Multicellular aggregation of maltol-modified cells triggered by Fe 3+ ions

Article abstract of DOI:10.1039/c3cc43727b

The synthesis of a maltol-derived hydrazide is described which, once attached to a cell surface, induces rapid multicellular aggregation selectively in the presence of Fe³⁺ ions. Heterocellular aggregates are also reported. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc43727b

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Multicellular aggregation of maltol-modified cells
triggered by Fe3+ ions†
Cite this: Chem. Commun., 2013,
49, 10148
Alexander Ciupa, Paul A. De Bank and Lorenzo Caggiano*
Received 17th May 2013,
Accepted 12th September 2013
DOI: 10.1039/c3cc43727b
www.rsc.org/chemcomm
The synthesis of a maltol-derived hydrazide is described which, once bread, affording a sweet caramel-like odour.12 It is a bidentate
attached to a cell surface, induces rapid multicellular aggregation ligand and derivatives of maltol have found medicinal and clinical
selectively in the presence of Fe3+ ions. Heterocellular aggregates applications,13 such as deferiprone 1a used therapeutically as an
are also reported.
iron chelator.14–19 Macromolecular iron chelators have also been
reported20 and some maltol derivatives exhibit antimalarial activity21
and antiproliferative activity against tumour cells.22,23 We wished to
attach maltol derivatives to cells and investigate inducing cellular
aggregation through metal chelation as outlined in Scheme 1.
Owing to maltol’s affinity for Fe3+ ions, aggregation should
selectively occur in their presence and not with other ions found
in the cell culture medium.
Cells have been previously modified by chemoselective ligation
with hydrazides.4–6,24 Therefore, we explored the synthesis and use of
maltol hydrazide 6 to trigger multicellular aggregation in the
presence of Fe3+ ions. Following previous reports,14,15 maltol 1 was
initially protected as the benzyl ether 2 (87%), which upon treatment
with b-alanine gave the corresponding acid 3 in 70% yield
(Scheme 2). Transformation to the N-hydroxysuccinimide ester14,15
and reaction with NH2NH2 was examined, although we experienced
difficulties in purifying the product. Instead, conversion to the
methyl ester 4 (98% yield) and simple nucleophilic substitution with
NH2NH2 gave the hydrazide product 5 which could be readily
purified by silica gel column chromatography in 62% yield. Depro-
tection of the benzyl ether was achieved using H2 and Pd/C and gave
the desired maltol-derived hydrazide 6 in 84% yield. Only one
Tissues and organs are made of multiple cell types arranged in
three-dimensional (3D) structures. The development of model 3D
multicellular structures in vitro is of great interest as it has potential
applications in tissue engineering1 and as tumour models for drug
testing.2 A number of techniques have been used to generate 3D
cell aggregates3–8 and we now report that ionic cross-linking, which
has been extensively used to form hydrogels (e.g. alginate)9 and
induce self-assembly of collagen peptides,10,11 can selectively
generate multicellular aggregates in the presence of specific ions
using cells decorated with a maltol derivative.
Maltol (3-hydroxy-2-methyl-4-pyrone, 1, Scheme 1) is a commonly
used flavour enhancer found in many foodstuffs such as beer and
Scheme 1 Schematic representation of chelate-mediated cell aggregation.
Medicinal Chemistry, Department of Pharmacy and Pharmacology,
University of Bath, Claverton Down, Bath, BA2 7AY, UK.
E-mail: l.caggiano@bath.ac.uk; Tel: +44 (0)1225 385709
Scheme 2 Synthesis of maltol-derived hydrazide 6. (i) BnCl, NaOH, MeOH/H2O.
(ii) b-Alanine, NaOH, EtOH/H2O. (iii) AcCl, MeOH. (iv) NH2NH2, MeOH. (v) H2,
Pd/C, MeOH.
† Electronic supplementary information (ESI) available: Experimental proce-
dures, characterisation data, 1H and 13C NMR spectra and additional aggregation
images are available. See DOI: 10.1039/c3cc43727b
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Fig. 1 Representative images of maltol-modified HT29 cells in the presence of 50 mM FeCl3. Scale bars = 400 mm and the results are typical of three independent
experiments (provided in the ESI† together with images of unmodified cells in presence and absence of FeCl3).
purification by column chromatography was required overall and the
five-step synthesis can be readily performed on gram-scale.
With the maltol-derived hydrazide 6 in hand, we were able to
investigate its attachment to cells. Human colon carcinoma (HT29)
and highly metastatic human breast carcinoma (MDA-MB-231) cell
lines were chosen as model cells as they are well characterised and
have predictable growth properties. Since maltol derivatives exhibit
antiproliferative activities with tumour cells,22,23 the hydrazide 6 was
initially examined for activity in these cell lines using the MTS
assay.25 The results showed weak antiproliferative activity after
72 h in the cell lines examined, (IC50 > 200 mM in HT29 and
MDA-MB-231, ESI†) suggesting that further cell-based studies
should be conducted at a concentration of 200 mM or below.
The HT29 cell surface was modified following previous reports
using mild oxidation conditions (1 mM NaIO4, 10 min, 4 1C) to
generate non-native aldehydes from the sialic acid residues on the cell
surface.4–6,24,26 The cells were then exposed to maltol hydrazide 6
(100 mM, 60 min, 20 1C) to generate the corresponding hydrazone and
thereby attaching the maltol derivative to the cell surface (see ESI† for
experimental procedure). The unmodified and maltol-modified HT29
cells were resuspended in serum free medium in agarose-coated six-
well plates and then treated with FeCl3 in phosphate buffered saline
(PBS, 50 mM, 20 1C) with gentle agitation by rocking (Fig. 1 and ESI†).
After 10 min, multicellular aggregation of the maltol-modified cells
was observed only in the presence of Fe3+, with no significant
aggregates seen in the absence of Fe3+ or with the unmodified cells.
After 20 min of agitation, large multicellular aggregates had
formed with the maltol-modified cells in the presence of Fe3+ which
were visible to the naked eye. These results were obtained in serum-
Fig. 2 The effects of (i) Fe3+ concentration after 20 min agitation, and (ii)
agitation time with 50 mM Fe3+ on mean apparent aggregate area for unmodi-
fied and maltol-modified HT29 cells. Data shown as mean Æ standard error of
three independent experiments.
free culture medium and similar findings were also observed in PBS in smaller aggregate sizes, possibly due to Fe3+ saturation of the
(not shown). However, performing the experiments in complete maltol-derivatives. Due to toxicity associated with excess Fe3+,27,28
culture medium, containing 10% foetal bovine serum, resulted in 50 mM was chosen as the optimal concentration. Time was also
no cellular aggregation. Presumably iron-chelating proteins such as investigated and showed that longer times gave larger aggregates, as
transferrin present in the serum sequester the Fe3+ ions, preventing one would expect (Fig. 2ii). However, prolonged exposure (up to
metal-mediated aggregation. Therefore, all further experiments were 180 min) also led to some aggregation of the unmodified cells, but
performed in serum-free culture medium.
only in the presence of Fe3+. These aggregates were much smaller
Various Fe3+ concentrations and incubation times were and slower to form than observed with the maltol-modified cells
examined with both unmodified and maltol-modified HT29 cells (see ESI†) and may be due to weak Fe-chelation by the native sugars
in order to optimise the aggregation process (Fig. 2). The apparent present on the cell surface.
aggregate area was calculated from nine randomly selected images
Although selective for Fe3+, maltol derivatives can also chelate
from three independent experiments, using previous methods.5 In other metal ions such as Ru3+,29 Cu2+ and Zn2+.13 To investigate the
the presence of 0, 5 and 20 mM FeCl3 only small aggregates were selectivity of metal-mediated aggregation of maltol-modified cells,
observed after 20 min, although increasing the concentration to the metal chlorides (MClx) of these ions were examined under the
50 mM resulted in larger aggregate sizes in only the maltol-modified optimised aggregation conditions (ESI†). The results demonstrate
HT29 cells (Fig. 2i). Increasing Fe3+ concentration to 200 mM resulted that only in the presence of 50 mM of Fe3+ were multicellular
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In summary, these results describe the efficient synthesis of
the novel maltol-derived hydrazide 6 and its attachment to two
different cancer cell lines. Preliminary experiments show that
multicellular aggregation can be selectively achieved with Fe3+
ions, even in the presence of other ions, generating 3D aggre-
gates on demand which could find various applications.
Although cancer cells were used in this study, this approach
could be applied to any cell type possessing sialic acid residues
on the surface. It could be used with cells which do not naturally
aggregate or to template layers of different cells to create
predictable multicellular structures for use in tissue engineering.
We are grateful to the University of Bath for providing a
studentship for AC and also wish to acknowledge RCUK and the
University of Bath for the fellowship to LC.
Fig. 3 Optical density of MTS assays with unmodified and maltol-modified
HT29 cells in the absence (white/black) and presence (shaded) of 50 mM Fe3+
after 24, 48 and 72 h incubation. Data shown as mean Æ standard error of three
independent experiments.
Notes and references
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10150 Chem. Commun., 2013, 49, 10148--10150
This journal is The Royal Society of Chemistry 2013