ЈЈ-H2), 3.14–3.29 (m, 8 H,
1Ј-H2, 1ЈЈ-H2, 3ЈЈ-H2, 5ЈЈ-H2), 6.73–6.77 (m, 2 H, 2ϫ 5-HAr), 7.15–
7.19 (m, 2 H, 2ϫ 6-HAr), 7.25–7.28 (m, 2 H, 2ϫ 2-HAr), 7.92–7.99
(m, 2 H, 8Ј-NH, 8ЈЈ-NH), 8.09, 8.15 (2ϫ tcis/trans, JNH,1 = 5.6 Hz,
1 H, 1Ј-NH), 8.26–8.32 (m, 3 H, 1ЈЈ-NH, 4ЈЈ-NH2+), 9.10 (br. s,
2 H, 2ϫ 3-OHAr), 9.50 (br. s, 2 H, 2ϫ 4-OHAr) ppm. 13C NMR
(125 MHz, [D6]DMSO): δ = 21.7, 23.4, 25.2, 26.1, 26.5, 28.1, 36.6,
37.1, 38.1, 38.6, 42.9, 43.7, 44.7, 45.2, 46.9, 73.9, 115.3, 115.4,
115.5, 119.2, 119.4, 125.9, 126.3, 145.3, 148.7, 148.9, 167.1, 169.6,
169.9, 170.0, 175.4 ppm. HRMS (ESI): calcd. for C36H53N6O12 [M
+ H]+ 761.3716; found 761.3712.
JNH,8 = 5.4 Hz, 1 H, 8Ј-NH), 8.30, 8.36 (2 ϫ tcis/trans, JNH,1
=
5.5 Hz, 1 H, 1Ј-NH), 12.10 (br. s, 1 H, CO2Hcitr) ppm. 13C NMR
(75 MHz, [D6]DMSO): δ = 21.0, 21.3, 26.3, 27.6, 28.9, 36.7, 38.2,
42.8, 43.0, 46.1, 48.0, 67.8, 69.9, 70.2, 73.3, 113.3, 120.5, 127.3,
127.5, 127.7, 127.8, 128.4, 136.9, 137.0, 147.6, 150.6, 165.3, 165.6,
169.0, 169.4, 171.1, 172.4 ppm. HRMS (ESI): calcd. for
C39H48N3O10 [M – H]– 718.3340; found 718.3344.
1-{N1Ј-[3ЈЈЈ,4ЈЈЈ-Bis(benzyloxy)benzoyl]-N4Ј-acetyl-spermidinyl}-3-
{N1ЈЈ-[3ЈЈЈ,4ЈЈЈ-bis(benzyloxy)benzoyl]-N4ЈЈ-benzyloxycarbonyl-
spermidinyl}-2-isopropyl Citrate (20): To a solution of acid 19
(0.70 g, 0.97 mmol) in anhydrous THF (5 mL) was added N-hy-
droxysuccinimide (167 mg, 1.46 mmol) and DIC (275 μL,
1.75 mmol). The mixture was stirred at room temperature for 16 h,
then the solvents were evaporated to dryness to afford the crude N-
hydroxysuccinimide ester, which was used for the next step without
further purification.
1,3-Bis-{N1Ј-[3ЈЈ,4ЈЈ-bis(benzyloxy)benzoyl]-N4Ј-benzyloxycarbonyl-
spermidinyl}-2-isopropyl Citrate (21): Synthesis of bis(pNP) ester:
To a solution of 2-isopropyl citrate (17, 800 mg, 3.42 mmol)[32,35]
in anhydrous CH3CN (34 mL) were added p-nitrophenol (1.23 g,
8.80 mmol) and DCC (1.83 g, 8.80 mmol) at 0 °C, and the mixture
was then stirred at room temperature for 20 h. After complete for-
mation of the active ester was observed by TLC (Rf = 0.40; CH2Cl2/
MeOH, 10:1), the solvent was removed under reduced pressure and
the yellow residue was purified by flash chromatography (SiO2;
CH2Cl2/MeOH, 10:1) to give the bis(pNP) ester (1.22 g, 74%).
To a mixture of amine 12 (TFA salt, 1.24 g, 1.75 mmol) and Et3N
(2.43 mL, 17.5 mmol) in anhydrous CH2Cl2 (10 mL) was added a
solution of the crude NHS ester in anhydrous dioxane (7 mL). The
mixture was stirred at room temperature for 48 h, then evaporated
to dryness. The residue was dissolved in EtOAc and extracted with
aq. HCl (1 m), aq. NaOH (1 m), and brine. The organic phase was
dried, filtered, and concentrated. The residue was purified by flash
chromatography (SiO2; CH2Cl2/MeOH, 20:1Ǟ10:1) to give petrob-
actin derivative 20 (628 g, 50%, 2 steps) as an amorphous solid. Rf To a solution of amine 12 (TFA salt, 0.43 g, 0.60 mmol) in anhy-
= 0.35 (CH2Cl2/MeOH, 10:1). 1H NMR (500 MHz, [D6]DMSO): drous DMF (2 mL) was added Et3N (0.14 mL, 1.0 mmol) at 0 °C
δ (cis/trans rotamers were observed) = 1.13 (d, J = 6.3 Hz, 6 H, 2ϫ
and the mixture was stirred for 15 min. This solution was added
dropwise to a solution of the bis(pNP) ester (122 mg, 0.250 mmol)
CH3iPr), 1.27–1.53 (m, 8 H, 6Ј-H2, 6ЈЈ-H2, 7Ј-H2, 7ЈЈ-H2), 1.64–1.80
(m, 4 H, 2Ј-H2, 2ЈЈ-H2), 1.93, 1.99 (2ϫ scis/trans, 3 H, CH3Ac), 2.44– in anhydrous DMF (2 mL) in the presence of molecular sieves
2.50, 2.55–2.62 (2ϫ m, each 2 H, 2ϫ CH2citr), 2.95–3.06 (m, 4 H, (3 Å), and the mixture was stirred at room temperature for 18 h.
8Ј-H2, 8ЈЈ-H2), 3.16–3.29 (m, 12 H, 1Ј-H2, 1ЈЈ-H2, 3Ј-H2, 3ЈЈ-H2, 5Ј- The mixture was filtered, evaporated to dryness, and the residue
H2, 5ЈЈ-H2), 4.84 (sept, J = 6.2 Hz, 1 H, CHiPr), 5.02–5.08 (m, 2 H,
CH2Cbz), 5.16, 5.19 (2ϫ br. s, 8 H, 4ϫ CH2Bn), 5.73 (s, 1 H, OHcitr),
7.09–7.14 (m, 2 H, 2ϫ 5-HAr), 7.29–7.48 (m, 27 H, 5ϫ Ph, 2ϫ 6-
HAr), 7.54–7.57 (m, 2 H, 2ϫ 2-HAr), 7.90–8.04 (2ϫ mcis/trans, 2 H,
was dissolved in EtOAc, extracted with satd. aq. Na2CO3 (2ϫ), aq.
HCl (1 m), and brine. The organic layer was dried, and the residue
obtained after evaporation was purified by flash chromatography
(SiO2; CH2Cl2/MeOH, 20:1) to give protected petrobactin 21
8Ј-NH, 8ЈЈ-NH), 8.25–8.36 (2 ϫ mcis/trans, 2 H, 1Ј-NH, 1ЈЈ- (240 mg, 69%) as an amorphous solid. Rf = 0.30 (CH2Cl2/MeOH,
1
NH) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 21.2, 21.3, 25.7, 10:1). H NMR (400 MHz, [D6]DMSO): δ = 1.12 (d, J = 6.3 Hz,
26.2, 26.4, 27.6, 28.6, 36.9, 37.9, 38.1, 42.4, 43.2, 44.2, 44.7, 45.6,
47.6, 66.0, 67.6, 69.8, 70.2, 73.8, 113.2, 113.3, 120.5, 120.6, 127.2,
6 H, CH3iPr), 1.28–1.36 (m, 4 H, 2ϫ 7Ј-H2), 1.42–1.51 (m, 4 H,
2ϫ 6Ј-H2), 1.69–1.77 (m, 4 H, 2ϫ 2Ј-H2), 2.40 (d, J = 14.6 Hz, 2
2
Eur. J. Org. Chem. 2014, 426–435
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
433

N. Bugdahn, M. Oberthür
FULL PAPER
H, CHaHcitr), 2.58 (d, J = 14.6 Hz, 2 H, CHHbcitr), 2.95–3.06 (m, 172.6 ppm. HRMS (ESI): calcd. for C69H84N6O13Na [M + Na]+
2
4 H, 2ϫ 8Ј-H2), 3.17–3.28 (m, 12 H, 2ϫ 1Ј-H2, 2ϫ 3Ј-H2, 2ϫ 5Ј- 1227.5989; found 1227.6001.
H2), 4.83 (quint, J = 6.3 Hz, 1 H, CHiPr), 5.01–5.08 (m, 2 H,
1,3-Bis-{N1Ј-[3ЈЈ,4ЈЈ-bis(hydroxy)benzoyl]-N4Ј-acetyl-spermidinyl}
CH2Cbz), 5.15 (s, 4 H, 2ϫ CH2Bn), 5.19 (s, 4 H, 2ϫ CH2Bn), 5.73
Citrate (5): Protected di-N4Ј-acetyl petrobactin 22 (200 mg,
(s, 1 H, OHcitr), 7.11 (d, J5,6 = 8.5 Hz, 2 H, 2ϫ 5-HAr), 7.25–7.47
166 μmol) was dissolved in a mixture of THF/H2O (15 mL, 2:1)
(m, 32 H, 6ϫ Ph, 2ϫ 6-HAr), 7.56 (br. s, 2 H, 2ϫ 2-HAr), 7.90–
and cooled to 0 °C. LiOH (11 mg, 0.25 mmol) was added and the
mixture was stirred for 40 min. Strongly acidic ion exchange resin
(Dowex 50WX8-100, 50–100 mesh) was added and the mixture was
7.95 (br. s, 2 H, 2ϫ 8Ј-NH), 8.26–8.32 (br. s, 2 H, 2ϫ 1Ј-NH) ppm.
13C NMR (100 MHz, [D6]DMSO): δ = 21.3, 25.0, 25.6, 26.3, 27.9,
28.6, 36.9, 38.1, 43.2, 44.3, 44.7, 46.0, 46.4, 66.0, 67.6, 69.8, 70.2,
stirred for 10 min, filtered, and the solvents evaporated. The crude
73.8, 113.2, 113.3, 120.6, 127.3, 127.4, 127.5, 127.6, 127.8, 128.3,
monoacid (200 mg) was used for the next step without further puri-
128.4, 136.9, 137.0, 147.7, 150.6, 155.3, 165.5, 169.3, 172.6 ppm.
fication.
HRMS (ESI): calcd. for C81H92N6O15NNa [M + Na]+ 1411.6513;
The monoacid was dissolved in EtOH/H2O (20 mL, 10:1), Pd/C
found 1411.6526.
(10%, 120 mg) was added, and the mixture was stirred vigorously
at room temperature for 36 h under a H2 atmosphere (45 bar). Af-
1,3-Bis-{N1Ј-[3ЈЈ,4ЈЈ-bis(hydroxy)benzoyl]spermidinyl} Citrate (Pe-
trobactin, 1): Protected petrobactin 21 (124 mg, 89.7 μmol) was dis-
solved in a mixture of THF/H2O (10 mL, 2:1) and cooled to 0 °C.
After LiOH (6.0 mg, 0.13 mmol) was added, the mixture was
stirred for 40 min, then strongly acidic ion exchange resin (Dowex
50WX8-100, 50–100 mesh) was added, and the mixture was stirred
for 10 min, filtered, and the solvents evaporated to dryness to give
the crude monoacid (90 mg).
ter filtration through a pad of Celite, the mixture was concentrated
and the residue was purified by preparative HPLC (column: C18,
4.60 mmϫ250 mm; solvent: CH3CN/H2O/0.1% TFA, flow rate:
1.10 mLmin–1, gradient: 5Ǟ35% CH3CN over 35 min). Appropri-
ate fractions (tR = 8.0–9.1 min) were lyophilized to give di-N4Ј-
acetyl petrobactin
5 (85 mg, 64%) as an amorphous solid.
1H NMR (600 MHz, [D6]DMSO): δ (cis/trans rotamers were ob-
served) = 1.28–1.45 (m, 6 H, 2ϫ 7Ј-H2, 2ϫ 6Ј-Ha), 1.52–1.59 (m,
2 H, 2ϫ 6Ј-Hb), 1.60–1.68, 1.72–1.80 (2ϫ mcis/trans, each 2 H, 2Ј-
H2), 1.94, 1.98 (2ϫ scis/trans, 6 H, 2ϫ CH3Ac), 2.45–2.52, 2.54–2.66
(2ϫ mcis/trans, each 2 H, 2ϫ CH2citr), 2.98–3.08 (m, 4 H, 2ϫ 8Ј-
H2), 3.14–3.28 (m, 12 H, 2ϫ 1Ј-H2, 2ϫ 3Ј-H2, 2ϫ 5Ј-H2), 5.32
(br. s, 1 H, OHcitr), 6.74 (d, J5,6 = 8.3 Hz, 2 H, 2ϫ 5-HAr), 7.16,
7.17 (2ϫ ddcis/trans, J5,6 = 8.3, 4J2,6 = 2.1 Hz, 2 H, 2ϫ 6-HAr), 7.25,
The monoacid was dissolved in EtOH/H2O (10 mL, 10:1) and Pd/
C (10%, 10 mg) was added. The mixture was stirred vigorously at
room temperature for 36 h under a positive H2 pressure (55 bar).
After filtration through a pad of Celite, the mixture was concen-
trated under reduced pressure and the residue was purified by pre-
parative HPLC (column: C18, 25 mmϫ250 mm; solvent: CH3CN/
H2O/0.1% TFA, flow rate: 16 mLmin–1, gradient: 5–10% CH3CN
over 10 min, 10–50% CH3CN over 30 min). The appropriate frac-
tions (tR = 14.4 min) were lyophilized to give petrobactin (1; 40 mg,
62%, two steps) as an amorphous solid. The NMR and HRMS
data were identical to those obtained previously.[15]
4
7.26 (2ϫ dcis/trans, J2,6 = 2.3 Hz, 2 H, 2ϫ 2-HAr), 7.92–7.96 (m, 2
H, 2ϫ 8Ј-NH), 8.08, 8.14 (2ϫ tcis/trans, JNH,1 = 5.6 Hz, 2 H, 2ϫ
1Ј-NH), 9.06 (br. s, 2 H, 2ϫ 3-OHAr), 9.40 (br. s, 2 H, 2ϫ 4-OHAr),
12.52 (br. s, 1 H, CO2Hcitr) ppm. 13C NMR (125 MHz, [D6]
DMSO): δ = 21.2, 24.7, 25.6, 26.3, 26.4, 27.6, 28.6, 36.6, 37.9, 38.2,
42.4, 43.1, 44.2, 45.7, 47.6, 73.5, 114.8, 114.9, 115.0, 118.7, 118.8,
125.8, 125.9, 144.8, 148.2, 165.9, 166.2,169.1, 169.4, 169.5,
174.9 ppm. HRMS (ESI): calcd. for C38H55N6O13 [M + H]+
803.3822; found 803.3804.
1,3-Bis-{N1Ј-[3ЈЈ,4ЈЈ-bis(benzyloxy)benzoyl]-N4Ј-acetyl-spermidinyl}-
2-isopropyl Citrate (22): To an ice-cooled solution of 16 (TFA salt,
0.32 g, 0.52 mmol) in anhydrous DMF (4 mL) was added Et3N
(74 μL, 1.0 mmol), and the mixture was stirred for 15 min. This
solution was added dropwise to a solution of the bis(pNP) ester of
17 (0.12 g, 0.25 mmol, prepared as described for the preparation of
21) in anhydrous DMF (2 mL) that contained molecular sieves
(3 Å). After stirring at room temperature for 18 h, the mixture was
filtered and the solvents evaporated. The residue was dissolved in
EtOAc, extracted with satd. aq. Na2CO3 (2ϫ), aq. HCl (1 m), and
brine. The organic layer was dried, filtered, and evaporated, and
the residue was purified by flash chromatography (SiO2; CH2Cl2/
MeOH, 10:1) to give the protected di-N4Ј-acetyl petrobactin 22
(231 mg, 77%) as an amorphous solid. Rf = 0.30 (CH2Cl2/MeOH,
10:1). 1H NMR (600 MHz, [D6]DMSO): δ (cis/trans rotamers were
observed) = 1.14 (d, J = 6.2 Hz, 6 H, CH3iPr), 1.27–1.52 (m, 8 H,
2ϫ 6Ј-H2, 2ϫ 7Ј-H2), 1.67, 1.76 (2ϫ quintcis/trans, J = 7.2 Hz, 4 H,
2ϫ 2Ј-H2), 1.94, 1.99 (2ϫ scis/trans, 6 H, 2ϫ CH3Ac), 2.45–2.49,
2.55–2.61 (2ϫ mcis/trans, 4 H, 2ϫ CH2citr), 2.96–3.08 (m, 4 H, 2ϫ
8Ј-H2), 3.17–3.29 (m, 12 H, 2ϫ 1Ј-H2, 2ϫ 3Ј-H2, 3ЈЈ-H2, 2ϫ 5Ј-
H2, 5ЈЈ-H2), 4.84 (sept, 1 H, J = 6.2 Hz, CHiPr), 5.16, 5.19 (2ϫ s,
each 4 H, 4ϫ CH2Bn), 5.74 (br. s, 1 H, OHcitr), 7.11, 7.12 (2ϫ d,
J5,6 = 8.6 Hz, 2 H, 2ϫ 5-HArcis/trans), 7.30–7.47 (m, 22 H, 4ϫ Ph,
2ϫ 6-HAr), 7.55, 7.56 (2ϫ dcis/trans, 2J2,6 = 2.0 Hz, 2ϫ 2-HAr), 7.91–
7.98 (m, 2 H, 2ϫ 8Ј-NHc), 8.28, 8.33 (2ϫ tcis/trans, JNH,1 = 5.6 Hz,
2 H, 2ϫ 1Ј-NH) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 21.2,
21.3, 21.4, 24.8, 25.7, 26.2, 26.4, 27.6, 28.6, 36.7, 36.8, 37.9, 38.1,
42.4, 43.2, 44.2, 45.6, 47.6, 67.7, 68.8, 70.2, 73.8, 113.2, 113.3,
120.5, 120.6, 127.2, 127.3, 127.5, 127.6, 127.8, 128.2, 128.3, 128.4,
136.9, 137.1, 147.6, 150.6, 165.3, 165.7, 169.0, 169.2, 169.3, 169.4,
Determination of Iron Binding Affinities; General Remarks: An Ul-
trospec 3100 pro spectrophotometer was used for the EDTA com-
petition experiments. A UV/Vis range from 200 to 700 nm with a
data pitch of 1.0 nm and a scan speed of 1800 nm/min was used.
The samples were mixed and equilibrated for at least 16 h prior to
the competition experiment. All operations prior to the measure-
ment were performed under strict safe-light conditions.
Determination of the pFeIII Values: For petrobactin derivatives 1–5,
a spectrophotometric EDTA competition assay was performed by
following the general protocol developed by Abergel et al.[38] The
petrobactin derivatives were dissolved in HEPES buffer and an
equimolar FeIII chloride solution was added, giving a final concen-
tration of 0.2 mm. The UV/Vis measurements were performed as
a batch assay by adding different EDTA concentrations (EDTA
amounts of 1:10 to 25:1) of the same volume and equilibration for
at least 16 h. The composition of each batch was determined by
measuring a complete UV/Vis spectrum (buffer/EDTA as back-
ground) and deconvolution by using the UV/Vis spectra of the pure
iron complexes of each derivative. A plot of log[EDTA]/[sid] (sid
= petrobactin derivative) against log[Fe-EDTA]/[Fe-sid] directly
provided the difference of pFeIII values of both ligands, ΔpFeIII
=
pFeIII (sid) – pFeIII (EDTA), when the data is fitted to Equation (1)
derived by Abergel et al.[38] and the known pFeIII value of EDTA
(23.42) is used.
log[Fe-EDTA]/[Fe-sid] = log[EDTA]/[sid] – ΔpFeIII
(1)
434
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 426–435

Bacillus anthracis Siderophore Petrobactin and Analogues
[19]
[20]
Although bacillibactin is the major siderophore produced by
B. anthracis, petrobactin is important for a successful survival
within the mammalian host because it can act as a “stealth”
siderophore that evades the host defense system during an an-
thrax infection, see: R. J. Abergel, M. K. Wilson, J. E. Ar-
ceneaux, T. M. Hoette, R. K. Strong, B. R. Byers, K. N. Ray-
mond, Proc. Natl. Acad. Sci. USA 2006, 103, 18499–18503.
During our investigation, the same uptake system was iden-
tified based on sequence homologies by the group of Raymond,
which supports the validity of our approach: A. M. Zawadzka,
Y. Kim, N. Maltseva, R. Nichiporuk, Y. Fan, A. Joachimiak,
K. N. Raymond, Proc. Natl. Acad. Sci. USA 2009, 106, 21854–
21859.
F. D. Doorneweerd, W. A. Henne, R. G. Reifenberger, P. S.
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R. A. Gardner, R. Kinkade, C. Wang, O. Phanstiel IV, J. Org.
Plots of log[EDTA]/[sid] against log[Fe-EDTA]/[Fe-sid] that were
used for the determination of pFeIII of the different ligands are
included in the Supporting Information (Figure S1).
Fluorescence Spectroscopy: General experimental details and the
calculation of the protein-binding constants were performed as de-
scribed previously.[15] A plot of the fluorescence intensity of FpiA
against the concentration of ligand 3 is included in the Supporting
Information (Figure S2).
Supporting Information (see footnote on the first page of this arti-
cle): 1H and 13C NMR spectra of all new compounds. For com-
1
pound 16, a H/13C-HSQC spectrum is shown because of the peak
broadening observed in the 13C spectrum. Plots of log[EDTA]/[sid]
against log[Fe-EDTA]/[Fe-sid] for ligands 1–5. Fluorescence inten-
sity plot of FpiA against ligand 3.
[21]
[22]
[23]
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Acknowledgments
This research was funded in part by the Deutsche Forschungsge-
meinschaft (DFG) (to M. O.). The authors thank Prof. Mohamed
M. Marahiel and Dr. Florian Peuckert, Fachbereich Chemie, Phil-
ipps-Universität Marburg, for valuable discussions and for provid-
ing purified petrobactin binding protein FpiA.
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Published Online: November 7, 2013
Eur. J. Org. Chem. 2014, 426–435
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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