Diphenyl-benzo[1,3]dioxole-4-carboxylic acid pentafluorophenyl ester: A convenient catechol precursor in the synthesis of siderophore vectors suitable for antibiotic Trojan horse strategies

Article abstract of DOI:10.1039/c3ob41990h

Catechols are components of many metal-chelating compounds, including siderophores that are naturally occurring iron(iii) chelators excreted by microorganisms. Catechol derivatives are poorly soluble in organic media and the synthesis of catechol-containing molecules requires the use of protected catechol precursors with improved organic solubility. We therefore developed 2,2-diphenyl-benzo[1,3]dioxole-4-carboxylic acid pentafluorophenyl ester. This activated ester reacts with an amine functionalized scaffold to generate chelators in which the catechol functions are protected in the form of diphenyl-benzodioxole moieties. The catechol can subsequently be deprotected, at the end of the synthesis, with trifluoroacetic acid (TFA). This strategy was applied to the synthesis of two catechol compounds functionalized with a terminal propargyl extension. These two compounds were shown to promote iron uptake in Escherichia coli and Pseudomonas aeruginosa. These two compounds are suitable for use as vectors in antibiotic Trojan horse approaches, as they could be conjugated with azide-functionalized antibiotics using the Huisgen dipolar 1,3-cycloaddition.

Full text of DOI:10.1039/c3ob41990h

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Diphenyl-benzo[1,3]dioxole-4-carboxylic acid
pentafluorophenyl ester: a convenient catechol
precursor in the synthesis of siderophore vectors
suitable for antibiotic Trojan horse strategies†
Cite this: Org. Biomol. Chem., 2014,
12, 749
Etienne Baco, Françoise Hoegy, Isabelle J. Schalk and Gaëtan L. A. Mislin*
Catechols are components of many metal-chelating compounds, including siderophores that are natu-
rally occurring iron(III) chelators excreted by microorganisms. Catechol derivatives are poorly soluble in
organic media and the synthesis of catechol-containing molecules requires the use of protected catechol
precursors with improved organic solubility. We therefore developed 2,2-diphenyl-benzo[1,3]dioxole-
4-carboxylic acid pentafluorophenyl ester. This activated ester reacts with an amine functionalized
scaffold to generate chelators in which the catechol functions are protected in the form of diphenyl-
benzodioxole moieties. The catechol can subsequently be deprotected, at the end of the synthesis, with
trifluoroacetic acid (TFA). This strategy was applied to the synthesis of two catechol compounds
functionalized with a terminal propargyl extension. These two compounds were shown to promote iron
uptake in Escherichia coli and Pseudomonas aeruginosa. These two compounds are suitable for use as
vectors in antibiotic Trojan horse approaches, as they could be conjugated with azide-functionalized anti-
biotics using the Huisgen dipolar 1,3-cycloaddition.
Received 3rd October 2013,
Accepted 12th November 2013
DOI: 10.1039/c3ob41990h
www.rsc.org/obc
combination of bidentate moieties, for example catechol,
hydroxamate or hydroxy-acid, attached to a peptide or a
Introduction
Although iron is one of the major components of the earth’s peptide-like scaffold. Catechol is the most common bidentate
crust, its bioavailability is drastically limited by the low solubi- moiety found in the siderophores described to date. Catechol
lity of iron(III) at physiological pH under aerobic conditions. siderophores, and their synthetic analogues, have been used in
Indeed, the iron(III) concentration in the environment is esti- recent decades for a variety of applications in human health.3,4
mated to be 10−9 M and in human biological fluids it is However, the use of catechol siderophores as vectors for anti-
around 10−18 M.1a Many microorganisms require an iron(III) biotic Trojan horse strategies is probably one of the more strik-
concentration in the micromolar range for an optimal prolifer- ing possibilities.5 The low permeability of membranes is the
ation. Under iron limitation, almost all microorganisms syn- first defense of bacteria against antibiotics.6 Siderophore-
thesize and secrete siderophores so that they can assimilate dependent iron uptake pathways are gates through this barrier
this essential element.1 Siderophores chelate iron(III) in the and therefore possible routes for introducing antibiotic com-
extracellular medium and the resulting ferric–siderophore– pounds into the bacteria.7 Several groups have described the
iron(III) is then translocated by a multiprotein system through synthesis of conjugates between siderophores and antibiotics
the membranes into the cytoplasm.2 Siderophores are a broad with this aim.5,8–22 Many of the siderophore vectors used
family of natural compounds with substantial structural diver- contain, at least, one catechol group to promote iron
sity, but nevertheless optimized during evolution towards a chelation.8–11 We have developed the bis-catechol 1 and tris-
common purpose: the efficient chelation of iron(III).1 Although catechol vector 2 carrying a terminal alkyne to facilitate the
there are some notable exceptions, most siderophores are a subsequent conjugation of azide-functionalized antibiotics
using the Huisgen dipolar 1,3-cycloaddition reaction (Fig. 1).
Catechol containing siderophores are generally synthesized
Team “Transports Membranaires Bactériens”, UMR 7242 Université de Strasbourg-
by a common pathway. A peptide, or peptidomimetic, scaffold
is synthesized first, then catechol precursors are grafted onto
amine functions present on the scaffold. The catechol precur-
sors described to date are all derived from the commercially
CNRS. Boulevard Sébastien Brant, 67400 Illkirch, France. E-mail: mislin@unistra.fr;
Fax: +33 368854829; Tel: +33 368854727
†Electronic supplementary information (ESI) available: 1H-NMR and HRMS
spectra of compounds 1, 2 and 7. 19F-NMR of compound 7. See DOI: 10.1039/
c3ob41990h
available 2,3-hydroxybenzoic acid
3 (Fig. 2). In these
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In compound 7, the catechol moiety is protected in the
form of a diphenyl-dioxole. It has been reported that the cate-
chol function can be released from this structure easily under
mild acidic conditions.26 This new catechol precursor, com-
pound 7, was used in the synthesis of the bis-catechol and tris-
catechol compounds 1 and 2, respectively.
Results and discussion
Organic synthesis
The title compound 7 was prepared from commercially avail-
able 2,3-dihydroxybenzoic acid 3. This acid was first reacted
with thionyl chloride and the resulting acyl chloride was esteri-
fied by a methanolic solution of triethylamine. The expected
ester 8 was isolated with a yield of 99%. The purified methyl-
ester 8 was then heated at 160 °C in a slight excess of dichloro-
diphenylmethane to produce a dioxole 9;26 as this compound
cannot easily be purified, the crude mixture was used as it is
for the next step. The methylester function of the dioxole 9
was converted into the corresponding carboxylate 10, using
potassium trimethylsilanolate (TMSOK) in refluxing THF.27
The expected carboxylate 10 was thus obtained from methyl-
ester 8 with a yield of 59% over two steps. Other methods,
including those described in the literature for similar mole-
cules,26 were tested but were unsuccessful: in general, they led
to partial hydrolysis of the dioxole moiety during the work-up.
The carboxylate function of compound 10 was then reacted
with pentafluorophenol in the presence of various carbodi-
imide coupling reagents. The best results were obtained using
diisopropyl-carbodiimide (DIC),28 which gave a yield of 85%.
Under these conditions the expected catechol precursor 7 was
obtained from the 2,3-dihydroxybenzoic acid 3 by a multigram
four-step process with an overall yield of 50% (Scheme 1).
During the development of the precursor 7, di-(4-methoxy-
phenyl)-dioxole was tested first as the protecting group for
Fig. 1 Structures of bis-catechol and tris-catechol vectors 1 and 2.
Fig. 2 Structures of 2,3-dihydroxybenzoic acid 3 and of catechol pre-
cursors 4 to 7.
precursors, the carboxylate is usually activated in the form of
an acyl-chloride; the two hydroxyl groups are protected.
Protecting groups prevent side reactions, and avoid problems
of solubility in organic media and tedious purification steps.
The last step of the siderophore synthesis is usually the clea-
vage of these protecting groups to restore the chelation ability
of the final compound. Several catechol synthons have been
described in recent decades. In most of these compounds, the
two hydroxyls of the catechol are protected by alkylation. Syn-
thons 4, 5 and 6 have been the most widely used for the syn-
thesis of siderophores and their analogues. The methyl and
isopropyl protecting groups of synthons 5 and 4 can be cleaved
by strong Lewis acids, for example BCl3 or BBr3, although in
some cases the yields are low.8,23,24 Also, the use of such acids
may affect many other organic functions. The benzyl protect-
ing groups of precursor 6 can be released by catalytic reduction
in the presence of hydrogen.9,10,24,25 However, this cleavage
strategy is not compatible with many chemical functions, and
especially the alkyne moiety present on the expected chelators
1 and 2. To avoid these problems, we synthesised diphenyl-
benzo[1,3]dioxole-4-carboxylic acid pentafluorophenyl ester 7,
a new catechol precursor; this precursor can be used for the
synthesis of chelating molecules that are sensitive to both
strong Lewis acids and hydrogenolysis (Fig. 2).
Scheme 1 Synthesis of diphenyl-benzo[1,3]dioxole-4-carboxylic acid
pentafluorophenyl ester 7. i. A. SOCl2 reflux. B. MeOH, NEt3, 0 °C to
25 °C. ii. Cl2CPh2, neat, 160 °C. iii. TMSOK, THF, 70 °C. iv. DIC, C6F5OH,
acetonitrile–pyridine, 25 °C.
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the catechol moiety. However, this protecting group was presence of HBTU. The reaction led to the expected amide 16
too unstable and undesired cleavage occurred under many isolated in 86% yield. Compound 16 was then treated with a
synthetic conditions. The diphenyl-dioxole proved to be a solution of diethylamine in methanol to cleave the Fmoc
more suitable protecting group for catechol. The activation group. The resulting free amine was coupled to another Boc-
of the carboxylate in the form of an N-hydroxysuccinimide Dap-Fmoc unit using the conditions described above. The
ester was also investigated. The yield of the activation expected molecule 17 was thereby isolated over two combined
reactions was identical for both N-hydroxysuccinimide steps from compound 16 at a yield of 63%. Further treatment
and pentafluorophenol, but pentafluorophenol was found to of compound 17 with diethylamine, followed by coupling to
be more easily removed from the crude mixture after Boc-N-glycine in the presence of HBTU, led to the expected
the reaction between the activated ester and the siderophore tricarbamate scaffold 18 at a yield of 44%. Treatment of
scaffold.
compound 18 with diluted TFA led to the corresponding
The catechol synthon 7 was then used in the synthesis of triamine; this triamine was then reacted with the catechol
the two catechol compounds 1 and 2. Compound 1 is an precursor 7. The protected tris-catechol compound 19 was
analogue of azotochelin, one of the three catechol sidero- isolated, and the yield over two steps from the tricarbamate 18
phores of Azotobacter vinelandii, a Gram-negative bacterium was 41%. The protecting groups were cleaved by treatment
and a member of the Pseudomonadaceae family.29–31 The syn- with diluted TFA in the presence of triisopropylsilane (TIS),
thesis of this compound starts with the commercially available leading to the expected free tris-catechol molecule 2. The com-
lysine methylester dihydrochloride 11: this lysine derivative pound 2 was easily purified by partition in a water–diethyl-
was treated with the catechol precursor 7 in the presence of oxide mixture and was thereby isolated with a quantitative
DIPEA leading to the expected diamide 12 which was isolated yield (Scheme 3).
in quantitative yield. The methyl ester function of compound
Catechol compounds 1 and 2 may be suitable for use
12 was then quantitatively saponified using the LiOH–H2O2 to vectorize antibiotics as part of a Trojan horse strategy.
system in THF.32 The corresponding free acid 13 was To evaluate the potential of these two compounds in this
coupled to propargylamine in the presence of HBTU, leading type of application, iron uptake experiments were used to
to the protected vector 14, isolated with a yield of 87%. assess whether they are recognized as siderophores by target
The diphenyl-dioxole groups protecting the catechol functions bacteria.
of molecule 14 were cleaved using diluted TFA in the
presence of triisopropylsilane (TIS). Using this procedure,
the bis-catechol compound 1 was isolated in 95% yield
(Scheme 2).
The second target chelator, the tris-catechol compound 2,
was synthesized using commercially available Boc-3-(Fmoc-
amino)-L-alanine (Boc-Dap-Fmoc) 15 as the starting material.
Boc-Dap-Fmoc 15 was first coupled to propargylamine in the
Scheme 3 Synthesis of tris-catechol vector 2. i. Propargylamine, HBTU,
DIPEA, CH2Cl2, 25 °C. ii. Diethylamine, MeOH, 0 °C to 25 °C. iii. 15,
Scheme 2 Synthesis of bis-catechol vector 1. i. DIPEA, CH2Cl2, 20 °C.
ii. LiOH, H2O2, THF, 24 °C. iii. Propargylamine, HBTU, DIPEA, CH2Cl2,
22 °C. iv. TFA–CH2Cl2 10%, TIS, 24 °C.
HBTU, DIPEA, CH2Cl2, 24 °C. iv. Boc-N-Glycine, HBTU, DIPEA, CH2Cl2,
24 °C. v. TFA–CH2Cl2 25%, 24 °C. vi. 7, DIPEA, THF, 24 °C. vii. TFA–
CH2Cl2 10%, TIS, 24 °C.
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Iron uptake experiments
uptake assays, the bacteria were incubated in the presence of
100 nM enterobactin-55Fe (Fig. 3A and 3B), 1-55Fe (Fig. 3C and
The ability of catechol compounds 1 and 2 to promote 55Fe(III) 3D) or 2-55Fe (Fig. 3E and 3F) and the radioactivity incorpor-
uptake by E. coli and P. aeruginosa was tested. To simplify ated into the bacteria monitored. Enterobactin was used as a
these analyses, strains not producing endogenous sidero- reference siderophore. The experiments were repeated with
phores were used for the uptake assays: E. coli strain BW25113 cells pretreated with the protonophore CCCP: this compound
entB::KanR,33 defective for the biosynthesis of enterobactin, inhibits the proton motive force (PMF) across the bacterial cell
and P. aeruginosa strain PAD07, a pyoverdine and pyochelin- membrane and therefore inhibits TonB-dependent iron uptake
deficient strain.34 Llamas et al. showed that the expression of (Fig. 3).37
some TonB-dependent siderophore transporters in P. aerugi-
Enterobactin-55Fe, 1-55Fe and 2-55Fe were taken up by both
nosa is upregulated by the absence of iron and the presence of P. aeruginosa and E. coli (Fig. 3). Moreover, 55Fe incorporation
the corresponding siderophore in the culture medium.35,36 by P. aeruginosa was more efficient with catechol compound 1
Therefore, both strains were grown in the presence of the and that by E. coli with catechol compound 2. The absence of
tested siderophore (catechol 1 or 2). Afterwards for the iron 55Fe accumulation in the presence of CCCP indicates that 1
Fig. 3 Enterobactin-55Fe, 1-55Fe and 2-55Fe uptake by PAD07 (P. aeruginosa) and BW25113entB::KanR (E. coli). PAD07 (A, C and E) and
BW25113entB::KanR (B, D and F) cells at an OD600 of 1 were incubated for 15 min in 50 mM Tris-HCl (pH 8.0) buffer. Transport assays were then
started by adding enterobactin-55Fe (A and B), 1-55Fe (C and D) or 2-55Fe (E and F). Aliquots (100 μL) of the suspension were removed at various
△
times and filtered, and the radioactivity retained was counted. As controls, similar experiments were repeated in the presence of 200 μM CCCP:
experiment in the presence of the tested siderophores and no CCCP; experiment in the presence of the tested siderophores and CCCP. All experi-
ments were repeated three times, with comparable results.
,
▲
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and 2 are transported through one or more TonB-dependent solution was diluted with non-radioactive FeCl3 to 9.4 Ci g−1
.
transporters, which are PMF-dependent (Fig. 3). 55Fe uptake Tetracycline hydrochloride was purchased from Euromedex.
occurred for compounds 1 and 2 only when cells were grown The protonophore CCCP (carbonyl cyanide m-chlorophenyl-
in the presence of the tested iron chelator. When the cells hydrazone), kanamycin, streptomycin sulphate and entero-
were grown in the absence of the tested chelator, no uptake bactin were purchased from Sigma-Aldrich.
was observed (data not shown) indicating that like many
Chromatography
siderophores used by P. aeruginosa,35,36 compounds 1 and 2
induce the expression of the TonB-dependent transporters All reactions were monitored by thin-layer chromatography
involved in their uptake.
(TLC) using Merck aluminium sheets precoated with silica gel
60F254 (0.25 mm). Column chromatography purifications were
performed using Merck silica gel 60 (63–200 µm). In some pro-
tocols, silica should be demetallated prior to use. For this
purpose commercial silica was suspended and stirred 10 h in
Conclusions
We report the synthesis of diphenyl-benzo[1,3]dioxole-4-car- 1 N HCl. Silica gel was filtered and washed several times with
boxylic acid pentafluorophenyl ester 7, a new catechol precur- bidistillated water, dried under reduced pressure and then in
sor suitable for the synthesis of siderophores and other an oven (110 °C) for at least 72 h.40
chelators containing the catechol moiety. Its synthetic versati-
Instrumentation
lity was illustrated by the efficient synthesis of the bis-catechol
and tris-catechol compounds 14 and 19 in which the catechol NMR spectra were recorded on Bruker Avance 400 (1H:
moiety is protected in the form of a 2,2-diphenyl-1,3-dioxole. 400 MHz, 13C: 100 MHz), using the residual non-deuterated
The acetylenic function of these two molecules was not chemi- solvent as a reference. The chemical shifts (δ) and coupling
cally altered during the deprotection of catechol function constants (J) are expressed in ppm and hertz (Hz) respectively.
under acidic conditions. The deprotected bis- and tris-catechol Multiplicities were indicated as s (singlet), d (doublet), t
compounds 1 and 2 enabled efficient iron(III) uptake by both (triplet), q (quadruplet) and m (multiplet). A broad signal is
E. coli and P. aeruginosa, two Gram-negative bacterial species. mentioned with br preceeding the multiplicity. Mass spectra
The bis-catechol 1 proved to be more efficient than the tris- were recorded in the Service Commun d’Analyse (SCA) de la
catechol 2 for 55Fe incorporation into P. aeruginosa whereas Faculté de Pharmacie de l’Université de Strasbourg and were
tris-catechol 2 was more efficient in E. coli. However, the iron measured after calibration in ES-TOF experiments on a Bruker
acquisition systems involved in the uptake of ferric complexes Daltonic MicroTOF mass spectrometer. Countings of radio-
of 1 and 2 have not yet been identified. Nevertheless, these active samples were performed on a Packard TriCarb 2100TR.
results pave the way for the use of catechol compounds 1 and
Methyl 2,3-dihydroxybenzoate 8. A solution of 2,3-dihydroxy-
2 as vectors in antibiotic Trojan horse strategies. For this benzoic acid 3 (5.00 g, 32.44 mmol) in thionyl chloride
purpose, the terminal alkyne function of protected catechol (15 mL) was heated at 78 °C (reflux). When the gases emission
compounds 14 and 19 will be linked to azide-functionalized stopped, excess thionyl chloride was eliminated under reduced
antibiotics by regioselective copper(I)-catalyzed 1,3-dipolar pressure. At 0 °C, the residue was very slowly dissolved in a
cycloaddition.38,39 This “click” reaction will be followed by mixture of methanol (17 mL) and triethylamine (6 mL). The
treatment with diluted TFA to release the catechol groups. resulting solution was stirred for 16 hours at 25 °C. The reac-
Many antibiotics are resistant to mild conditions of this type, tion mixture was adsorbed on silica gel before being purified
such that their structures should be unaffected. This work is by chromatography on a silica gel column (cyclohexane–
underway, with the aim of developing novel tools for antibiotic CH2Cl2 1 : 1 then pure CH2Cl2). The expected methylester 8
therapy against pathogenic E. coli and P. aeruginosa strains.
was isolated in the form of white crystals (5.40 g, 32.10 mmol,
yield: 99%). Rf 0.41 (cyclohexane–CH2Cl2 1 : 1). 1H-NMR
(400 MHz, CDCl3, 25 °C): δ 7.34 (d, J = 8.1 Hz, 1H); 7.09 (d, J =
7.9 Hz, 1H); 6.77 (t, J = 8.0 Hz, 1H); 3.93 (s, 3H). 13C-NMR
(100 MHz, acetone d6) δ 171.8, 150.9, 147.0, 121.6, 121.0,
120.0, 113.5, 53.0. MS (ESI) m/z: 169.0 [M + H+].
Experimental section
General
All reactions were carried out under argon purchased from Air
2,2-Diphenylbenzo[d][1,3]dioxole-4-carboxylic acid 10. 2,3-
Liquide. Solvents used for reaction were of analytical grade Dihydroxybenzoic acid methyl ester 8 (5.40 g, 32.10 mmol) and
purity (>99.9%). When necessary, and specified in the proto- dichloro-diphenylmethane (7.4 mL, 9.10 g, 38.50 mmol) were
cols, solvents were purchased extra-dry from Aldrich, Acros heated neat at 160 °C. When the gases emission stopped, the
and Alfa-Aesar companies. Amines were distilled and stored on viscous crude mixture was cooled down to room temperature
KOH before use. All other chemicals were obtained from com- under argon before being dissolved in ethyl acetate (200 mL).
mercial suppliers (Aldrich, Acros or Alfa-Aesar) and were used The organic layer was washed three times with an aqueous
as received, unless otherwise stated. 55FeCl3 was obtained from solution saturated in NaHCO3 and finally with brine. After fil-
Perkin Elmer Life and Analytical Sciences (Billerica, MA, USA) tration and solvent removal under reduced pressure the result-
with a specific activity of 93.76 Ci g−1. This radioactive 55Fe ing pale yellow powder (compound 9) was dissolved in THF
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(150 mL) and potassium trimethylsilanolate tech. (90%) 112.2, 112.0, 52.7, 39.8, 32.6, 29.6, 22.8. MS (ESI) m/z 761.3
(21.00 g, 160.60 mmol) was added. The solution was then [M + H+], 783.3 [M + Na+]. HRMS (ESI): C47H40N2O8: calcd
heated to reflux for 2 hours. THF was then removed under 760.2785, found 760.2792.
reduced pressure and the resulting residue was diluted in an
(R)-2,6-Bis(2,2-diphenylbenzo[d][1,3]dioxole-4-carboxamido)-
aqueous solution saturated with citric acid. After several extrac- hexanoic acid 13. To a solution of methyl ester 12 (785 mg,
tions with CHCl3, the organic layers were collected, washed 1.03 mmol) in THF (5 mL) was added, at 0 °C, a solution of
with brine, dried over Na2SO4, and filtered before being LiOOH (5 mL; 2.7 M of LiOH solution in 30% H2O2). The
adsorbed on silica gel. A purification by chromatography on a mixture was stirred at 24 °C overnight for 16 hours. After com-
silica gel column (cyclohexane–CH2Cl2 1 : 1 then pure CH2Cl2 pletion of the reaction, an aqueous solution saturated in citric
and finally CH2Cl2–EtOH 9 : 1) afforded the acid 10 (6.00 g, acid was added and THF was evaporated under reduced
19.00 mmol, yield: 59% over two steps) isolated as a pale pressure. The aqueous residue was extracted three times with
yellow powder. Rf 0.40 (CH2Cl2–EtOH 96 : 4). 1H-NMR chloroform. Organic layers were collected, dried over Na2SO4,
(400 MHz, CDCl3): δ 7.63–7.60 (m, 4H), 7.47 (d, J = 8.2 Hz, 1H), filtered and the solvent was removed under reduced pressure.
7.40–7.35 (m, 6H), 7.06 (d, J = 7.7 Hz, 1H), 6.87 (dd, J = 7.7, The expected acid 13 (770 mg, 1.03 mmol, yield: 100%) was
8.2 Hz, 1H). 13C-NMR (100 MHz, CDCl3): 169.5, 149.0, 148.6, isolated as a white foamy solid. Rf 0.50 (CH2Cl2–EtOH 92 : 8).
139.8, 129.6, 128.6, 126.6, 123.5, 121.6, 118.5, 113.4, 112.3. MS 1H-NMR (400 MHz, CDCl3): δ 8.03 (d, J = 7.3 Hz, 1H), 7.62 (d,
(ESI) m/z: 319.1 [M + H+], 341.1 [M + Na+]. HRMS (ESI): J = 8.0 Hz, 2H), 7.57–7.48 (m, 8H), 7.35–7.20 (m, 12H), 7.19 (t,
C20H14O4: calcd 318.0892, found 318.0888.
J = 5.8 Hz, 1H), 7.02 (dd, J = 7.5, 13.9 Hz, 2H), 6.9 (td, J = 6.6,
Pentafluorophenyl 2,2-diphenylbenzo[d][1,3]dioxole-4-car- 8.0 Hz, 2H), 4.86 (q, J = 6.5 Hz, 1H), 3.50–3.34 (m, 2H),
boxylate 7. To a solution of the dioxole 10 (1.00 g, 3.14 mmol) 2.11–2.08 (m, 1H), 1.96–1.94 (m, 2H), 1.68–1.63 (m, 2H),
in a mixture of acetonitrile (9 mL) and pyridine (1 mL) were 1.54–1.50 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 174.6, 163.8,
successively added pentafluorophenol (726 mg, 3.95 mmol) 147.6, 147.4, 145.0, 139.7, 139.6, 139.2, 129.9, 129.7, 129.6,
and diisopropylcarbodiimide (614 µL, 3.95 mmol). The 128.7, 126.6, 126.2, 122.7, 122.4, 122.3, 115.8, 115.4, 112.3,
mixture was stirred at 25 °C under argon for 3 hours. The 112.1, 52.8, 40.0, 32.2, 29.6, 22.7. MS (ESI) m/z 747.2 [M + H+],
crude mixture was adsorbed on silica gel before being purified 770.0 [M + Na+].
by chromatography on a silica gel column (cyclohexane–
N,N′-(6-Oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)bis(2,2-
CH2Cl2 1 : 1). The expected catechol synthon 7 (1.29 g, diphenylbenzo[d][1,3]dioxole-4)carboxamide) 14. To a solu-
2.67 mmol, yield: 85%) was isolated as a white solid. Rf 0.50 tion of acid 13 (200 mg, 0.27 mmol) in anhydrous CH2Cl2
(cyclohexane–CH2Cl2 1 : 1).1H NMR (400 MHz, CDCl3):
δ
(5 mL) were added successively HBTU (112 mg, 0.29 mmol),
7.71–7.67 (m, 4H), 7.59 (d, J = 8.3 Hz, 1H), 7.46–7.39 (m, 6H), DIPEA (133 µL, 0.80 mmol) and propargylamine (43 µL,
7.18 (d, J = 7.8 Hz, 1H), 6.96 (dd, J = 7.7, 8.3 Hz, 1H). 13C NMR 0.67 mmol). The reaction was stirred under argon at 22 °C for
(100 MHz, CDCl3): δ 160.5, 149.6, 148.9, 139.7, 136.9, 129.6, 16 hours. The mixture was diluted with CH2Cl2 and succes-
128.6, 126.5, 123.4, 121.9, 119.0, 114.2, 109.8. 19F NMR sively washed with an aqueous solution saturated in NaHCO3,
(100 MHz, CDCl3): δ −152.0 (d, 2F) −158.1 (t, 1F), −162.4 (m, with water and finally with a saturated aqueous citric acid solu-
2F). MS (ESI): m/z 485.0 [M + H+]. HRMS (ESI): C26H13F5O4: tion. The organic layer was dried over Na2SO4, filtrated and
calcd 484.0734, found 484.0735.
adsorbed on silica gel. A purification by chromatography on a
(R)-Methyl N2,N6-bis(2,2-diphenylbenzo[d][1,3]dioxole-4-car- silica gel column (CH2Cl2 then CH2Cl2–EtOH 9 : 1) afforded
bonyl)lysinate 12. Lysine methyl ester di-hydrochloride 11 the propargylamide 14 (183 mg, 0.23 mmol, yield: 87%) iso-
(239 mg, 1.03 mmol) was dissolved in a mixture of DIPEA lated as a white solid. Rf 0.33 (cyclohexane–AcOEt 1 : 1)
(2.05 mL, 12.40 mmol) and anhydrous CH2Cl2 (10 mL). To this 1H-NMR (400 MHz, acetone d6): δ 8.33 (d, J = 7.3 Hz, 1H), 8.10
solution was added dropwise a solution of pentafluorophenyl- (t, J = 5.2 Hz, 1H) 7.89 (d, J = 8.7 Hz, 2H), 7.82 (d, J = 8.0 Hz,
ester 7 (1000 mg, 2.06 mmol) in anhydrous CH2Cl2 (10 mL). 2H), 7.75–7.70 (m, 4H), 7.67 (t, J = 6.7 Hz, 1H), 7.58–7.43 (m,
The mixture was stirred overnight for 16 hours at 24 °C before 14H), 7.25 (d, J = 7.7 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.05 (dt,
being adsorbed on silica gel and further purified by chromato- J = 6.5 Hz, 7.7 Hz, 2H), 4.90 (dt, J = 5.1, 7.1 Hz, 1H), 4.18 (br s,
graphy on a silica gel column (CH2Cl2 then CH2Cl2–EtOH 2H), 3.54 (q, J = 7.4 Hz, 2H), 2.75 (s, 1H), 2.20–2.11 (m, 2H),
9 : 1). The diamide 12 (785 mg, 1.03 mmol, yield: 100%) was 2.06–1.96 (m, 2H), 1.82–1.74 (m, 2H), 1.68–1.61 (m, 2H).
isolated as a white foamy solid. Rf 0.35 (CH2Cl2–EtOH 96 : 4). 13C-NMR (100 MHz, acetone d6): δ 172.2, 171.0, 163.6, 163.0,
1H-NMR (400 MHz, CDCl3): δ 8.01 (d, J = 7.4 Hz, 1H), 7.66 (dd, 148.3, 145.8, 145.6, 140.9, 140.8, 140.5, 130.4, 130.3, 129.5,
J = 2.0, 7.2 Hz, 2H), 7.6 (dd, J = 2.3, 7.5 Hz, 2H), 7.56–7.50 (m, 129.4, 127.1, 126.9, 126.8, 123.2, 123.0, 122.9, 118.9, 118.8,
6H), 7.43–7.33 (m, 12H), 7.19 (t, J = 5.8 Hz, 1H), 7.02 (dd, J = 117.7, 117.2, 112.6, 112.2, 81.1, 72.4, 53.9, 40.3, 34.1, 30.4,
7.9, 10.7 Hz, 2H), 6.93 (t, J = 8.0 Hz, 2H), 4.88 (dt, J = 5.5, 29.3, 23.4. MS (ESI) m/z 784.2 [M + H+], 807.2 [M + Na+]. HRMS
6.9 Hz, 1H), 3.75 (s, 3H), 3.53–3.38 (m, 2H), 2.11–2.02 (m, 1H), (ESI): C49H41N3O7: calcd 783.2945, found 783.2960.
1.96–1.87 (m, 1H), 1.71–1.67 (m, 2H), 1.52–1.44 (m, 2H).
N,N′-(6-Oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)bis(2,3-
13C-NMR (100 MHz, CDCl3): δ 172.8, 164.1, 163.4, 147.5, 147.4, dihydroxybenzamide) 1. Compound 14 (105.5 mg, 0.14 mmol)
145.3, 144.9, 139.7, 139.6, 139.2, 129.9, 129.7, 129.6, 128.7, was dissolved in a mixture of TFA (400 µL, 1.53 mmol) in
126.5, 126.2, 122.6, 122.4, 122.3, 118.6, 118.4, 115.9, 115.4, CH2Cl2 (3.6 mL). After 16 hours at 24 °C, the mixture was
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evaporated under reduce pressure and diethyl-oxide was as a white solid. Rf 0.70 (CH2Cl2–EtOH 9 : 1). 1H-NMR
added. The gum formed was triturated in diethyl-oxide. The (400 MHz, DMSO d6): δ 8.27 (br s, 1H), 7.93 (br s, 1H) 7.89 (d,
solvent was removed by filtration and the resulting solid was J = 7.6 Hz, 2H), 7.68 (br s, 2H), 7.41 (dd, J = 6.7, 8.0 Hz, 2H),
finally dissolved in acetonitrile before being adsorbed on 7.32 (dd, J = 6.7, 7.7 Hz, 2H), 7.23 (br s, 1H), 6.82 (d, J = 7.4 Hz,
demetellated silica gel and purified by chromatography on a 1H), 6.67 (d, J = 7.9 Hz, 1H), 4.28 (br s, 2H), 4.22–4.19 (m, 1H),
demetalled silica gel column (CH2Cl2, then CH2Cl2–EtOH 4.00 (br s, 1H), 3.83 (br s, 2H), 3.08 (br s, 1H) 1.36 (s, 18H),
9 : 1). The expected bis-catechol compound
1
(58 mg, 1.25 (br s, 2H). 13C-NMR (100 MHz, DMSO d6): δ 170.5, 170.0,
0.13 mmol, yield: 95%) was isolated as a brownish solid. Rf 156.3, 155.1, 143.8, 140.7, 127.6, 127.1, 125.1, 120.1, 80.9, 78.4,
1
0.50 (CH2Cl2–EtOH 96 : 4). H-NMR (400 MHz, CD3CN): δ 7.49 73.0, 65.6, 54.9, 54.2, 53.6, 46.6, 42.2, 40.5, 28.0. MS (ESI) m/z
(d, J = 7.5 Hz, 1H), 7.39–7.35 (m, 1H), 7.19 (dd, J = 1.7, 8.2 Hz, 672.2 [M + Na+]. HRMS (ESI): C34H43N5O8: calcd 649.3112,
1H), 7.10 (t, J = 5.9 Hz, 1H), 7.06 (dd, J = 1.6, 8.2 Hz, 1H), 6.96 found 649.3111.
(ddd, J = 1.6, 7.9, 12.2 Hz, 2H), 6.72 (dt, J = 8.0, 9.6 Hz, 2H),
{2-[2-tert-Butoxycarbonylamino-3-(2-tert-butoxycarbonyl
4.51 4.45 (m, 1H), 3.92 (dd, J = 2.6, 5.9 Hz, 2H), 3.36 (dq, J = amino-acetylamino)-propionylamino]-1-prop-2-ynylcarbamoyl-
1.9, 7.1 Hz, 2H), 1.97–1.89 (m, 1H), 1.86–1.76 (m, 1H), ethyl}-carbamic acid tert-butyl ester 18. To a solution of com-
1.69–1.56 (m, 2H), 1.51–1.39 (m, 2H), 1.26 (s, 1H). 13C-NMR pound 17 (500 mg, 0.77 mmol) in MeOH (5 mL) at 0 °C was
(100 MHz, CD3CN): δ 171.9, 171.0, 170.9, 150.1, 150.0, 146.5 added diethylamine (793 µL, 7.70 mmol). The resulting solu-
(2), 119.3, 119.1, 118.9, 118.8, 118.1, 117.5, 115.2, 114.9, 80.7, tion was stirred at 24 °C for 3 hours. The mixture was evapor-
71.5, 54.1, 39.4, 31.7, 29.1, 28.9, 23.4. MS (ESI) m/z 456.0 [M + ated under reduced pressure and the crude amine (ca. 500 mg)
H+]. HRMS (ESI): C23H25N3O7: calcd 455.1693, found 455.1701. was used as it is, without any further purification, in the fol-
(9H-Fluoren-9-yl)methyl tert-butyl (3-oxo-3-(prop-2-yn-1-yl- lowing synthetic step. The crude amine was dissolved in a
amino)propane-1,2-diyl)dicarbamate 16. Boc-DAP-Fmoc-OH mixture of anhydrous CH2Cl2 (8 mL) and DIPEA (580 µL,
15 (1000 mg, 2.35 mmol) was dissolved in anhydrous CH2Cl2 3.51 mmol). To this solution was added, at 24 °C, a solution of
(15 mL). HBTU (978 mg, 2.58 mmol), DIPEA (1.16 mL, N-Boc-glycine (205 mg, 1.17 mmol) and HBTU (488 mg,
7.03 mmol) and propargylamine (0.15 mL, 2.35 mmol) were 1.29 mmol) in CH2Cl2 (5 mL). After 16 hours at 24 °C, the
added successively to this solution at 24 °C. The reaction mixture was adsorbed on silica gel and a purification of the
mixture was then stirred at 24 °C for 16 hours under argon. crude material by a chromatography on a silica gel column
The solution was adsorbed on silica gel and a chromato- afforded the expected tricarbamate 18 (200 mg, 0.34 mmol,
graphic purification on a silica gel column (CH2Cl2, then overall yield on two combined steps: 44%) isolated as a pale
CH2Cl2–EtOH 96 : 4) afforded propargylamide 16 (940 mg, yellow powder. Rf 0.26 (CH2Cl2–EtOH 96 : 4). 1H NMR
2.03 mmol, yield: 86%) isolated as a white powder. Rf 0.50 (400 MHz, MeOD): δ 4.19 (t, J = 5.3 Hz, 1H), 4.11 (t, J = 4.4 Hz,
(CH2Cl2–EtOH 96 : 4). 1H-NMR (400 MHz, CDCl3): δ 7.45 (d, J = 1H), 3.98–3.96 (m, 2H), 3.72 (d, J = 3.0 Hz, 2H), 3.68–3.65 (m,
7.6 Hz, 2H), 7.3 (d, J = 7.4 Hz, 2H), 7.08 (t, J = 7.4 Hz, 2H), 6.99 1H), 3.52 (d, J = 4.4 Hz, 2H), 3.38–3.34 (m, 1H), 2.6 (t, J =
(t, J = 7.4 Hz, 2H), 4.06 (d, J = 6.9 Hz, 2H), 3.89 (t, J = 7.0 Hz, 2.4 Hz, 1H), 1.46 (s, 9H), 1.44 (s, 18H). 13C NMR (100 MHz,
2H), 3.66 (d, J = 2.5 Hz, 2H), 3.17 (dd, J = 5.0, 13.9 Hz, 1H), MeOD): δ 173.7 (2), 173.4 (2), 172.5 (2), 158.6, 157.6, 81.0, 80.9
2.04 (t, J = 2.5 Hz, 1H), 1.11 (s, 9H). 13C-NMR (100 MHz, (2), 72.5, 57.0, 55.7, 44.8, 42.3, 41.9, 29.7, 28.8, 28.7. MS (ESI)
CDCl3): δ 143.5, 141.0 (2), 127.3, 126.7, 124.6, 119.6, 71.1, 66.7, m/z 607.2 [M + Na+]. HRMS (ESI): C26H44N6O9: calcd: 584.3170,
54.5, 46.8, 42.4, 28.4, 27.5. MS (ESI) m/z 486.2 [M + Na+]. found 584.3175.
HRMS (ESI): C26H29N3O5: calcd 463.2107, found 463.2105.
N-(1-(2,2-Diphenylbenzo[d][1,3]dioxol-4-yl)-11-(2,2-diphenyl
[1-(2-tert-Butoxycarbonylamino-2-prop-2-ynylcarbamoyl-ethyl benzo[d][1,3]dioxole-4-carboxamido)-1,4,8,12-tetraoxo-2,5,9,13-
carbamoyl)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethyl]- tetraazahexadec-15-yn-7-yl)-2,2-diphenyl benzo[d][1,3]dioxole-
carbamic acid tert-butyl ester 17. The propargylamide 16 4-carboxamide 19. The tricarbamate 18 (100 mg, 0.171 mmol)
(900 mg, 1.94 mmol) was dissolved in MeOH (15 mL). To this was dissolved in a mixture of TFA (250 µL, 3.42 mmol) in
solution was added, at
0 °C, diethylamine (1.60 mL, CH2Cl2 (750 µL). This solution was stirred for 16 hours at
15.62 mmol). The resulting solution was stirred at 24 °C for 24 °C before being evaporated under reduced pressure. The
3 hours and then the mixture was evaporated under reduced resulting crude triamine was used without any further purifi-
pressure. Crude amine (ca. 470 mg) was used for the next syn- cation for the next synthetic step. The crude triamine was dis-
thetic step without any further purification. The crude product solved in a mixture of DIPEA (360 µL, 2.18 mmol) and
was dissolved in a mixture of DIPEA (966 µL, 5.84 mmol) and anhydrous THF (3 mL). To this solution, at 24 °C, under
anhydrous CH2Cl2 (15 mL). To this solution was added, at argon, was added a solution of the pentafluorophenyl-ester 7
24 °C, a solution of HBTU (873 mg, 2.14 mmol) and Boc- (249 mg, 0.51 mmol) in anhydrous THF (5 mL). The mixture
DAP-Fmoc-OH 15 (831 mg, 1.95 mmol) in CH2Cl2 (20 mL). The was stirred for 3 hours at 24 °C. The reaction mixture was
mixture was finally stirred for 72 hours at 24 °C under argon. adsorbed on silica gel and purified by chromatography on a
The mixture was adsorbed on silica gel and a purification by silica gel column (CH2Cl2, then CH2Cl2–EtOH 92 : 8). The
chromatography on a silica gel column (CH2Cl2 then CH2Cl2– expected triamide 19 (84 mg, 0.07 mmol, overall yield over two
EtOH 9 : 1) led to the expected tricarbamate 17 (793 mg, steps: 41%) was isolated as a white powder. Rf 0.45 (CH2Cl2–
1
1.22 mmol, overall yield on two combined steps: 63%) isolated EtOH 96 : 4). H-NMR (400 MHz, CDCl3): δ 8.58 (d, J = 6.8 Hz,
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1H), 8.37 (d, J = 6.5 Hz, 1H), 8.21 (t, J = 5.4 Hz, 1H), 8.04 (t, Iron uptake experiments
1H), 7.93 (t, J = 5.2 Hz, 1H), 7.88 (t, 1H), 7.73 (dt, J = 1.5,
Iron uptake assays were carried out as previously reported for
the FpvA/pyoverdine system.42 After an overnight culture
(20 h), bacteria were prepared in 50 mM Tris-HCl (pH 8.0) at
OD600 nm of 1, and incubated at 37 °C. Transport assays were
started by adding 100 nM of enterobactin-55Fe(III), 1-55Fe(III) or
2-55Fe(III). Enterobactin-55Fe(III) and the two catecholate-55Fe(III)
complexes were prepared at concentrations of 10 µM of 55Fe(III)
with a siderophore : iron (mol : mol) ratio of 20 : 1. The solu-
tions were prepared using a 10 mM solution of enterobactin, 1
or 2 (in DMSO). To 2 µL of this solution were added 4 µL of a
solution of 55FeCl3 (250 µM, 9.4 Ci g−1 in HCl 0.5 N), obtained
by dilution of the stock solution, plus 96 μL of 50 mM Tris-HCl
pH 8.0. To separate siderophore-55Fe transported into P. aerugi-
nosa cells from unbound siderophore-55Fe, aliquots (100 µL) of
the suspensions were removed at different times, filtered
(0.45 μm, cellulose nitrate membrane filters, Whatman), and
washed with 5 mL 50 mM Tris-HCl (pH 8.0) and the radio-
activity counted. As controls, the experiments were repeated
with cells incubated at 0 °C in the presence of 200 μM CCCP.
6.7 Hz, 2H), 7.68–7.65 (m, 6H), 7.55 (dt, J = 1.8, 8.0 Hz, 4H),
7.44 (d, J = 8.2 Hz, 1H), 7.40–7.25 (m, 20H), 6.97 (d, J = 7.8 Hz,
2H), 6.9 (d, J = 7.8 Hz, 1H), 6.79 (t, J = 8.0 Hz, 1H), 6.77–6.72
(m, 2H), 4.84 (dt, J = 5.2, 6.7 Hz, 1H), 4.73 (dt, J = 6.8, 5.8 Hz,
1H), 4.17 (d, J = 5.5 Hz, 2H), 4.08–4.07 (m, 2H), 3.96–3.84 (m,
2H), 3.71–3.68 (m, 2H), 2.01 (t, J = 2.5 Hz, 1H). 13C-NMR
(100 MHz, CDCl3, 25 °C): δ 170.6, 170.1, 169.6, 164.4, 164.0,
163.5, 147.4, 145.3, 139.6, 139.1, 129.5, 128.5, 126.4, 126.1,
122.2, 122.0, 118.4, 115.1, 111.9, 79.3, 71.6, 62.6, 58.4, 54.3,
53.7, 53.4, 45.3, 44.0, 41.7, 29.3. MS (ESI) m/z 1185.2 [M + H+].
HRMS (ESI): C71H56N6O12: calcd 1184.3956, found 1184.3954.
N-(7-(2,3-Dihydroxybenzamido)-1-(2,3-dihydroxyphenyl)-
1,4,8,12-tetraoxo-2,5,9,13-tetraazahexadec-15-yn-11-yl)-2,3-di-
hydroxy benzamide 2. To a solution of protected tris-catechol
compound 19 (40 mg, 0.04 mmol) in anhydrous CH2Cl2
(900 µL) was added triisopropylsilane (21 µL, 0.10 mmol) and
TFA (100 µL). The mixture was stirred for 16 hours at 24 °C
before being evaporated under reduced pressure. The resulting
residue was suspended in diethyl-oxide leading to the for-
mation of a gummy solid. The diethyl-oxide was removed and
this solid was dissolved in water. The lyophilization afforded a
slight grey solid which needed to be rinsed several times with Acknowledgements
diethyl-oxide. The expected tris-catechol compound 2 (23 mg,
We thank the Agence National pour la Recherche (ANR-11-BS07-
0.034 mmol, yield: 100%) was isolated as a white powder. Rf
0.39 (CH2Cl2–EtOH 90 : 10). 1H NMR (400 MHz, DMSO d6): δ
12.24 (s, 1H), 12.05 (s, 1H), 11.98 (s, 1H), 9.22 (br s, 3H), 9.01
(t, J = 5.6 Hz, 1H), 8.80 (d, J = 7.2 Hz, 1H), 8.75 (d, J = 7.2 Hz,
1H), 8.54 (t, J = 5.1 Hz, 1H), 8.36 (t, J = 5.8 Hz, 1H), 8.14 (t, J =
6.3 Hz, 1H), 7.30–7.26 (m, 3H), 6.94–6.91 (m, 3H), 6.71–6.65
(m, 3H), 4.55–4.49 (m, 2H), 3.93–3.80 (m, 4H), 3.60–3.57 (m,
2H), 3.44–3.35 (m, 2H), 3.09 (s, 1H). 13C NMR (100 MHz,
DMSO d6): δ 170.3, 169.6, 169.4, 169.1, 149.2, 149.0, 146.1,
146.0, 118.9, 118.0, 117.7, 115.4, 115.3, 115.2, 80.8, 73.1, 53.8,
53.6, 42.3, 40.4, 30.7, 28.2. MS (ESI) m/z 693.2 [M + H+], 716.0
[M + Na+]. HRMS (ESI): C32H32N6O12: calcd 692.2078, found
692.2091.
007-01) for financial support and for the postdoctoral fellow-
ship attributed to E.B. We also warmly thank Vaincre la Mucov-
iscidose (the French cystic fibrosis association) for repeated
financial support for the antibiotic Trojan horse project. We
thank the Centre National de la Recherche Scientifique (CNRS)
for general financial support. In addition, the research leading to
some of these results was conducted as part of the Translocation
consortium (http://www.translocation.com) and has received
support from the Innovative Medicines Joint Undertaking
under Grant Agreement no. 115525, resources which are com-
posed of financial contribution from the European Union’s
seventh framework programme (FP7/2007-2013) and EFPIA
companies in kind contribution.
Bacterial strains
The pyochelin- and pyoverdine-deficient Pseudomonas aerugi-
nosa strain PAD07,34 and the enterobactin-deficient Escherichia
coli strain BW25113entB::Kanr,33 have been described
previously.
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position in g L−1 is: K2HPO4, 6.0; KH2PO4, 3.0; (NH4)2SO4, 1.0;
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presence of 50 μg mL−1 tetracycline and 100 μm mL−1 strepto-
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amino acids for BW25113entB::Kanr. PAD07 and BW25113entB::
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