Carbohydrate functionalized iron(iii) complexes as biomimetic siderophores

Article abstract of DOI:10.1039/c2cc16656a

Catechol functionalized iron(iii) glycodendrimers have been prepared using a self-assembly process for targeting a specific strain of E. coli bacteria and have been shown to exhibit carbohydrate-lectin mediated iron delivery in growth promotion assay. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc16656a

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COMMUNICATION
Carbohydrate functionalized iron(III) complexes as biomimetic siderophoresw
Rohan Yadav and Raghavendra Kikkeri*
Received 26th October 2011, Accepted 25th November 2011
DOI: 10.1039/c2cc16656a
Catechol functionalized iron(III) glycodendrimers have been
prepared using a self-assembly process for targeting a specific strain
of E. coli bacteria and have been shown to exhibit carbohydrate–
lectin mediated iron delivery in growth promotion assay.
because they had previously been proven to bind and doesn’t bind
to towards E. coli ORN178.1b Synthesis of the first generation
mannose-substituted catecholic ligands (Scheme 1) 10 involved
coupling of 2,3-dihydroxybenzyl benzoic acid and 20-aminoethyl
mannoside 8 in the presence of a coupling reagent, followed by
hydrogenolysis with 10% palladium–carbon and deacetylation
with sodium methoxide to yield 10. The second and third
generation catechol substituted glycodendrons were prepared
starting from compound 16. Following treatment of 8 or 22 to
yield 17 or 23, respectively, and finally deacetylation with
Targeting carbohydrate-binding lectins is an attractive approach
for cell and bacteria directed therapies. Specific binding of glycans
to bacterial cell surface lectins results in cell-specific binding and
endocytosis.1,2 However, monomeric glycan binding affinities
for carbohydrate–lectin interactions are typically in the milli- to
micromolar range and thus, multivalent interactions are often
required to exert biological effects. Different strategies such as
glyconanoparticles, glycopolymers, metalloglyco-dendrimers and
glycodendrimers3 have been employed to achieve multivalency.
Amongst these, self-assembled metallo-glycodendrimers are
pre-eminent as carbohydrate–protein interactions can be
traced with high sensitivity and spacial resolution with optical,
electrochemical and radioactive signals.3,4 In addition to probing
these interactions, metal chelating glycodendrons can be used to
increase the local concentration of essential elements to amplify
the growth and other functions in living species. For example,
microorganisms synthesize and release iron chelators, termed
siderophores, to overcome iron deficiency.5 Many siderophores
are secreted by the bacterium into the environment, bind iron,
and then re-enter the cell. Others are found on the cell wall where
they bind iron and transport it into the bacterium. Moreover,
these siderophores are highly specific to the chirality of the metal
complexes and types of ligands in the binding sites.6 Herein,
for the first time we report catechol confined glycodendrons as
biomimetic siderophores to bind Fe(III) by a self-assembly process.
Self-assembly of biomimetic siderophores was evaluated by
spectroscopic techniques. Finally, we show the interactions of
Fe(III)–glycodendrimers with concanavalin A lectin and a specific
E. coli strain, inducing iron mediated growth promotion.
To obtain biomimetic analogs that could serve as iron(III)
delivery tools in a species specific manner, we attached 2,3-
dihydroxybenzoic acid to mannose and galactose glycodendrimers,
at a site that does not interfere with sugar receptor binding.
These two congeners (mannose and galactose) were selected
Scheme 1 Synthesis of Fe(III)–glycodendrimers 2–4: (a) DCM/TFA
(3 : 1), 1 h, RT; DCM/TEA, 12 h, RT 58%; (b) DCM/TFA (3 : 1),
1 h, RT; 2,3-bis(benzyloxy)-benzoic acid, DIC, HOBT, DCM, rt, 12 h,
56%; NaOMe, MeOH, 2 h, 56% (b) H2, Pd/C, MeOH, 12 h, 64%;
(c) NaOMe, MeOH, 2 h; H2, Pd/C, MeOH, 12 h, 43%; (d) tert-
butoxycarbonyl-3-{tris[3-[2-ethoxy-2,3,4,6-tetra-O-acetyl-a-D-manno-
pyranoside-ethoxy]methyl] methylamide}-3-b-alanine 22, DCM/TFA
(3 : 1), 1 h, rt; TEA, DCM, rt, 12 h, 65%; NaOMe, MeOH, 2 h, rt; H2,
Pd/C, rt, MeOH, 12 h, 69%; (e) FeCl3, MeOH.
Indian Institute of Science Education and Research,
Sai Trinity Building, Pashan, Pune 411021, India.
E-mail: rkikkeri@iiserpune.ac.in; Fax: +91-20-25899790;
Tel: +91-20-25908207
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc16656a
c
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Fig. 2 Turbidity analysis–absorption change of compound 2 (D),
3 (}), 4 (&) and 7 (J) (1.0 mM) at 700 nm upon addition of
ConA (1 mg mLꢀ1) in Hepes buffer with 1 mM of CaCl2 and 1 mM
MgCl2. Mannose (100 mM) was added after 25 min.
Fig. 1 Structures of enterobactin 1 and catecholic glycodendrimers
2–7 iron(III) complexes.
Fig. 3 Fe(III)–glycodendrimer dependent growth promotion under
iron limitation. Mannose positive mutants of E. coli (ORN178) were
grown in medium containing 0.1 mM of iron chelator 2,20-dipyridyl
(U), 1 (’), 50 mM of complex 2 (m), 3 (K), 4 (E), 5 (&), 6 (J) and 7
sodium methoxide and hydrogenolysis afforded compound
19 or 25. Galactose substituted catecholic glycodendrons
13, 20 and 26 were also prepared in analogy to the process
employed for 10, 19 and 25 respectively.
(
}
). A600 value represents the means ꢁ standard deviations from the
parallel cultures.
UV/vis titration of the sugar embedded catechol derivatives
(10, 19 and 25) and with ferric chloride established the
formation of Fe(III) complexes with a 1 : 3 stoichiometry, as
reflected by their absorption around 490 nm at high pH
(Fig. S1, ESIw).7 The metallo-glycodendrimers 2–7 were prepared
by refluxing stoichiometric amounts of glycodendrons with ferric
chloride in methanol.
(positive control) having a mannose receptor (FimH) and
mutant E. coli strain ORN208 as a negative control. We first
tested the biological activity of complexes 1–7 as siderophores
in growth promotion assays. As shown in Fig. 3, the medium
containing the iron chelator 2,20-dipyridyl no longer supports
growth. Growth of ORN 178 was restored by adding 50 mM of
natural siderophore 1, whereas the equal amount of Fe(III)–
glycodendrimers 2 and 3 had significant effect on growth.
However, growth of ORN178 decreased substantially from
complex 3 to 4, in support of the notion that carbohydrate
multivalency encapsulate the iron(III) complex stops effective
endocytosis. Complexes 4–7 bearing b-galactopyranoside
served as a negative control and did not show any growth.
A similar experiment with ORN208 strain was neither initiated
by mannose or galactose glycodendrimers 2–7 (Fig. S5, ESIw).
To evaluate the receptor mediated iron delivery process, the
inhibition assay was performed with NaN3 (Fig. S4, ESIw). As
seen from the results, compounds 1–3 are significantly affected
by NaN3 indicative of the participation of the energy-driven
processes in the Fe(III) uptake (Fig. 4). A similar inhibition
experiment with complex 4 (high mannose) showed significant
To investigate the specific lectin–carbohydrate interactions
between these complexes, turbidity assay was carried out with
a concanavalin A (ConA) lectin model, since ConA selectively
binds to mannopyranosides. When aqueous solutions of Fe(III)
complex of 2–7 were added to solution of ConA in Hepes
buffer, complexes 3 and 4 showed an increase in turbidity of
the mixture, indicative of binding (Fig. 2). On the other hand,
complex 7 bearing b-galactopyranoside served as a negative
control and did not bind to ConA. In order to demonstrate
that the turbidity increase observed is due to specific protein–
carbohydrate interactions, a large excess of mannose was added to
inhibit dendrimer–ConA binding. Indeed, the turbidity disappeared
upon addition of mannose.
To demonstrate carbohydrate receptors on microbial cell
surfaces, as iron(III) transporter, we selected E. coli ORN178
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2 and 3 transported iron via a specific mannose receptor. This
was confirmed by growth promotion in the culture media and
LB plate assays. Moreover, these compounds were found to
exhibit high specificity by distinguishing between different
E. coli species (ORN 178 compared to ORN208). Current
investigations are aimed at preparing tailor made carbohydrate–
iron sequestering conjugates and applying them to target micro-
organisms in mammalian hosts.
R. K. and R. Y. thank IISER, Pune, Indo-German (DST-MPG)
programme, and CSIR, India, for financial support. We thank
Prof. Orndorff for providing the E. coli strains and Prof.
Abraham Shanzer for enterobactin template.
Notes and references
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limitation. Mannose positive mutants of E. coli (ORN178) were grown in
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are the best candidates for iron(III) delivery. Focusing on
E. coli as an indicator organism, we demonstrated that complexes
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Fig. 5 Tentative model for the uptake of Fe(III)–glycodendrimers (2 and 3)
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c
1706 Chem. Commun., 2012, 48, 1704–1706
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