Targeting cancer cells with folic acid-iminoboronate fluorescent conjugates

Article abstract of DOI:10.1039/c3cc47534d

Herein we present the synthesis of fluorescent 2-acetylbenzeneboronic acids that undergo B-N promoted conjugation with lysozyme and N-(2-aminoethyl) folic acid (EDA-FA), generating conjugates that are selectively recognized and internalized by cancer cell

Full text of DOI:10.1039/c3cc47534d

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Targeting cancer cells with folic acid–
iminoboronate fluorescent conjugates†
Cite this: Chem. Commun., 2014,
50, 5261
Pedro M. S. D. Cal,a Raquel F. M. Frade,a Vijay Chudasama,b Carlos Cordeiro,c
Stephen Caddickb and Pedro M. P. Gois*a
Received 1st October 2013,
Accepted 4th November 2013
DOI: 10.1039/c3cc47534d
www.rsc.org/chemcomm
Herein we present the synthesis of fluorescent 2-acetylbenzene-
boronic acids that undergo B–N promoted conjugation with lysozyme
and N-(2-aminoethyl) folic acid (EDA-FA), generating conjugates
that are selectively recognized and internalized by cancer cells that
over-express folic acid receptors.
Boronic acids are well-defined neutral, planar trivalent Lewis acids
that are readily converted into negatively charged tetravalent
borates by forming reversible complexes with Lewis base donors
such as amines.1 Appreciation of the unique B–N bond properties
triggered a burgeoning interest for this bonding motif.2 B–N bonds
Scheme 1 Protein modification via the formation of iminoboronates.
Proposed structure of N–B based cancer cell targeting fluorescent conjugates.
have been extensively exploited to construct self-assembled mole- develop into an innovative strategy to design conjugates that may
cularly defined nanostructures, polymeric materials and molecular selectively target and deliver cargo to cancer cells.7
sensors for the detection of carbohydrates.3 Recently, the isosterism
In this communication, we address for the first time, the
between B–N and C–C bonds was also recognized as a powerful tool synthesis and evaluation of cancer cell targeting fluorescent
to tune the stereo electronic properties of organic molecules.4 In conjugates, in which the recognition biomolecule is connected
this context, we used the B–N bond to prepare natural product- to the fluorescent motif via a B–N linkage (Scheme 1). In our
like structures and heterocycles with inhibitory activity against previous study, 2-acetylbenzeneboronic acid afforded the most
human neutrophil elastase.5 More recently, we found that this stable iminoboronate in buffer solutions (pH range 6–9).6 Conse-
bonding motif could be exploited to efficiently modify proteins at quently, this scaffold was selected to integrate the fluorescent
their N-terminal or at lysine’s e-amino group, via the formation of motifs as outlined in Scheme 1.
alkylic iminoboronates in aqueous media (Scheme 1).6 Despite
We initiated this study with the synthesis of fluorescent
their stability, these modifications were also shown to be reversible derivatives of 2-acetylbenzeneboronic acid. As shown in
in the presence of fructose, dopamine and glutathione, as they Scheme 2, 2,4-dihydroxyacetophenone was alkylated selectively
presumably induce hydrolysis of the iminoboronate by disruption with ethyl 4-bromobutyrate in a 5 g scale to yield compound 2
of the B–N bond.2 Based on this, we became very interested in in 87% isolated yield. After this, phenol 2 was converted to a
understanding if the B–N bond could also be used to assemble triflate, which then underwent a Miyaura borylation reaction
constructs that are able to internalize into cells. If so, this simple, with bis(pinacolato)diboron to afford 3 in 64% yield. Hydrolysis
selective and reversible methodology to functionalize proteins may of the ester with TFA in aqueous media led to acid 4, which
became the core structure for connecting fluorescent probes to
targeting molecules. Dansyl and nitrobenzofurazan fluorescent
a
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´
Instituto de Investigaçao do Medicamento (iMed.ULisboa), Faculdade de Farmacia,
Universidade de Lisboa Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
E-mail: pedrogois@ff.ul.pt; Fax: +351 217946470; Tel: +351 217946400
motifs were modified with ethylenediamine and ethanolamine,
respectively, and each subsequently reacted with 4. Removal of
the pinacol group using a boronic acid resin under acidic
conditions afforded the final fluorescent compounds 5 and 6
in moderate yields (Scheme 2). Once prepared, compounds 5
and 6 were immediately tested for their ability to conjugate with
lysozyme. Gratifyingly, both compounds retained their ability
b Department of Chemistry, University College London, 20 Gordon St, London, UK
c
´
´
´
´
Centro de Quımica e Bioquımica, Departamento de Quımica e Bioquımica,
ˆ
Faculdade de Ciencias da Universidade de Lisboa, 1749-016 Lisboa, Portugal
† Electronic supplementary information (ESI) available: Experimental proce-
dures, full DFT reference list, atomic coordinates for the optimized species,
and spectral data for all compounds. See DOI: 10.1039/c3cc47534d
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Scheme 2 (a) Ethyl 4-bromobutyrate, K2CO3, NaI, acetone, reflux, 20 h;
(b) PhNTf2, TEA, DMF, rt, 2 h; (c) (Bpin)2, PdCl2(dppf), NaCH3CO2, dioxane,
95 1C, 4 h; (d) TFA, H2O, 90 1C, 3 h; (e) NHS-OH, DCC; THF, rt, 16 h;
(f) 2-dansylaminoethylamine, THF, rt, 2 h; (g) CH3CN : HCl (1 M) (9 : 1), 20 h, rt,
20 h; (h) DMAP, DCC; DCM, 0 1C, 0.5 h; (i) 4-ethanolamine-7-nitrobenzo-
furazan, DCM, rt, 22 h. UV spectra: see ESI† for compounds 5 and 6.
Scheme 3 Reaction of lysozyme (10 mM) with 5 (200 mM) in acetate buffer
(50 mM, pH 7) at room temperature. Zoom of the (7+) charge state of the
ESI-FTICR-MS spectrum. Reaction of EDA-FA (100 mM) with compound 5
(100 mM) in acetate buffer (50 mM, pH 7) to obtain 8 confirmed by HRMS.
HEK (A) and NCI-H460 (B) cells were incubated with compound 8 at 20 mM
for 4 h. Images show an overlay of the emission of compound 8 in the
460–580 nm region upon excitation at 458 nm (green), and the emission
of the membrane marker in the 650–700 nm region upon excitation at
514 nm (red).
to functionalize the protein, yielding the expected constructs in
ammonium acetate buffer (50 mM, pH 7.0) at room temperature
as confirmed by ESI-FTICR-MS (Schemes 3 and 4). Encouraged
by these results, we next evaluated the possibility of designing a
cancer cell targeting conjugate, in which the fluorescent motif is
linked to the targeting molecule by a B–N bond.
Folic acid is essential for cell functioning, however, a consider-
able percentage of cancer cell lines over-express receptors for this
small vitamin due to their fast cell division and growth.8 Conse-
quently, folic acid has been extensively used as a recognition
moiety in many conjugates.9 Based on this, the conjugation of 5
and 6 with N-(2-aminoethyl) folic acid (EDA-FA) was evaluated. As
expected, upon addition of 100 mM of 5 or 6 in DMSO to EDA-FA
in ammonium acetate buffer (50 mM, pH 7.0) at room tempera-
ture, their corresponding iminoboronates were formed readily,
as characterized by high resolution mass spectrometry – HRMS
(Schemes 3 and 4). Following this, the ability of the conjugates
to differentiate between cancer and non-cancer cell lines was
studied. To do this, conjugate 8 was generated prior to exposure
to cells and tested against human non-small lung cancer cells
(NCI-H460) and non-cancer human kidney embryonic cells
(HEK 293). Interestingly, laser scanning confocal images of
HEK 293 and NCI-H460 treated with 8 revealed that only the
cancer cell line that over-expressed the FA receptor up took
fluorescence, which suggests a selective folate-receptor mediated
internalization of the B–N construct 8.
To further elucidate this mechanism, the cancer cell line
NCI-H460 was treated with 6 without EDA-FA. In agreement with
a folate-receptor mediated internalization mechanism, without Scheme 4 Reaction of lysozyme (10 mM) with 6 (200 mM) in acetate
buffer (50 mM, pH 7) at room temperature. Zoom of the (7+) charge state
the recognition moiety, no fluorescence was detected inside the
cells. In contrast, when the same compound 6 was allowed to
form a conjugate with EDA-FA, the previously generated B–N
of the ESI-FTICR-MS spectra after 30 min and 2 h of reaction. Reaction of
EDA-FA (100 mM) with compound 6 (100 mM) in acetate buffer (50 mM,
pH 7) to obtain 10 as confirmed by HRMS. NCI-H460 (C) and NCI-H460
construct 10 smoothly underwent internalization as shown in
Scheme 4. Finally, the NCI-H460 cells were treated with EDA-FA compound 10. The procedure is described in Scheme 3.
without (D) and with pre-treatment with EDA-FA (E) were incubated with
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highlight the contribution of boronic acid to imine stabili-
zation and internalization of the conjugates.
In this communication we show for the first time that the B–N
dative bond may be used to synthesize conjugates that selectively
target cancer cells. Fluorescent 2-acetylbenzeneboronic acid deriva-
tives were successfully prepared and conjugated via a B–N linkage
with lysozyme and N-(2-aminoethyl) folic acid, generating conju-
gates that were selectively recognized and internalized by NCI-H460
cancer cells, which over-express folic acid receptors. The ability of
these iminoboronates to undergo a receptor mediated internaliza-
tion, and their efficiency to promote the selective and reversible
functionalization of proteins, highlights that these constructs have
a promising future in the design of conjugates that selectively target
and deliver cargo to cancer cells.
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ˆ
Fundaçao para a Ciencia e Tecnologia (PTDC/QUI-QUI/118315/
2010; Pest-OE/SAU/UI4013/2011; SFRH/BD/72376/2010: REDE/1501/
REM/2005) is acknowledged for financial support.
Scheme 5 (a) Ethyl 4-bromobutyrate, K2CO3, NaI, acetone, reflux, 20 h; (b)
TFA, H2O, 90 1C, 3 h; (c) DMAP, EDC; DCM, 0 1C, 1 h; (d). NBDNH(CH2)2OH,
DCM, rt, 5 h. UV spectrum: see ESI† for compound 14. Reaction of lysozyme
(10 mM) with 14 (200 mM) in acetate buffer (50 mM, pH 7) at room
temperature. Zoom of the (7+) charge state of the ESI-FTICR-MS spectra
after 30 min and 2 h of reaction. Image F shows NCI-H460 treated with
compound 16. Incubation and imaging conditions are likewise described
in Scheme 3.
Notes and references
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prior to the addition of the conjugate 10. In this case, the
internalization of the conjugate 10 was considerably impaired
due to the initial exposure to folic acid that reduced the cell’s
need for this vitamin. These results clearly stress the importance
of the folate moiety to mediate the internalization process.
Finally, to determine the contribution of the B–N bond to
the formation and stability of these conjugates, the fluorescent
compound 14, featuring no boronic acid, was prepared via
alkylation of 4-hydroxyacetophenone with ethyl 4-bromobutyrate,
followed by hydrolysis and DMAP catalysed esterification with
4-ethanolamine-7-nitrobenzofurazan (NBDNH(CH2)2OH). Once
prepared, 14 was solubilised in DMSO and combined with EDA-
FA in an ammonium acetate buffer solution (50 mM, pH 7.0) at
room temperature to form conjugate 16, as confirmed by HMRS. A
mixture containing the conjugate was then used to treat the NCI-
H460 cancer cells. This resulted in no fluorescence being detected
inside the cells, presumably due to the poor stability of conjugate
16 under these conditions. Therefore, we studied the ability of
compound 14 to modify lysozyme. As depicted in Scheme 5, the
compound lacking the boronic acid functionality hardly
formed the expected constructs with the protein after 30 min
in ammonium acetate buffer (50 mM, pH 7.0). Even the ones
that did form seemed to hydrolyse in only 2 h under these
reaction conditions. In stark contrast, under the same condi-
tions, boronated compound 6 readily afforded the constructs
with lysozyme within 30 min and the modifications were
persistent after 2 h in ammonium acetate buffer (50 mM,
pH 7.0) at room temperature (Scheme 4). These results clearly
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