Modular fluorescent-labeled siderophore analogues

Article abstract of DOI:10.1021/jm970581b

Biomimetic analogues 1 of the microbial siderophore (iron carrier) ferrichrome were labeled via piperazine with various fluorescent markers at a site not interfering with iron binding or receptor recognition (compounds 10- 12). These iron carriers were bu

Full text of DOI:10.1021/jm970581b

J . Med. Chem. 1998, 41, 1671-1678
1671
Mod u la r F lu or escen t-La beled Sid er op h or e An a logu es
Raphael Nudelman,† Orly Ardon,‡ Yitzhak Hadar,‡ Yona Chen,§ J acqueline Libman,†,| and Abraham Shanzer*,†
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel, and Department of Plant Pathology
and Microbiology and The Otto Warburg Center for Agricultural Biotechnology, Department of Soil and Water Sciences,
The Hebrew University of J erusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot 76100, Israel
Received September 2, 1997
Biomimetic analogues 1 of the microbial siderophore (iron carrier) ferrichrome were labeled
via piperazine with various fluorescent markers at a site not interfering with iron binding or
receptor recognition (compounds 10-12). These iron carriers were built from a tetrahedral
carbon symmetrically extended with three strands, each containing an amino acid (G ) glycyl,
A ) alanyl, L ) leucyl and P ) phenylalanyl) and terminated by a hydroxamic acid, which
together define an octahedral iron-binding domain. A fourth exogenous strand provided the
site for connecting various fluorescent markers via a short bifunctional linker. Iron(III)
titrations, along with fluorescence spectroscopy, generated quenching of fluorescence emission
of some of the probes used. The quenching process fits the Perrin model which reinforces the
intramolecular quenching process, postulated previously.1 All tested compounds, regardless
of their probe size, polarity, or the linker binding them to the siderophore analogue, promote
growth of Pseudomonas putida with the same efficacy as the nonlabeled analogues 1, with the
added benefit of signaling microbial activity by fluorescence emission. All G derivatives of
compounds 10-12 were found to parallel the behavior of natural ferrichrome, whereas A
derivatives mediated only a modest iron(III) uptake by P. putida. Incubation of various
Pseudomonas strains with iron(III)-loaded G derivatives resulted in the build-up of the labels’
fluorescence in the culture medium to a much larger extent than from the corresponding A
derivatives. The fluorescence buildup corresponds to iron utilization by the cells and the release
of the fluorescent labeled desferrisiderophore from the cell to the media. The fact that the
microbial activity of these compounds is not altered by attachment of various fluorescent
markers via a bifunctional linker proposes their application as diagnostic tools for detecting
and identifying pathogenic microorganisms.
In tr od u ction
appears to be the most common one, involves a trans-
porter or permease for intact iron(III)-siderophore
complexes. The outer membrane transversing occurs
via a high-affinity active transport mechanism. After
transport, iron is removed from the complex, and the
desferri chelate is either secreted back to the medium
and possibly recycled there, or is hydrolyzed inside the
cell. The other two mechanisms involve extracellular
iron reduction followed by its uptake.
Iron is a trace element essential for all organisms with
only a few exceptions.2 It is required for basic cellular
functions such as respiration and DNA synthesis. In
aerobic environments iron exists as Fe(III) ion, forming
extremely insoluble oxide and hydroxide salts (Ksp
)
10-38). Siderophores are chelating agents produced by
microorganisms in iron deficient environments, which
solubilize and mobilize iron into their cells by ferri-
siderophore complexation.3 Microorganisms invading
the circulating blood produce siderophores which com-
pete for iron with the human transport protein trans-
ferrin, thus constituting one aspect of virulence and
pathogenicity.4-6 In bacteria the iron uptake system
is usually specific and involves a number of membranal
proteins activated under iron stress.7
The extensively studied ferrichrome transporter in
Escherichia coli is regulated by the Ton system.7,9-11
Particular gating loops in the outer membrane were
shown to be responsible for high specificity and molec-
ular recognition.11,12 Recently, it has been shown that
gated-porin channels open and close during membrane
transport.13 It is assumed that ferrichrome uptake
mechanisms for other bacteria are similar to the well-
defined mechanism found in E. coli.
Three different iron(III) uptake mechanisms are
known for siderophore-mediated iron transport across
microbial cell membranes.8 The first mechanism, which
Our studies have demonstrated that synthetic fer-
richrome analogues simulate natural ferrichrome as
growth promoters of P. putida.14 Small neutral fluo-
rescent labels have been attached to synthetic sidero-
phores1,15-21 and their potential as powerful tools for
iron(III) transport and uptake processes in microorgan-
isms has been shown.1,18-22 Moreover, some of these
compounds distinguish between different Pseudomonas
species with high specificity.1 However, it was not clear
whether fluorescent probes of substantial size and/or
* Corresponding author. Tel: +972-8-934-3954. Fax: +972-8-934-
2917. E-mail: coshanzr@weizmann.weizmann.ac.il.
† Weizmann Institute of Science.
‡ Department of Plant Pathology and The Otto Warburg Center for
Agricultural Biotechnology, The Hebrew University of J erusalem.
§ Department of Soil and Water Sciences, The Hebrew University
of J erusalem.
| Dr. J acqueline Libman, a main contributor to the work described
in this article, passed away prematurely on March 30, 1997. This
article is dedicated to her memory.
S0022-2623(97)00581-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/14/1998

1672 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10
Nudelman et al.
F igu r e 1. Schematic presentation of ferrichrome analogues 1 and fluorescent ferrichrome analogues 10-12.
charge could penetrate via particular siderophore trans-
port systems.
phobic leucyl (R ) i-Bu) and phenylalanyl (R ) Bn)
derivatives (L and P analogues in Figure 1), which do
not promote growth,25,26 were used as reference com-
pounds. The L derivative was used in microbial iron-
(III) utilization studies, whereas L and P derivatives
were used in fluorescence quenching measurements.
In earlier experiments it was shown that attachment
of the chemically inert anthracene to the carbon anchor
of ferrichrome analogues did not interfere with recogni-
tion by iron(III)-ferrichrome-specific uptake systems or
with the affinity of the iron(III) binding to the sidero-
phores.1 Analogous compounds 10-12 were prepared
by linking various fluorescent probes to the tetrahedral
carbon of ferrichrome analogues 1, via piperazine as a
bifunctional linker. This methodology, which facilitated
the insertion of a variety of fluorescent probes, enabled
the selection of probes suitable for available spectro-
scopic devices, such as light sources and filters used in
fluorescence microscopy, as well as the adaptation of
commercial probes. The strategy for choosing the
fluorescent probes was to determine whether charac-
teristic structural factors such as polarity and size of
the fluorescent probes effect the siderophores’ cell
transmembranal transport. NBD and the fluorescein
derivative diMe-FITC have similar optical properties
but differ in size, whereas lissamine rhodamine B (LRB)
and diMe-FITC are of similar size but differ in polarity
(Figure 1). Thus cell penetration characteristics of the
fluorescent ferrichrome analogues, as a function of size
and polarity, could be perused. The fluorescence inten-
sity of these probes was found to be stronger than the
autofluorescence of the bacterial culture. Another
aspect considered in the choice of probes was the
differences in their quenching properties. The fluores-
cence emission of NBD and of diMe-FITC are quenched
in the presence of iron(III), whereas that of LRB is not.
These optical properties enabled both in vitro monitor-
ing of iron(III) binding and in vivo follow-up of the
In this work we introduce a series of bioactive
ferrichrome analogues labeled with various fluorescent
probes at a site that does not interfere with iron binding
or with receptor recognition. The synthetic methodology
is based on piperazine as a bifunctional linker, which
connects the probes to the ferrichrome analogues. We
have found that in the presence of iron(III), divergent
fluorescent behavior is displayed depending on the
nature of the probe. Compounds 10 and 11 underwent
fluorescent quenching upon iron(III) binding and re-
gained their fluorescence upon ligand exchange with a
competing chelator or via iron removal by the microbial
cell. These analogues were detected in the extracellular
medium as a result of microbial iron uptake. The
fluorescence of compounds 12 was completely unaffected
by the presence or absence of iron, thereby facilitating
the monitoring of the iron uptake pathway in fungal
cells.23 All the synthesized compounds, independent of
the probe’s size or charge, display microbial activity in
P. putida similar to that of the biomimetic analogues
lacking the probe.
Resu lts a n d Discu ssion
Design . A series of fluorescent ferrichrome ana-
logues was synthesized by linking fluorescent probes to
bioactive ferrichrome analogues 1, at a site not interfer-
ing with the ferrichrome receptor binding (Figure 1).24
The glycyl ferrichrome analogue 1G (R ) H), which fully
mimics natural ferrichrome as an iron(III) carrier and
growth promoter, is taken up by specific receptor
recognition in P. putida.14 However, the alanyl ana-
logue 1A (R ) Me) inhibits the action of the natural
iron(III) carrier by competing for a specific site in the
uptake system.14 Siderophore analogue 1A alone pro-
motes some growth, but at a much lower level than the
natural ferrichrome or the 1G analogue. The hydro-

Modular Fluorescent-Labeled Siderophore Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10 1673
Sch em e 1. Synthesis of Fluorescent Ferrichrome
Sch em e 2. Synthesis of Piperazinyl-Fluorescent Probe
Analogues
Conjugates
microbial iron(III) uptake process. The fate of the iron
carriers after iron delivery could be monitored optically
by using the quenchable probes, and the iron uptake
pathway in eukaryote cells could be followed using the
nonquenchable probe.23
Syn th esis. The labeled ferrichrome analogues were
prepared by coupling the C3-symmetric tetraphenolate
2 with equivalent amounts of the piperazinyl-fluorescent
probe conjugates 3-5, followed by coupling with 3 equiv
of hydroxamate-bearing amino acid residues 9 (Scheme
1). This two-step synthesis gave yields significantly
higher than the one-pot reaction procedure described
previously, by eliminating statistically possible byprod-
ucts.1
that the fluorescent probe be in proximity to its quench-
er (the iron binding domain in our case).28 Indirect
evidence21,29,30 indicates that electron transfer is indeed
involved in these processes. Furthermore, the diversity
of spectral properties of the fluorescent probes influ-
enced the quenching of fluorescence emission (Table 1).
Upon titration with iron(III), the fluorescence emis-
sion of the NBD derivatives 10 (Figure 2) and the diMe-
FITC derivatives 11 (data not shown) was quenched.
The observed fluorescence quenching, φ/φo, of these
ferrichrome analogues was not linear with quencher
[iron(III)] concentration and thus failed to obey the
Stern-Volmer model of dynamic quenching (Figure 3,
top). However, the ln φ/φo values were linear with
quencher concentration, in agreement with the Perrin
model of static quenching (Figure 3, bottom), suggesting
that all the added quencher is bound to the ligand, and
the quenched chromophore lies within the quencher’s
active sphere.31 This agreement with the Perrin model
further supports the suggestion that fluorescence quench-
ing for systems of this type occurs intramolecularly.1
The fluorescence quenching efficiency values of com-
pound 10 and 11, in the Perrin equation were k′ ≈ 2 ×
105 M-1 and 1 × 105 M-1, respectively. The fluorescence
emission of LRB derivatives of compound 12 was not
quenched at all by iron(III) chelation (data not shown).
This observation implies a relationship between the
optical properties of a fluorescent probe and its quench-
ing ability.
The piperazinyl-fluorescent probe conjugates were
prepared by condensing NBD chloride or LRB sulfonyl
chloride with piperazine, yielding monosubstituted pip-
erazinyl-NBD 3 and piperazinyl-LRB 5, respectively.
Condensation of fluorescein-5-isothiocyanate (FITC iso-
mer I) with N-t-Boc-piperazine followed by methylation
gave the methyl ester, methyl ether of fluorescein (diMe-
FITC) (Scheme 2). This alkylation, which was neces-
sary for the purification of the compounds, reduced
fluorescence intensity. (Etherification of both phenolic
groups of fluorescein locks the molecule into its non-
fluorescent lactone form.27) However, the fluorescence
intensity of fluorescein’s monoether derivative was
found to be adequate for this work, because of its high
quantum yield. Final removal of the Boc resulted in
the synthesis of the desired piperazinyl-(diMe-FITC)
conjugate 4.
Ir on (III) Bin din g P r oper ties. Fluorescence quench-
ing can occur by electron transfer, energy transfer, or
by intersystem crossing. Earlier studies suggested that
fluorescence quenching by electron transfer requires

1674 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10
Ta ble 1. Spectral Characteristics of Fluorescent Ferrichrome Analogs
Nudelman et al.
fluorescent
quenching
efficiencies (kq)
compd
no.
R (amino acid
derivative)
fluorescent
probe
excitation
(λmax nm)
ꢀ (×103
emission
(λmax nm)
cm2 M-1
)
10G
10A
10L
10P
H (glycyl)
Me (alanyl)
i-Bu (leucyl)
Bn (phenylalanyl)
NBD
474
23
540
2 × 105 M-1
11G
11A
H (glycyl)
Me (alanyl)
diMe-FITC
LRB
457
530
25
53
522
606
1 × 105 M-1
∼0
12G
12A
12L
H (glycyl)
Me (alanyl)
i-Bu (leucyl)
F igu r e 2. Fluorescence iron(III) titration curves with labeled
ferrichrome analogue 10G. Aliquots of stock solutions of 10G
in MeOH were treated with aliquots of methanolic solutions
of FeCl3 (0, 0.2, 0.4, 0.6, 0.8, and 1.0 equiv) and diluted with
MeOH (80%)-0.1 N aqueous NaOAc (20%) to a final ligand
concentration of 50 µM.
Biology
Micr obia l Gr ow th P r om otion in Liqu id Med iu m .
The growth-promoting effects of iron(III)-loaded fer-
richrome and its synthetic analogues were studied in
liquid media. The growth was tested with P. putida
J M218, a non-siderophore-producing mutant; therefore,
no ligand exchange could occur between exogenous and
native siderophores. Growth of such sid- mutants is
therefore proportional to the bioavailability of the iron
source supplied. Here we have shown that all G
ferrichrome derivatives, irrespective of the size or
polarity of the fluorescent probes linked to them,
promoted the growth of P. putida with the same
efficiency as the nonlabeled analogue 1G and as the
natural ferrichrome. However, all A derivatives pro-
moted growth to a lesser extent than both the G
derivatives and the natural ferrichrome, while retaining
efficacy regardless of the probe attached. Figure 4
presents the growth of compounds 10G and 10A in
comparison to ferrichrome, 1G and 1A. Similar results
were observed for compounds 11G, 11A, 12G, and 12A
(data not shown).
F igu r e 3. Plots for dynamic quenching (top) and static
quenching (bottom) of the fluorescent-labeled ferrichrome
analogues 10G, 10A, 10L, 10P , 11G, and 11A by iron(III). The
conditions were as described in the legend of Figure 2.
medium after delivering iron(III) to the bacterial cells.1
Therefore, analogues whose fluorescence was quenched
by iron(III) binding regained their fluorescence and were
detected in the culture medium in accordance with the
microbial uptake of iron(III). P. putida J M218 was
incubated with iron(III) complexes of 10G, 11G, 10A,
11A, and 10L, and the appearance of the free ligands
fluorescence in the growth media culture was monitored
(Figure 5). The fluorescence obtained from the G
Micr obia l Ir on (III) Up ta k e. Natural ferrichrome
and its synthetic analogues are released to the culture

Modular Fluorescent-Labeled Siderophore Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10 1675
F igu r e 4. Growth promotion of P. putida J M218 in liquid
medium in the presence of iron(III)-loaded ferrichrome and
its synthetic analogues, 1G, 1A, 10G, and 10A.
F igu r e 6. The fluorescence appearance in cultures of P.
putida J M218 amended with 10G (1 µM) in the presence of
other ferrichrome analogues (1 µM).
Addition of sodium azide to the medium drastically
reduced the increase of fluorescence in the supernatant
as well as the 55Fe(III) incorporation to the cell, indicat-
ing that iron(III) uptake in P. putida is an energy driven
process, as shown previously.1,14
Our experiments indicated that neither size nor
polarity of the attached fluorescent probe interferes with
the recognition of the siderophore and its transport in
P. putida. The G ferrichrome analogues were found to
be better promoters of iron(III) uptake than the corre-
sponding A analogues just as their parent nonlabeled
ferrichrome analogues do.14
Con clu sion s
F igu r e 5. Emergence of fluorescence signals derived from
ferrichrome analogues in cultures of P. putida J M218.
Biomimetic siderophore analogues were found to be
useful tools for studying iron uptake in bacteria and
fungi.14,22,33-40 Attaching a fluorescent derivative to a
site which does not interfere either with the recognition
of the siderophore by the cell or the transport of the iron
to it, enables the selection of these compounds in iron
uptake studies. Earlier results1 along with the present
work demonstrated the usefulness of these novel tools
in studying the ferrichrome uptake system in Pseudomo-
nas sp. The possible attachment of fluorescent labels
to the siderophore via short bifunctional linkers enables
to choose the most suitable probe for each specific
microbial system (e.g., the avoidance of autofluorescence
of the cells). These modular compounds facilitate the
use of different fluorescence techniques with the avail-
able laboratory equipment (e.g., different filters in
fluorescent microscopes). The preferential iron uptake
of microorganisms may lead to the development of
species-specific libraries of siderophore analogues. The
uniqueness of the compounds described herein is that
they can serve as diagnostic tools for the presence of
living cells and particularly pathogenic ones in a physi-
ologically active form or stage.
derivatives indicated more efficient iron(III) utilization
than from the A analogues, whereas the lack of any
fluorescence from the hydrophobic L derivative indi-
cated that iron(III) was not utilized. The data were
compared to that obtained from two other Pseudomonads
(Pseudomonas fluorescens S680 and Pseudomonas fluo-
rescens WCS 3742). In ferrichrome analogues where no
piperazine ring linked the fluorescent probe (an-
thracene) to the fourth exogenous strand, selectivity was
exhibited between these Pseudomonads.1 In the present
study, similar specificity toward the amino acid deriva-
tive (G or A) was observed regardless the fluorescent
probe conjugated, but no selectivity was detected be-
tween the different strains (data not shown). This lack
of selectivity resembles that of the natural ferrichrome’s
toward these Pseudomonads.32
Fluorescence of the nonquenchable ferrichrome ana-
logues in the cultures’ medium is obviously not an
indication of iron uptake. Therefore, to show that
nonquenchable analogues compete for a common recep-
tor in the uptake system in P. putida, the fluorescence
emission of 10G (a quenchable ferrichrome analog) in
the culture medium was measured in the presence of
compounds 12G and 12A (nonquenchable ferrichrome
analogues), and 1G and 1A (nonlabeled ferrichrome
analogues). As predicted, 12G and 1G equally de-
creased the fluorescence emission intensity of 10G to a
larger extent than 12A and 1A, suggesting an inhibition
of bacterial iron utilization from 10G (Figure 6).
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were measured with a
Fischer-J ones apparatus and are uncorrected. IR spectra were
recorded on a Nicolet 510 FTIR spectrometer. UV/vis was
measured with a Hewlett-Packard model 8450A diode array
spectrophotometer. Molar extinction coefficients ꢀ are given
in units of cm2 M-1. Fluorescence spectra were recorded on a

1676 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10
Nudelman et al.
SLM-AMINCO MC200 monochromator fluorospectrometer. In
biological experiments fluorescence was measured with a SLM
Instruments fluorometer (model 4800). 1H NMR spectra were
measured on a Bruker AMX-400 MHz spectrometer or on a
Bruker DPX-250 MHz spectrometer with TMS as the internal
reference. All J values are given in hertz. Positive fast atomic
bombardment mass spectrometry (FABMS) was measured on
a VG Autospec Fison Instruments mass spetrometer. Flash
chromatography was performed using Merck 230-400 mesh
silica gel. Thin-layer chromatography (TLC) on silica gel 60
F-254 on aluminum was visualized by UV light, by ninhydrin,
by iodine, or by FeCl3/EtOH.
Solven ts a n d Ma ter ia ls. MeCN and CH2Cl2 were dried
by filtration through basic alumina. Acrylonitrile was purified
prior to use by filtration over neutral alumina. THF was
distilled from Na/benzophenone under Ar. All reagents unless
otherwise mentioned were purchased from Sigma and were
used without further purification. Piperazine was dried under
vacuum over P2O5. Compound 21 and N-t-Boc-protected 941
were prepared according to our earlier procedures. The
following abbreviations have been used: DIPCI ) diisopro-
pylcarbodiimide, PCP ) pentachlorophenol, pip ) piperazine,
NBD chloride ) 4-chloro-7-nitrobenzofurazane, FITC ) fluo-
rescein isothiocyanate, LRB ) lissamine rhodamine B, TM-
SCHN2 ) trimethylsilyldiazomethane.
P ip er a zin yl-NBD (3). A solution of NBD chloride (Fluka,
2.00 g, 10 mmol) in THF (80 mL) was slowly added to an ice
cold solution of piperazine (2.68 g, 31 mmol) in THF. The color
of the solution changed instantaneously to orange and within
a few seconds became red. The solvent was evaporated, the
crude product was dissolved in a minimal amount of MeOH/
acetone (1:1), and the solution was dripped into CHCl3 (500
mL). The mixture was extracted with water, dried (Na2SO4),
and concentrated to give 3 as a red powder (2.5 g, 10 mmol,
∼100% yield). 1H NMR (acetone-d6): δ 8.47, 6.63 (two d, J )
9.2, 2H, ArH), 4.16 (t, J ) 5.0, 4H, CH2NAr), 3.07 (t, J ) 5.0,
4H, CH2NH).
(157 mg, 0.112 mmol, 56%). 1H NMR (CDCl3): δ 8.41, 6.24
(two d, J ) 8.9, 2H, ArH), 4.16, 4.01, 3.92, 3.80 (four br t, 8 H,
piperazine CH2’s), 3.80 (t, J ) 6.0, 6H, OCH2CH2COO), 3.75
(t, J ) 6.5, 2H, OCH2CH2CON), 3.45 (s, 6H, CH2OCH2CH2-
COO), 3.43 (s, 2H, CH2OCH2CH2CON), 2.92 (t, J ) 6.0, 6H,
CH2COO), 2.64 (t, J ) 6.5, 2H, CH2CON).
DiMe-FITC-pip-COCH2CH2OCH2C(CH2OCH2CH2COOC6-
Cl5)3 (7). DMAP (27 mg, 0.22 mmol) and 4 (110 mg, 0.218
mmol) were added to 2 (618 mg, 0.436 mmol) in THF, and the
solution was stirred overnight under Ar. Evaporation of the
solvent and flash chromatography (CHCl3/MeOH, 48:1) yielded
7 as a yellow waxy solid (75 mg, 0.045 mmol, 10%). 1H NMR
(CDCl3): δ 9.84 (s, 1H, NH), 8.23 (d, J ) 2.1, 1H, CHC-
COOMe), 7.75 (dd, J 1 ) 8.0, J 2 ) 2.1, 1H, CHCNH), 7.10 (d, J
) 9.1, 1H, CHCHCOMe), 7.09 (d, J ) 8.0, 1H, CHCHCNH),
6.95 (d, J ) 2.4, 1H, CCHCOMe), 6.77 (dd, J 1 ) 9.1, J 2 ) 2.4,
1H, CHCHCOMe), 6.63 (d, J ) 9.6, 1H, CHCHCO), 6.45 (d, J
) 1.9, 1H, CCHCO), 5.96 (dd, J 1 ) 9.6, J 2 ) 1.9, 1H,
CHCHCO), 4.26, 4.15, 3.82, 3.76 (four br t, 8 H, piperazine
CH2’s), 3.80 (t, J ) 6.0, 6H, CH2CH2COO), 3.75 (t, J ) 6.5,
2H, CH2CH2CON), 3.46 (s, 6H, CH2OCH2CH2COO), 3.44 (s,
2H, CH2OCH2CH2CON), 2.92 (t, J ) 6.0, 6H, CH2COO), 2.64
(t, J ) 6.5, 2H, CH2CON).
LRB-p ip -COCH 2CH2OCH2C(CH2OCH2CH2COOC6Cl5)3
(8). DMAP (20 mg, 0.16 mmol) and 5 (100 mg, 0.16 mmol)
were added to 2 (454 mg, 0.32 mmol) in MeCN in an ice bath,
and the solution was stirred overnight under Ar. Evaporation
of the solvent and flash chromatography (CH2Cl2/ether/MeOH,
17:2:1) yielded 8 as a purple waxy solid (65 mg, 0.036 mmol,
23%). 1H NMR (CDCl3): δ 8.58 (s, 1H, CHCSO3), 8.40 (d, J )
7.8, 1H, CHCHSO2), 7.29 (d, J ) 7.8, 1H, CHCHSO2), 7.13 (d,
J ) 9.5, 2 H, CHCHCN), 6.87 (dd, J 1 ) 9.5, J 2 ) 2.3, 2H,
CHCHCN), 6.73 (d, J ) 2.3, 2H, CCHCN), 3.80 (t, J ) 6.1,
6H, CH2CH2COO), 3.62 (m, 8H, CH2CH2CON + CH2Me), 3.51,
3.36 (two br s, 8 H, piperazine CH2’s), 3.43, 3.36 (two s, 8H,
CCH2O), 2.91 (t, J ) 6.1, 6H, CH2COO), 1.34 (t, J ) 7.0, 12H,
CH3).
P ip er a zin yl-d iMe-F ITC (4). A solution of FITC isomer I
90% (400 mg, 0.924 mmol) and Boc-piperazine (172 mg, 0.924
mmol) in THF was stirred under Ar for 2 h. TMSCHN2 (0.92
mL of 2.0 M solution in hexanes, 1.84 mmol) was added, and
the solution was stirred overnight. The solvent was evapo-
rated, and flash chromatography (CHCl3/MeOH 98:2) of the
residue provided the Boc-protected product of 4. The Boc was
removed by stirring in TFA/CH2Cl2 (1:2) for 20 min, the
product was neutralized with [(diethylamino)methyl]polysty-
rene, filtered, and flash chromatographed (CHCl3/MeOH 4:1),
yielding 4 as a yellow waxy solid (110 mg, 0.22 mmol, 24%).
1H NMR (CDCl3): δ 9.84 (s, 1H, NH), 8.23 (d, J ) 2.1, 1H,
CHCCOOMe), 7.75 (dd, J 1 ) 8.0, J 2 ) 2.1, 1H, CHCNH), 7.10
(d, J ) 9.1, 1H, CHCHCOMe), 7.09 (d, J ) 8.0, 1H, CHCH-
CNH), 6.95 (d, J ) 2.4, 1H, CCHCOMe), 6.77 (dd, J 1 ) 9.1, J 2
) 2.4, 1H, CHCHOMe), 6.63 (d, J ) 9.6, 1H, CHCHCO), 6.45
(d, J ) 1.9, 1H, CCHCO), 5.96 (dd, J 1 ) 9.6, J 2 ) 1.9, 1H,
CHCHCO), 4.15 (t, J ) 5.2, 4H, CH2NCS), 3.89 (s, 3H,
COOCH3), 3.62 (t, J ) 5.2, 4H, CH2NCO), 3.48 (s, 3H, COCH3),
1.48 (s, 9H, t-Bu).
P ip er a zin yl-LRB (5). A solution of LRB sulfonyl chloride
(Molecular Probes, 500 mg, 0.87 mmol) and piperazine (373
mg, 4.33 mmol) in DMF was stirred overnight under Ar. The
solvent was evaporated, and flash chromatography (MeCN/
MeOH/NH4OH, 79:20:1) of the residue yielded 5 (230 mg, 0.37
mmol, 42%) as a purple powder. 1H NMR (CD3OD): δ 8.54
(d, J ) 1.7, 1H, CHCSO3), 8.24 (dd, J 1 ) 7.9, J 2 ) 1.7, 1H,
CHCHSO2), 7.56 (d, J ) 7.9, 1H, CHCHSO2), 7.09 (AB q, J )
9.5, ∆ ) 8.8, 4H, CHCHCN, CHCHCN is also coupled to
CCHCN, J ) 2.2), 6.98 (d, J ) 2.2, 2H, CCHCN), 3.68 (q, J )
7.1, 8 H, CH2Me), 2.90, 2.64 (two t, J ) 4.8, 8H, CH2CH2N),
1.30 (t, J ) 7.1, 12H, CH3).
Gen er a l P r oced u r e for P r ep a r in g F lu or escen t F er -
r ich r om e An a logu es. The Boc-protective group was re-
moved from the N-methylhydroxamates by stirring in TFA/
CH2Cl2 (1:2) for 20 min and neutralized with [(diethyl-
amino)methyl]polystyrene, which was then removed by filtra-
tion, yielding compounds 9. N-Methylhydroxamates 9 (0.33
mmol) were added to THF solutions of the fluorescent-probe-
piperazine-triphenolate compounds 6, 7, and 8 (0.10 mmol),
respectively. “DMAP” on polystyrene was added to the solu-
tions and stirred overnight. Basic pH was maintained by
adding “DMAP” on polystyrene and/or [(diethylamino)methyl]-
polystyrene. TLC analysis and the disappearance of the IR
active ester absorption at 1783 cm-1 were indications that the
reaction was completed. The basic polymers were removed
by filtration, the filtrates were evaporated, and the residual
products were purified by preparative TLC. Unless stated
otherwise all compounds were obtained as viscous oils.
NBD-p ip -COCH2CH2OCH 2C(CH2OCH2CH2CONHCH 2-
CON(OH)Me)3 (10G). Elution CHCl3/MeOH (9:1), yield 61%.
1H NMR (CD3OD): δ 8.49, 6.53 (two d, J ) 9.0, 2H, ArH), 4.28,
4.17, 3.94, 3.89 (four br t, 8H, piperazine CH2’s), 4.11 (s, 6H,
NHCH2CO), 3.67 (t, J ) 6.2, 2H, NCOCH2CH2O), 3.61 (t, J )
6.1, 6H, NHCOCH2CH2O), 3.38, 3.36 (two s, 8H, OCH2C), 3.17
(s, 9H, NCH3), 2.69 (t, J ) 6.2, 2H, NCOCH2), 2.46 (m, 6H,
NHCOCH2). FABMS: 914.19 MH+. UV/vis: λmax 474 nm, ꢀ
) 27 240. Anal. (C36H55N11O17‚2.5H2O) C, H, N.
NBD-p ip -COCH 2CH 2OCH 2C(CH 2OCH 2CH 2CONH CH -
MeCON(OH)Me)3 (10A). Elution CHCl3/MeOH (87:13), yield
48%. 1H NMR (CD3OD): δ 8.41, 6.48 (two d, J ) 8.9, 2H,
ArH), 4.98 (m, 3H, CH), 4.29, 4.18, 3.99, 3.94 (four br t, 8H,
piperazine CH2’s), 3.72 (t, J ) 6.0, 2H, NCOCH2CH2O), 3.63
(t, J ) 5.8, 6H, NHCOCH2CH2O), 3.38, 3.35 (two s, 8H,
OCH2C), 3.20 (s, 9H, NCH3), 2.74 (t, J ) 6.0, 2H, NCOCH2),
2.45 (t, J ) 5.8, 6H, NHCOCH2), 1.32 (d, J ) 7.0, 9H, CHCH3).
FABMS: 955.11 M+. Fe(III) complex: UV/vis: λmax 476 nm,
ꢀ ) 11 920. Anal. (C39H61N11O17‚4H2O) C, H; N: calcd, 14.99;
found, 14.31.
N B D -p ip -C O C H 2C H 2O C H 2C (C H 2O C H 2C H 2C O O C 6-
Cl5)3 (6). DMAP (24 mg, 0.2 mmol) and 3 (50 mg, 0.20 mmol)
were added to 2 (570 mg, 0.40 mmol) in THF, and the solution
was stirred for 2 h. Evaporation of the solvent and flash
chromatography (CHCl3) yielded 6 as an orange waxy solid

Modular Fluorescent-Labeled Siderophore Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10 1677
NBD-p ip -COCH2CH2OCH2C(CH2OCH2CH2CONHCH(i-
Bu )CON(OH)Me)3 (10L). This compound was prepared in
a slightly different manner than described in the general
procedure. Compounds 2, 3, and 9 (R ) i-Bu) were dissolved
in THF at a 1:1:3 ratio and stirred overnight yielding 10L,
along with the dihydroxamate-diNBD and monohydroxamate-
triNBD. (This procedure gave much lower yields than achieved
when using the general procedure). Elution toluene/i-PrOH
(7:3), yield 2.2%. 1H NMR (CD3OD): δ 8.46, 6.52 (two d, J )
9.0, 2H, ArH), 5.12 (t, J ) 5.8, 3H, NHCH), 4.29, 4.18, 3.97,
3.92 (four br t, 8H, piperazine CH2’s), 3.71 (t, J ) 6.3, 2H,
NCOCH2CH2O), 3.62 (m, 6H, NHCOCH2CH2O), 3.37, 3.35 (two
s, 8H, OCH2C), 3.18 (s, 9H, NCH3), 2.73 (t, J ) 6.3, 2H,
NCOCH2), 2.45 (m, 6H, NHCOCH2), 1.69 (m, 3H, i-Bu CH’s),
1.51 (m, 6H, i-Bu CH2’s), 0.93 (d, J ) 6.9, 18H, i-Bu CH3’s).
FABMS: 1105 MNa+. UV/vis: λmax 474 nm, ꢀ ) 14 220.
NBD-p ip -COCH 2CH 2OCH 2C(CH 2OCH 2CH 2CONH CH -
(Bn )CON(OH)Me)3 (10P ). Elution CHCl3/MeOH (23:2), yield
13%. 1H NMR (CD3OD): δ 8.44, 6.47 (two d, J ) 9.0, 2H,
ArH), 7.20 (m, 15H, Ph), 5.28 (m, 3H, CH), 4.23, 4.13, 3.89,
3.89 (four br t, 8H, piperazine CH2’s), 3.62 (t, J ) 6.0, 2H,
NCOCH2CH2O), 3.48 (t, J ) 5.6, 6H, NHCOCH2CH2O), 3.22
3.20 (two s, 8H, OCH2C), 3.19 (s, 9H, NCH3), 2.67 (t, J ) 6.0,
2H, NCOCH2), 2.36 (t, J ) 5.6, 6H, NHCOCH2). FABMS:
1181.66 (M - H2)+. UV/vis: λmax 481 nm, ꢀ ) 22 580.
DiMe-FITC-pip-COCH2CH2OCH2C(CH2OCH2CH2CONH-
CH2CON(OH)Me)3 (11G). Elution CHCl3/MeOH (17:3), yield
21%. 1H NMR (CD3OD): δ 8.54 (br s, 1H, NHCS), 8.31 (d, J
) 2.1, 1H, CHCCOOMe), 7.86 (dd, J 1 ) 8.2, J 2 ) 2.1, 1H,
CHCNH), 7.36 (d, J ) 8.2, 1H, CHCHCNH), 7.25 (d, J ) 2.4,
1H, CCHCOMe), 7.20 (d, J ) 9.0, 1H, CHCHCOMe), 7.18 (d,
J ) 9.6, 1H, CHCHCO), 6.96 (dd, J 1 ) 9.0, J 2 ) 2.4, 1H,
CHCHOMe), 6.60 (dd, J 1 ) 9.6, J 2 ) 2.0, 1H, CHCHCO), 6.51
(d, J ) 2.0, 1H, CCHCO), 4.16 (s, 6H, CH2CONOH), 4.14 (br
d, 4H, CH2NCS), 3.99 (s, 3H, COOCH3), 3.83 (m, 4H, CH2-
NCO), 3.71 (t, J ) 6.1, 2H, CH2CH2CON), 3.67 (t, J ) 6.0,
6H, CH2CH2CONH), 3.60 (s, 3H, COCH3), 3.42 (s, 8H, CCH2O),
3.21 (s, 9H, NCH3), 2.72 (t, J ) 6.1, 2H, CH2CON), 2.52 (t, J
) 6.0, 6H, CH2CONH). FABMS: 1169.12 MH+. Fe(III)
complex: UV/vis: λmax 458 nm, ꢀ ) 8864.
DiMe-FITC-pip-COCH2CH2OCH2C(CH2OCH2CH2CONH-
CHMeCON(OH)Me)3 (11A). Elution CHCl3/MeOH (9:1),
yield 37%. 1H NMR (CD3OD): δ 8.30 (d, J ) 2.1, 1H,
CHCCOOMe), 7.86 (dd, J 1 ) 8.2, J 2 ) 2.1, 1H, CHCNH), 7.35
(d, J ) 8.2, 1H, CHCHCNH), 7.24 (d, J ) 2.4, 1H, CCHCOMe),
7.19 (d, J ) 9.0, 1H, CHCHCOMe), 7.16 (d, J ) 9.6, 1H,
CHCHCO), 6.95 (dd, J 1 ) 9.0, J 2 ) 2.4, 1H, CHCHCOMe),
6.59 (dd, J 1 ) 9.6, J 2 ) 2.0, 1H, CHCHCO), 6.50 (d, J ) 2.0,
1H, CCHCO), 5.00 (t, J ) 7.0, 3H, CHMe), 4.18, 4.07 (two br
t, 4H, CH2NCS), 3.98 (s, 3H, COOCH3), 3.82 (m, 4H, CH2NCO),
3.71 (t, J ) 6.1, 2H, CH2CH2CON), 3.64 (t, J ) 6.0, 6H, CH2-
CH2CONH), 3.59 (s, 3H, COCH3), 3.40, 3.39 (two s, 8H,
CCH2O), 3.20 (s, 9H, NCH3), 2.71 (t, J ) 6.1, 2H, CH2CON),
2.46 (t, J ) 5.6, 6H, CH2CONH), 1.32 (d, J ) 7.0, 9H, CHCH3).
FABMS: 1210.18 M+. UV/vis: λmax 457 nm, ꢀ ) 16 040.
7.09 (AB q, J ) 9.5, ∆ ) 10.2, 4 H, CHCHCN, CHCHCN also
coupled to CCHCN, J ) 2.0), 6.98 (d, J ) 2.0, 2H, CCHCN),
5.01 (m, 3H, CHCONOH), 3.68 (q, J ) 7.1, 8H, CH2Me), 3.61,
3.60 (m, 8H, CH2CH2O), 3.50, 3.45 (two br t, 4H, SO2NCH2
{piperazine}), 3.34, 3.33 (two s, 8H, CCH2O), 3.19 (s, 9H,
NCH3), 3.05, 2.92 (two br t, 4H, CH2NCO {piperazine}), 2.58
(t, J ) 7.0, 2H, NCOCH2CH2O), 2.43 (m, 6H, NHCOCH2CH2O),
1.30 (m, 21H, CH2CH3 + CHCH3). FABMS: 1356.75 (MH-
Na)+. Anal. (C60H88N10O20S2‚5H2O) C, H; N: calcd, 9.84;
found, 7.86.
LRB-p ip -COCH2CH2OCH2C(CH2OCH2CH2CONHCH(i-
Bu )CON(OH)Me)3 (12L). Elution CHCl3/MeOH (9:1), 10%
yield. This compound was prepared by using the procedure
described for compound 10L and not by the general procedure.
1H NMR (CD3OD): δ 8.54 (d, J ) 1.5, 1H, CHCSO3), 8.27 (dd,
J 1 ) 7.8, J 2 ) 1.5, 1H, CHCHSO2), 7.59 (d, J ) 7.8, 1H,
CHCHSO2), 7.09 (AB q, J ) 9.5, ∆ ) 13.5, 4 H, CHCHCN,
CHCHCN is also coupled to CCHCN J ) 2.2), 6.97 (d, J )
2.2, 2H, CCHCN), 5.13 (t, J ) 6.9, 3H, CHCONOH), 3.67, 3.63,
3.61 (m, 16H, CH2Me + CH2CH2O), 3.42 (br t, 4H, SO2NCH2-
{piperazine}), 3.37, 3.36 (two s, 8H, CCH2O), 3.20 (s, 9H,
NCH3), 3.05, 2.92 (two br t, 4H, CH2NCO {piperazine}), 2.60
(t, J ) 6.2, 2H, NCOCH2CH2O), 2.48 (t, J ) 6.0, 6H,
NHCOCH2CH2O), 1.70 (m, 3H, i-Bu CH’s), 1.53 (dd, J ) 6.9,
6H, i-Bu CH2’s), 1.30 (t, J ) 6.9, 12H, CH2CH3), 0.95 (d, J )
6.6, 18H, i-Bu CH3’s). FABMS: 1427.3 (M - O2)+. UV/vis:
λmax 556 nm, ꢀ ) 52 800.
Ba cter ia l Str a in s. The sid- mutants P. putida J M218, P.
fluorescens S680 and P. fluorescens WCS 3742 were kindly
provided by L. C. van Loon, Utrecht, The Netherlands.
In Vivo F lu or escen ce Stu d ies. The bacteria were grown
in LMKB medium42 overnight at 28 °C on a rotary shaker at
180 rpm. Bacterial cultures were centrifuged for 15 min at
2500 rpm, resuspended in fresh half-strength standard suc-
cinate medium (SSM) to a final absorbance of 0.6 (at 620 nm),
and incubated for 60 min at 28 °C. In some experiments NaN3
was added 30 min prior to the addition of the siderophores to
a final concentration of 5 mM. The Fe-siderophore complexes
were added to the bacterial suspensions to a final concentra-
tion of 1 or 5 µM. Aliquots of 1 mL were centrifuged, and the
supernatants were collected. Experiments were performed in
duplicate.
Gr ow th Cu r ve Stu d ies. The 100 mL flasks were washed
with 6 N HCl followed by thorough rinsing with double-
distilled water, prior to autoclaving, and filled with 10 mL of
Chelite-treated SSM. Fe-siderophores were added to a final
concentration of 1 µM. The flasks were inoculated with
bacteria and shaken on a rotary shaker at 180 rpm at 28 °C.
Samples were taken at specific intervals, and turbidity and
absorbance at λ ) 620 nm were recorded. Experiments were
performed in duplicate.
Ack n ow led gm en t. The authors thank Mrs. Rahel
Lazar for her skillful technical assistance. Financial
support from the U.S.-Israel Binational Science Foun-
dation, the Dutch-Israeli Agricultural Research Pro-
gram (DIARP), the Minerva Foundation, and the Israel
Academy of Sciences and Humanities is greatly ac-
knowledged. A. Shanzer is holder of the Singfried and
Irma Ullman Professorial Chair.
LR B-p ip -COCH 2CH 2OCH 2C(CH 2OCH 2CH 2CONH CH 2-
CON(OH)Me)3 (12G). Elution CHCl3/MeOH (17:3), yield
81%. 1H NMR (CD3OD): δ 8.54 (d, J ) 1.5, 1H, CHCSO3),
8.27 (dd, J 1 ) 7.9, J 2 ) 1.5, 1H, CHCHSO2), 7.59 (d, J ) 7.9,
1H, CHCHSO2), 7.11 (AB q, J ) 9.5, ∆ ) 10.2, 4 H, CHCHCN,
CHCHCN also coupled to CCHCN, J ) 2.1), 6.98 (d, J ) 2.1,
2H, CCHCN), 4.15 (s, 6H, CH2CONOH), 3.69 (q, J ) 7.0, 8H,
CH2Me), 3.63 (t, J ) 6.0, 8H, CH2CH2O), 3.47, 3.43 (two br m,
4H, SO2NCH2 {piperazine}), 3.36, 3.35 (two s, 8H, CCH2O),
3.20 (s, 9H, NCH3), 3.15, 2.95 (two br m, 4H, CH2NCO
{piperazine}), 2.53 (t, J ) 6.0, 2H, NCOCH2CH2O), 2.43 (t, J
) 6.0, 6H, NHCOCH2CH2O), 1.32 (t, J ) 7.0, 12H, CH2CH3).
FABMS: 1290.83 M+, 1312.81 MNa+. Anal. (C57H82N10O20S2‚
10H2O) C, H, N.
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