Molecular recognition of bisphosphonate-based drugs by di-zinc receptors in aqueous solution and on gold nanoparticles

Article abstract of DOI:10.1039/d0dt00930j

Metal-based anion receptors have several important applications in sensing, separation and transport of negatively charged species. Amongst these receptors, di-zinc(ii) complexes are of particular interest for the recognition of oxoanions, in particular phosphate derivatives. Herein we report the synthesis of a di-zinc(ii) receptor and show that it has high affinity and selectivity for bisphosphonates such as alendronate and etidronate - which are used to treat a number of skeletal disorders as well as showing interesting anticancer properties. The binding mode of the di-zinc(ii) receptor with alendronate and etidronate has been unambiguously established by single crystal X-ray crystallography. In addition, by modifying the backbone of the receptor, we show that the drug-loaded receptor can be attached onto gold nanoparticles as potential drug-delivery vehicles.

Full text of DOI:10.1039/d0dt00930j

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14 March 2017
Pages 3073-3412
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Molecular recognition of bisphosphonate-based drugs by di-
zinc receptors in aqueous solution and on gold nanoparticles
Aaron Torres-Huerta,1 Tiffany G. Chan, 1 Andrew J.P. White, Ramon Vilar*1.
1Department of Chemistry, Imperial College London, White City Campus, London W12
OBZ (UK)
Corresponding author: r.vilar@imperial.ac.uk
Abstract
Metal-based anion receptors have several important applications in sensing, separation and
transport of negatively charged species. Amongst these receptors, di-zinc(II) complexes are
of particular interest for the recognition of oxoanions, in particular phosphate derivatives.
Herein we report the synthesis of a di-zinc(II) receptor and show that it has high affinity and
selectivity for bisphosphonates such as alendronate and etidronate – which are used to treat
a number of skeletal disorders as well as showing interesting anticancer properties. The
binding mode of the di-zinc(II) receptor with alendronate and etidronate has been
unambiguously established by single crystal X-ray crystallography. In addition, by
modifying the backbone of the receptor, we show that the drug-loaded receptor can be
attached onto gold nanoparticles as potential drug-delivery vehicles.
1

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1.
Introduction
Over the past two decades, there has been great interest in the development of molecular
receptors for oxoanions such as carboxylates, phosphates and nitrates, amongst several
others. 1 Such receptors have been designed for a wide range of applications such as sensing,2
removal of pollutants from solutions3 and to transport anionic guests across biological
membranes.4 One large family of such receptors is based on metal complexes with the ability
to coordinate reversibly to the target anionic guest.5 Depending on the number and nature of
the metal ion as well as its coordination environment, it is possible to tune the selectivity of
metallo-receptors for a given oxoanion.6 For example, di-zinc(II) complexes have been
shown to display high affinity for phosphate derivatives and, by tuning the coordination
environment, display good selectivity for a given phosphate over other anions.7
One class of phosphate derivatives that attracted our attention are bisphosphonates such as
etidronate and alendronate (see Scheme 1). These oxoanions act as inhibitors of osteoclastic
bone resorption and are therefore used in the treatment of a variety of skeletal disorders such
as Paget´s disease and osteoporosis.8 Recent studies have also revealed that this type of
bisphosphonates can induce apoptosis in tumour cells,9,10 are synergistic with antineoplastic
drugs11 and can act as anti-angiogenic agents.12,13 However, the use of bisphosphonates in
the clinic has been limited since less than 1% of an orally administered dose is taken up by
the cell.14 This has been attributed to their negative electrostatic charge, which hinders their
transport through the lipophilic cell membrane. Furthermore, bisphosphonates exhibit a short
plasma half-life and are rapidly eliminated in urine.15 To address these problems, liposomes,
nanopeptides, calcium phosphate-DNA nanoparticles and nanopolymers have been explored
as drug carriers.16-22
2

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With this in mind, the aim of the research herein presented was to study whether di-zinc(II)
complexes could act as good receptors for bisphosphonates and whether it would be possible
to attach such host-guest systems to gold nanoparticles (AuNP) as nanocarriers for future
biological applications. Thus, we report the synthesis of the di-zinc(II) molecular receptor 2
(see Scheme 1) and its interactions with etidronate and alendronate. The binding process has
31
been studied by spectroscopic techniques (i.e. UV-vis and P NMR spectroscopy) and the
exact binding mode of both bisphosphonates to the receptor has been established by X-ray
crystallography. Furthermore, the drug-loaded metallo-receptor was further functionalised
with dithiocarbamate and successfully attached onto AuNPs (see Scheme 2).
Results and Discussion
Synthesis and spectroscopic characterization of di-zinc(II) complex 2. The bis(zinc(II)-
dipicolylamine) motif has been used extensively in receptors for several oxoanions such as
phosphates and carboxylates.23, 24 There is also one previous example where this type of
complex has been incorporated into a receptor for bisphosphonates.25 Therefore, we
synthesised the di-zinc(II) receptor 2 (see Scheme 1) and explored its ability to selectively
recognise alendronate and etidronate over several other anions. In addition to the di-zinc
recognition moiety, the complex also contains a benzylamine substituent since it can be
readily converted into a dithiocarbamate which in turn would render the complex suitable for
attachment to gold nanoparticles (see below).
Ligand 1 was obtained following a synthetic procedure previously reported for similar bis-
dipicolylamine compounds (see Scheme S1).26 Metallo-receptor 2 was synthesized by
reacting 1 with two equivalents of Zn(OAc)2 and one equivalent of NaBF4 (see Scheme 1).
3

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HO
R
NH2
O
O
O
O
P
P
O
O
N
HO
P
R
O
O
N
N
HO
O
O
N
N
N
N
N
N
N
N
HO
O
N
HO
O
Zn
Zn
P
Zn
Zn
P
P
O
ONa
N
N
O
ONa
OH HO
O
N
OH
N
N
N
Zn(AcO)2
NaBF4
OH HO
O
Alendronate
MeOH
H2O
HO CH3
HN
HN
H2N
HO
O
P
P
O
ONa
OH HO
Etidronate
2
1
3
4
; R = CH3
R = CH2CH2CH2NH2
;
Scheme 1: Synthetic route for the preparation di-zinc(II) receptor 2 and its complexes with
bisphosphonate-based drugs (i.e. etidronate and alendronate). Complex 3 is electrostatically neutral
due to the protonation of the benzyl amine group while complex 4 is +1 due to the protonation of both
the benzylamine and the amine of alendronate.
The 1H and 13C NMR spectrum of complex 2 (as compared to 1) shows that the pyridine C/H
are in two different chemical environments due to the coordination to the zinc(II) centres (see
Figures S1 – S4 for 1H and 13C NMR spectra; S5 – S8 for 2D NMR spectra). In the 13C NMR
spectrum, ligand 1 shows singlets at 149.0 (C1-py), 136.6 (C3-py), 123.1 (C2-py), 122.1 (C4-
py) while complex 2 displays double signals for all of them, i.e. at 148.1 and 146.1 (C1-py),
139.1 and 137.7 (C3-py), 124.1 and 123.6 (C2-py), 122.7 and 121.0 (C4-py) – see ESI for
numbering of atoms). The observed splitting of the 1H and 13C NMR resonances is consistent
with what has been previously reported for other Zn-bis-dipicolylamine complexes.27
Indicator displacement assays (IDA) and UV/vis titrations. To study whether 2 could act
as a molecular receptor for bisphosphonate-based drugs, its interaction to etidronate and
alendronate was investigated via IDA. The interaction of 2 with several other anions which
3-
2-
2-
2-
2-
-
are physiologically relevant (i.e. HP2O7 , HPO4 , SO4 , SO3 , CO3 , NO3 , OH- and Cl-)
was also studied to establish its selectivity profile. This was performed using pyrocatechol
violet (PV) as an indicator, which is yellow when free in solution but blue when coordinated
4

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to zinc(II). Compound 2 (50 µM) was mixed with one molar equivalent of PV and the
resulting 2∙PV complex was individually incubated with one molar equivalent of each anion.
Upon mixing with etidronate and alendronate, an instantaneous colour change was observed,
indicating the displacement of the dye by the bisphosphonate drugs (Figure 1a). In contrast,
mixing 2∙PV with the other anions under study did not generate a significant change in the
colour of the original solution (Figure 1a). The same experiment was repeated using 10
equivalents of each anion; in this case, besides etidronate and alendronate, pyrophosphate
also induced a colour change (consistent with previous reports showing that at high
concentrations pyrophosphate binds to this type of di-zinc(II) receptors – see Figure S30).
Following this initial screening, the interaction between 2∙PV and the anions was further
analysed by UV-Vis spectroscopy, which confirmed the ability of receptor 2 to bind
etidronate and alendronate with high selectivity over all other anions investigated – except
pyrophosphate which also shows some binding at high concentrations (Figure 1b).
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2-
2-
Cl-
3-
2-
2-
-
OH-
SO4
SO3
Etidronate Alendronate HP2O7
HPO4
CO3
NO3
a)
c)
b)
Only PV+2
Etidronate
Alendronate
O[P(O)(OH)2]2
HNa2PO4
Na2SO3
Na2SO4
Na2CO3
NaNO3
NaOH
Only PV+2
Etidronate
Alendronate
O[P(O)(OH)2]2
HNa2PO4
Na2SO3
Na2SO4
Na2CO3
NaNO3
NaOH
1
0.8
0.6
0.4
0.2
0
1
Pyrophosphate
0.8
0.6
0.4
0.2
0
NaCl
NaCl
Etidronate
Alendronate
Etidronate
Alendronate
Pyrophosphate
350
450
550
650
350
450
550
650
Wavelength (nm)
Wavelength (nm)
Figure 1: Colorimetric response of 2∙PV to different anions in 10 mM HEPES solution at pH 7.4 at
room temperature. (a) With 1 eq of anions. (b) UV-Vis spectra of 50 µM 2∙PV in 10 mM HEPES
solution at pH = 7.4 and 1 eq of anions (c) UV-Vis spectra of 50 µM 2∙PV in 10 mM HEPES solution
at pH = 7.4 and 1 eq. of etidronate, alendronate and 10 equivalents of common inorganic anions.
Quantitative determination of the binding affinity between receptor 2 and the bisphosphonate
drugs was assessed by UV-Vis and 31P NMR titrations (Figure 2). The UV-Vis titration was
performed by adding increasing amounts of either etidronate or alendronate to a solution of
2∙PV (50 µM). The intensity of the UV-Vis bands centred at 455 nm increases while the band
centred around 650 nm decreases upon addition of the bisphosphonate anion (Figure 2b).
From this data, the association constants for 2 with etidronate (Ka = 2.42±0.15 x 105 M-1) and
alendronate (2.25±0.16 x 105 M-1) were calculated.
The interaction between 2 and the two drugs was also studied by 31P NMR spectroscopy in
D2O at room temperature (Figure 2d). Upon addition of alendronate to a solution of 2, a
resonance at 21.9 ppm (assigned to coordinated alendronate) appeared and increased with
subsequent additions of the anion until ca. 1 equivalent. Subsequent additions led to the
appearance of a second resonance (17.8 ppm) corresponding to free bisphosphonate (similar
6

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results were obtained for etidronate, Figure S31). The UV-Vis and 31P NMR spectroscopic
studies indicate that 2 binds to etidronate and alendronate in a 1:1 stoichiometry. To confirm
this, the method of continuous variation (i.e. Job’s plots) was applied; as can be seen in Figure
2c and S30c, these studies clearly show the presence of a 1:1 binding stoichiometry.
a)
d)
NH3+BF4
-
HO
O
SO3H
O
O
P
P
O
O
N
HO
O
O
N
N
N
Alendronate
Zn
Zn
O
O
N
N
NH2
O
N
N
N
N
HO
P
Zn
Zn
+
HO
O
P
N
N
O
ONa
2.0 eq
1.5 eq
1.0 eq
O
OH OH
H2N
HN
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
c)
b)
0.75 eq
0.5 eq
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
1.1
0.9
0.7
0.5
0.3
0.1
0.25 eq
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1
30
25
20
δ (ppm)
15
1
370
470
570
Wavelength (nm)
670
mole fraction of PV
Figure 2: (a) Indicator displacement assay between 2∙PV and alendronate; (b) UV spectra of 50 µM
2∙PV titrated with increasing amounts of 1.25 mM alendronate in 10 mM HEPES solution, pH = 7.4.
(c) Job´s plot of 50 µM 2∙PV and alendronate in 10 mM HEPES solution at pH = 7.4. (d) 31P NMR
(400 MHz, D2O) spectra showed the titration of 2 with alendronate.
Synthesis of complexes 3 and 4. Having established that receptor 2 interacts strongly with
etidronate and alendronate, host-guest complexes 3 and 4 were prepared, isolated and fully
characterized (including determination of their X-ray crystal structures – see below). The 1H
NMR spectra of these complexes showed the disappearance of the acetate groups (i.e. the
original anion in 2) and the appearance of new resonances at 1.64 ppm and between 1.87 and
3.05 ppm associated to etidronate and alendronate, respectively. In addition, the 31P NMR
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spectra of both these complexes in MeOD showed two doublets for the phosphorus of the
etidronate and alendronate in the range of 23.4-24.8 and 21.9-22.9 ppm, respectively. It is
interesting to note that there are differences in the 31P NMR spectra of 3 and 4 when recorded
in D2O (see Figure 2d) and MeOD (see Figures S10 and S12). In the former a single
resonance is observed, while in MeOD two distinct doublets are observed, which is consistent
with the two different phosphorous environments (coupling to each other). The formulation
and purity of these host-guest complexes were confirmed by mass spectrometry and
elemental analyses (see Experimental Details).
Single-crystal X-ray diffraction analysis. The molecular structures of compounds 3 and 4
were determined by single-crystal X-ray crystallography (Figures 3 and 4) confirming the
1:1 association between the receptor and bisphosphonates. Suitable crystals of compounds 3
and 4 were grown from acetone and methanol/toluene solutions respectively. In both of these
structures, the four negative charges of the bisphosphonate groups are delocalized between
the four oxygen atoms coordinated to the zinc(II) centres and the two remaining
uncoordinated oxygen atoms. This effect is evidenced in the relatively narrow distribution of
the P–O distances, from 1.510(2) Å to 1.546(3) Å and 1.518(3) Å to 1.543(3) Å for 3 and 4,
respectively. These values are intermediate to the standard bond lengths reported for P–O
(1.50 Å) and P=O (1.63 Å). The average values for the N–Zn, Odrug–Zn and Oligand–Zn bond
distances are 2.210 Å, 2.063 Å, 2.100 Å for 3, and 2.212 Å, 2.059 Å, 2.096 Å for 4. In both
structures the coordination geometry around the zinc(II) centres corresponds to a distorted
octahedron. In 4, the amine group of the coordinated alendronate is protonated giving an
overall positive charge to the complex.
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Figure 3.
The crystal structure of 3 (50% probability ellipsoids) showing the interaction
between the di-zinc(II) receptor and etidronate.
Figure 4.
The structure of the cation in the structure of 4 (50% probability ellipsoids) showing
the interaction between the di-zinc(II) receptor and alendronate.
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Functionalization of gold nanoparticles with receptors. The next aim of this work was to
explore the attachment of receptor 2 (loaded with bisphosphonates) onto gold nanoparticles
(AuNP) to yield a potential drug delivery system. As has been previously reported,
dithiocarbamates are efficient groups for the attachment of small molecules onto gold
nanoparticles. Thus, we functionalized the backbone of receptor 2 with a dithiocarbamate to
yield 5 as shown in scheme 2.
HO
R
-
-
BF4
BF4
O
O
P
P
O
O
O
O
HO
P
R
O
O
N
O
O
O
O
O
O
N
N
N
NaO
O
N
N
N
N
N
N
P
N
N
Zn
Zn
1) NaOH
2) 1 ml CS2
Zn
Zn
O
ONa
Zn
Zn
OH HO
N
N
N
O
N
N
N
O
O
H2O
EtOH
S
-
N
S
N
HN
-
S
Na+
S
Na+
6
7
; R = CH3
R = CH2CH2CH2NH2
5
2
;
Scheme 2: Synthetic route for the preparation of dithiocarbamate-modified di-zinc(II) receptor and
the corresponding complexes with etidronate and alendronate.
Subsequently, the modified receptor 5 was loaded with either etidronate or alendronate to
yield compounds 6 and 7 respectively. Due to the limited solubility of 6 and 7, it was not
1
possible to obtain H and 13C NMR spectra of good enough quality to assign all the
resonances. However, the 31P NMR spectra in MeOD of these complexes showed the
expected doublets for coordinated etidronate (δ = 22.1-24.9 ppm) and alendronate (δ = 22.1-
22.9 ppm). In addition, ESI(+)-MS showed the molecular ion peaks for both the host-guest
complexes: [M+H]+ and [M–BF4]+ at 1079.70 and 1120.12 a.m.u. for 6 and 7 respectively.
10

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These complexes were then attached to AuNPs which were also functionalised with thiolated
oligonucleotides labelled with fluorescein (FAM-DNA-SH) to give the system greater
biocompatibility as well as a fluorescent tag to help determining the DNA loading on the
AuNP (see below). It is well established that passivation of AuNPs with a dense layer of
oligonucleotides (often referred to as ‘spherical nucleic acids’) provides nanoconjugates with
good solubility profile, low cytotoxicity and cell permeability.28 Therefore, the following
systems were prepared and studied (see Figure 5): AuNPs without receptor 2 or drug (AuNP-
DNA – as a control), with receptor 2 but no drug (AuNP-DNA-5), and with drug-loaded
receptor complexes 6 or 7 (AuNP-DNA-6 and AuNP-DNA-7).
HO
R
O
O
O
P
P
FAM
O
FAM
O
O
N
N
N
N
Zn
Zn
N
N
O
(CH2)2
N
S
S
S
S
AuNP
AuNP-DNA-6
AuNP-DNA-7
; R = CH3
; R = CH2CH2CH2NH2
Figure 5: Schematic representation of nanoconjugates AuNP-DNA-6 and AuNP-DNA-7.
The AuNPs were synthesized following previously reported procedures.29 Briefly, they were
grown through the reduction of HAuCl4 with sodium citrate and tannic acid to yield quasi-
spherical, narrowly-dispersed AuNPs with an average core diameter of 10.59 ± 1.8 nm. The
characterization of the AuNPs was carried out through UV-Vis spectroscopy, dynamic light
scattering (DLS), and transmission electron microscopy (TEM) (Figure S32 and Table S1).30
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Three different methods were studied to passivate the AuNPs with thiolated fluorescein-
labelled oligonucleotides and the receptor-drug 6: (i) by first preparing AuNP-DNA and then
adding 6 to the nanoparticles; (ii) by adding FAM-DNA-SH and 6 at the same time to the
unfunctionalized AuNPs; (iii) by first adding 6 to the AuNPs and then the FAM-DNA-SH.
The changes observed in the hydrodynamic diameter (dh) and zeta potential (ζ) of the NPs
after functionalization (see Table S3) suggest that all three methods yield AuNPs passivated
with both the oligonucleotides and receptor-drug conjugate 6. However, a higher
oligonucleotide loading – which, in turn, increases the stability and biocompatibility of the
systems – was observed by adding the FAM-DNA-SH and receptor-drug complex together
– i.e. second method described above. The oligonucleotide loading was estimated by treating
the corresponding DNA-passivated AuNPs with a large excess of mercaptoethanol following
reported protocols.31 Incubation with mercaptoethanol displaces the fluorescently-labelled
oligonucleotides from the AuNP surface, allowing the number of oligonucleotides attached
per AuNP to be calculated.
Having established the best synthetic method, samples of AuNP-DNA-5 and AuNP-DNA-7
(in addition to AuNP-DNA-6 as described above) were prepared and fully characterised by
UV-Vis and fluorescence spectroscopy, DLS, TEM and inductively coupled plasma mass
spectrometry (ICP-MS). The coating of AuNPs with FAM-DNA-SH to yield AuNP-DNA
induces a change dh from 21.4 ± 0.2 nm to 45.7 ± 0.4 nm, and a change ζ from -34.8 ± 1.2
mv to -81.2 ± 0.9 mV. Addition of the complexes (6 and 7) makes ζ less negative which is
consistent with less DNA being present on the surface of the AuNPs (see Table S3 in the
ESI). The largest difference is observed for AuNP-DNA-7 (with ζ = -25.4 ± 1.6 cf. -81.2 ±
0.9 for AuNP-DNA) which can be attributed to alendronate’s positively charged alkyl
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ammonium group. Additionally, there were changes in the UV-Vis absorbance of the
nanoparticles before and after functionalisation, which reflects the changes of optical
properties of AuNPs caused by the coating process (Figure 6a).30 The TEM images showed
that the spherical morphology of the AuNPs is preserved after the formation of the AuNP-
DNA-6 and AuNP-DNA-7 (Figure 6c and 6d).
Figure 6: (a) UV-Vis spectra of AuNPs, AuNP-DNA, AuNP-DNA-5, AuNP-DNA-6 and AuNP-
DNA-7; (b) Size distribution of unfunctionalised AuNPs; and TEM of images of (c) AuNP-DNA-6
and (d) AuNP-DNA-7.
Subsequently, the number of FAM-DNA molecules attached to the AuNPs was determined
by fluorescence spectroscopy (as described above using mercaptoethanol) while the number
of attached host-guest complexes was indirectly determined by quantifying the amount of Zn
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via ICP-MS (see Table 1). These measurements indicate that there are 69 and 79 molecules
of etidronate and alendronate per AuNP in AuNP-DNA-6 and AuNP-DNA-7 respectively.
Interestingly, in the AuNP-DNA-5, AuNP-DNA-6 and AuNP-DNA-7 samples, a higher
oligonucleotide loading was observed in comparison with AuNP-DNA sample. This effect
could be attributed to the stabilization of the negative charge of oligonucleotides strands by
the presence of the complexes.
Table 1: Samples AuNP-DNA, AuNP-DNA-5, AuNP-DNA-6 and AuNP-DNA-7
AuNP-DNA AuNP-DNA-5
AuNP-DNA-6
AuNP-DNA-7
DNA-Concentration
DNA-molecules
0.42 µM
54
0.61 µM
78
0.64 µM
82
0.7 µM
90
Zn-Concentration
n/a
206.4 µg/L
(3.2 µM)
200
141.6 µg/L
(2.2 µM)
138
161.5 µg/L
(2.5 µM)
157
Zn-atoms
n/a
n/a
n/a
Drug-concentration
Drug-molecules
n/a
1.1 µM
69
1.2 µM
79
n/a
Note: The initial concentration of AuNPs was 12.1 nM. After addition of thiolated oligonucleotide and the
corresponding compounds, the final concentration of AuNPs was 7.8 nM.
Conclusions
In summary, we have shown that di-zinc(II) complex 2 binds bisphosphonates with high
affinity and good selectivity over many other physiologically-relevant anions. The X-ray
crystal structures of this receptor bound to alendronate and etidronate show for the first time
the exact binding mode of these oxoanions to a di-metallic receptor. We also show that by
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modifying the backbone of the receptor, it is possible to load the drug-loaded receptor onto
AuNPs that have also been passivated with oligonucleotides to make them water-soluble and
biocompatible. These new nanoconjugates are likely to be good vehicles for the delivery of
these drugs across the cell membrane. Future work will explore this possibility.
Experimental Section
General Information.
1H NMR, 13C NMR and 31P NMR spectra were recorded on either a Bruker Avance 400 MHz
Ultrashield NMR spectrometer or a Bruker Avance 500 MHz NMR spectrometer.
Electrospray ionisation mass spectra were obtained on a Bruker Daltonics Esquire 3000
spectrometer. The X-ray crystal structures where measured using an OD Xcalibur PX Ultra
Diffractometer (1.54184 Å). UV-Vis spectra were collected with a Perkin Elmer UV-Vis
Lambda 25 Spectrophotometer. Fluorescence measurements were made on a Cary Eclipse
Fluorescence Spectrophotometer. Hydrodynamic diameters and zeta potentials were
measured on a Malvern Zetasizer Nano ZS instrument. Nanoparticles images were taken
using a JEOL JEM-2100 Plus transmission electron microscope. All reagents were purchased
from commercial suppliers and used without further purification. Etidronate and alendronate
were purchased as etidronate disodium hydrate and alendronate sodium trihydrate,
respectively. The organic precursor d was prepared following a previously reported synthetic
procedure (Scheme S1).26 For numbering of the 1H NMR assignments shown below, please
see the corresponding spectra in the ESI.
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Synthesis of Compound 1: To a solution of d (0.815 g, 1.45 mmol) in methanol (20 mL)
benzaldehyde (0.177 mL, 1.74 mmol) was added dropwise. The reaction mixture was stirred
for 2 h at room temperature. NaBH4 (110g, 2.90 mmol) was then added to the reaction
mixture and after stirring it for 4 h, the solvent was removed under vacuum and the excess
NaBH4 neutralised with saturated NH4Cl aqueous solution (60 mL). The product was
extracted with dichloromethane and dried over Na2SO4 powder. After filtration, the solvent
was reduced under vacuum to yield a brown oil which was purified by silica column
chromatography with CHCl3/MeOH/Et3N (94:5:1) as eluent to afford ligand 1 as a yellow
oil. Yield: 87 % (0.819 g, 1.26 mmol). 1H NMR (400 MHz, CDCl3) δ 2.72 (t, J = 7.0 Hz, 2H,
H12), 2.82 (t, J = 7.0 Hz, 2H, H13), 3.74 (s, 2H, H14), 3.77 (s, 4H, H7), 3.85 (s, 8H, H6),
6.97, 7.02, 7.10, 7.22, 7.48, 7.54 (m, 18H, H of Ph, Ar and Py groups), 8.49 (s, 4H, H1),
13
10.89 (s, 1H, OH) ppm. C{1H} NMR (101 MHz, CDCl3) δ 35.5 (C12), 51.0 (C13), 54.0
(C14), 55.0 (C7), 60.0 (C6), 122.1, 123.1, 123.3, 124.2, 127.0, 128.2, 128.5, 129.5, 136.6,
140.2, 149.0, 154.4, 159.4 (C of ph, Ar and Py groups) ppm. High-resolution ES-MS for
C41H44N7O (calcd 650.3607): m/z (%) = 650.3625 (100) (M+H, 100; error 2.8 ppm).
Synthesis of Compound 2: To a mixture of 1 (0.465 g, 0.71 mmol) and zinc(II) acetate
dihydrate (0.313 mg, 1.43 mmol) methanol (30 mL) was added and left stirring for 2 h at
room temperature. NaBF4 (0.078g, 0.71 mmol) was subsequently added and the resulting
reaction mixture was stirred overnight. After this time, all the volatiles was removed under
reduce pressure. The remaining solid was washed three times with ethyl acetate (60 ml) and
the product was extracted with dichloromethane to afford compound 2 as a slightly yellow
solid. Yield: 90 % (0.630 g, 0.64 mmol). Elemental analysis (%) Calcd for
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C45H48BF4N7O5Zn2∙CH2Cl2 (1069.40 g·mol–1): C 51.66, H 4.71, N 9.17; Found: C 51.16, H
4.68, N 8.97. FT-IR (ATR) (cm–1)1604 (s), 1481 (m), 1414 (s), 1325 (w), 1295 (w), 1098
(m), 1053 (s), 1020 (s), 964 (w), 767 (m). 1H NMR (400 MHz, CDCl3) δ 2.02 (s, 6H, H20),
2.54 (m, 2H, H12), 2.81 (m, 2H, H13), 3.20 (d, J = 11.1 Hz, 2H, H6d), 3.32 (m, 4H, H7b,
NH), 3.50 (d, J = 16.1 Hz, 2H, H7a), 3.81 (d, J = 11.0 Hz, 2H, H6c), 3.95 (s, 2H, H14), 3.98
(d, J = 14.4 Hz, 2H, H6b), 4.51 (d, J = 14.4 Hz, 2H, H6a) 6.31 (d, J = 7.7 Hz, 2H, H4b), 6.49
(s, 2H, H10), 6.90 – 7.45 (m, 13H, H2b, H3b, H2a, H17, H18, H16, H4a), 7.83 (t, J = 8.5 Hz,
2H, H3a), 8,18 (d, J = 4.5 Hz, 2H, H1b), 8.83 (d, J = 4.5 Hz, 2H, H1a) ppm. 13C{1H} NMR
(101 MHz, CDCl3) δ 24.6 (C20), 33.4 (C12), 50.3 (C13), 52.7 (C14), 57.8 (C7), 60.1 (C6),
121.0, 122.7, 123.6, 123.9, 124.1, 125.8, 127.9, 128.8, 129.1, 131.2 (C4b, C4a, C2a, C2b,
C9, C15, C11, C16, C17, C18, C10), 137.7, 139.1 (C3a, C3b), 146.1, 148.1 (C1a, C1b),
154.6, 155.1 (C5a, C5b), 160.1 (C8), 178.8 (C19) ppm. ES-MS: m/z (%) = 872.2 (60) [M –
BF4 – CCH3]+.
Synthesis of Compound 3: To a solution of 2 (0.200 g, 0.20 mmol) in water (30 ml), a
solution of etidronate disodium hydrate (0.054 g, 0.20 mmol) in water (2 ml) was added
dropwise. The resulting reaction mixture was stirred overnight. After this time, the solvent
was reduced under vacuum to ca. 5 ml. At this point, a white precipitate formed which was
filtered and then washed with chloroform and acetone. Crystals of compound 3 were grown
from acetone at room temperature. Yield: 88 % (0.176 g, 0.20 mmol). Elemental analysis
(%) Calcd for C43H47N7O8P2Zn2∙2H2O (1018.61 g·mol–1): C 50.70, H 5.05, N 9.63; Found:
C 50.40, H 5.01, N 9.34. FT-IR (ATR) (cm–1)1608 (w), 1481 (w), 1442 (w), 1154 (m),
1053 (s), 1020 (s), 927 (w), 767 (w). 1H NMR (400 MHz, MeOD) δ 1.64 (t, J = 14.8 Hz, 3H),
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2.95–2.57 (m, 6H), 3.12 (m, 2H), 3.45 (m, 2H), 4.17–3.98 (m, 6H), 4.65 (m, 2H), 6.44–6.35
(m, 4H,), 7.41 (m, 13H), 7.88 (t, J = 7.6 Hz, 2H), 8.81 (m, 4H) ppm. 31P NMR (400 MHz,
MeOD) δ 23.4 (d, J = 36.5 Hz, 1P), 24.8 (d, J = 36.5 Hz, 1P) ppm. ES-MS: m/z (%) = 989.1
(20) [M–CH3+Na]+.
Synthesis of Compound 4: Compound 4 was synthesized using the same methodology
described for 3. Crystals of compound 4 were grown from methanol/toluene solution at room
temperature. Yield: 64 % (0.290 g, 0.26 mmol). Elemental analysis (%) Calcd for
C45H53BF4N8O8P2Zn2∙4H2O (1185.52 g·mol–1): C 45.59, H 5.19, N 9.45; Found: C 45.20, H
4.95, N 9.05. FT-IR (ATR) (cm–1)1608 (w), 1477 (w), 1442 (w), 1319(w), 1290 (w), 1078
(s), 1050 (s), 1020 (s), 933 (m), 763 (m). 1H NMR (400 MHz, MeOD) δ 1.87 (m, 3H), 2.30–
3.05 (m, 14H), 3.73–4.21 (m, 8H), 4.73 (m, 3H), 6.25–6.38 (m, 4H), 6.96–7.35 (m, 13H),
7.85 (m, 2H), 8.82–8.89 (m, 4H) ppm. 31P NMR (400 MHz, MeOD) δ 21.9 (d, J = 30.7 Hz,
1P), 22.9 (d, J = 31.1 Hz, 1P). ES-MS: m/z (%) = 1025.2 (15) [M–BF4]+.
Synthesis of Compound 5: To a solution of 2 (1.00 g, 1.01 mmol) in ethanol (10 ml) NaOH
(0.04 g, 4.04 mmol) was added and left stirring for one hour at room temperature.
Subsequently, an excess of CS2 (1 ml) was added to the mixture and a white precipitate
formed immediately. The reaction was left stirring for two more hours. After this time, the
solvent was removed under reduce pressure and the resulting crude solid dissolved in
acetone. The solution was then filtered and hexane added to the filtrate to yield a white
precipitated which was filtered and dried under vacuum. Yield: 71 % (0.777 g, 0.72 mmol).
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Elemental analysis (%) Calcd for C46H49BF4N7NaO5S2Zn2 (1084.61 g·mol–1): C 50.94, H
4.55, N 9.04; Found: C 50.41, H 4.54, N 8.80. FT-IR (ATR) (cm–1)1604 (s), 1578 (m),
1481 (m), 1418 (m), 1323, (w), 1295 (w), 1098 (m), 1057 (s), 1024 (s), 964 (w), 771 (m).
ES-MS: m/z (%) = 957.1 (25) [M–CH3–Na–BF4]+.
Synthesis of Compound 6: A solution of 5 in water (10 ml) was stirred for 30 minutes.
Subsequently, etidronate dissolved in 3 ml of water was added dropwise and the resulting
reaction mixture was left sitting for 4 hours. After this time, the solvent was reduced to 3 ml
and a white precipitate formed which was filtered and washed with acetone, diethyl ether and
hexane. Yield: 90 % (0.359 g, 0.33 mmol). Elemental analysis (%) Calcd for
C44H45N7NaO8P2S2Zn2∙2H2O (1115.73 g·mol–1): C 47.37, H 4.43, N 8.79; Found: C 47.20,
H 4.15, N 8.55. FT-IR (ATR) (cm–1) 1608 (w), 1477 (w), 1444 (w), 1318 (w), 1297 (w),
1102 (s), 1053 (s), 1024 (s), 983 (m), 919 (w), 767 (w). 31P NMR (400 MHz, MeOD) δ 22.1
(br s, 1P), 24.9 (br s, 1P) ppm. ES-MS: m/z (%) = 1078.1 (80) [M+H]+.
Synthesis of Compound 7: Compound 7 was synthesized using the same methodology
described for 6. Yield: 87 % (0.777 g, 0.72 mmol). Elemental analysis (%) Calcd for
C46H51BF4N8NaO8P2S2Zn2∙5H2O (1300.65 g·mol–1): C 42.48, H 4.73, N 8.62; Found: C
42.18, H 4.60, N 8.33. FT-IR (ATR) (cm–1) 1604 (w), 1477 (w), 1436 (w), 1319 (w), 1294
(w), 1154 (m), 1098 (s), 1072 (s), 1053 (s), 1020 (s), 957 (s), 919 (m), 886 (w), 767 (w). 31P
NMR (400 MHz, MeOD) δ 22.9 (d, J = 31.8 Hz, 1P), 22.1 (d, J = 30.0 Hz, 1P) ppm. ES-
MS: m/z (%) = 1122.1 (5) [M–BF4]+.
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Indicator displacement assays (IDAs). All samples were freshly prepared in 10 mM
HEPES buffer (pH 7.1) prior to measurements. All measurements were repeated 3 times. For
optical tests, 1 ml of 2∙PV (50 μM) was mixed with 5 μL (1 eq.) or 50 μL (10 eq.) of the
corresponding anion (10 mM) in 10 mM HEPES solution at pH 7.4 at room temperature. The
mixture was stirred gently and then left to stand for 10 min. To obtain the UV-Vis spectra,
700 µL 2∙PV were added to a UV-Vis cuvette and mixed with one or ten molar equivalent of
each anion under study in a buffer solution (10 mM HEPES at pH = 7.4) at room temperature.
To obtain Ka between pyrocatechol violet (PV) and compound 2, a solution of 50 µM PV
and 1.25 mM 2 were prepared. A series of solutions were prepared with increasing
concentrations of 2, keeping the indicator concentration constant. The solutions were
equilibrate for 10 min before measuring the absorbance at 445 nm. The obtained data was
fitted by a Benesi-Hildebrand plot. To obtain Ka between 2 with the etidronate and
alendronate, a fresh solution of 50 µM 2∙PV and 1 mM etidronate and 1 mM alendronate
were prepared. A series of solutions were prepared with increasing concentrations of
etidronate or alendronate, keeping the 2∙PV concentration constant. The association constant
were obtained based on the previously described equilibrium for competition assays.32
Synthesis of Gold Nanoparticles via a Seeded Growth Method. AuNPs of different sizes
were synthesized following protocols reported from Puntes et al.33 Sodium citrate (25 ml, 2.2
mM), tannic acid (17 µL, 2.5 mM) and potassium carbonate (170 µL, 150 mM) was heated
in a 50 mL three-necked round-bottom flask under vigorous stirring. When the temperature
reached 70 °C, HAuCl4 (0.41 mL, 10 mM) was injected, resulting in a color change from
colorless to pink. Stirring at 70 °C for a further 10 minutes generated a solution of seed
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AuNPs. To grow larger NPs, the seed solution was immediately diluted by removing 9.1 mL
of the solution and replacing with sodium citrate (9.1 mL, 2.2 mM). When the temperature
reached 70 °C again, two injections of HAuCl4 (0.2 mL, 10 mM) on a time interval of 10
minutes were done. This growing process (sample dilution followed by the injection of
HAuCl4) was followed by UV-Vis spectroscopy and was repeated until the nanoparticles
reached the desired size.
Functionalization of Gold Nanoparticles. Au NPs were functionalized with FAM-DNA-
SH and the corresponding complexes (i.e. 5, 6 or 7) based on general methodologies
previously described in the literature.34,35 FAM-TT-GGG-TTA-GGG-TTA-GGG-TTA-
GGG-TT-(CH2)6-SH (FAM-DNA-SH) oligonucleotide was selected with a thiol modifier on
the 3’ end and a fluorescein (as an optical label) amidite group on the 5’ end. Before use,
tris(2-carboxyethyl)phosphine) (TCEP) was added to FAM-DNA-SH in a 100x excess and
the mixture was shaken for 1 hr at room temperature. Final concentrations of TCEP and
FAM-DNA-SH were 5 mM and 50 μM respectively. 1 wt% Tween 20 (68.7 μL) was added
to the AuNPs (12.1 nM, 6.87 mL) and the mixture was stirred for 1 min. FAM-DNA-SH (50
μM) and the corresponding bimetallic zinc complex (500 μM) was added at a volume that
allowed for 90 and 350 oligonucleotide strands and complex per AuNP. NaCl (5 M) was then
added until its final concentration in the mixture reached 800 mM. The solution was
incubated for 1 h at 25 °C with gentle agitation and then centrifuged at 30,000 rpm for 15
min at 15 °C. The supernatant was removed and the NPs were redispersed in Milli-Q water.
This centrifugation process was repeated at least 3 times to ensure thorough washing of the
AuNPs.
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Determination of oligonucleotide loading. 200 µL of the corresponding AuNP sample (i.e.
AuNP-DNA, AuNP-DNA-5, AuNP-DNA-6 and AuNP-DNA-7) were dispersed in PBS
(66.6 µL) and mercaptoethanol (533.3 µL). The final concentration of mercaptoethanol was
30 mM. After stirring the reaction mixture at 25 °C for 48 h, it was centrifuged at 15,000
rpm for 15 min and the supernatant (containing the displaced oligonucleotides) was
separated. The fluorescence of the FAM-DNA-SH was measured and compared to a standard
curve; samples were excited at 495 nm and were scanned in the range of 500 to 650 nm. The
concentration of fluorescent oligonucleotides was divided by the initial concentration of NPs
to give the number of oligonucleotides attached per nanoparticle.
Crystal data for 3: C43H47N7O8P2Zn2·12.5(H2O), M = 1207.75, triclinic, P-1 (no. 2), a =
13.6466(11), b = 14.0282(7), c = 16.2009(9) Å, α = 91.859(4), β = 96.471(5), γ =
117.397(7)°, V = 2723.5(3) Å3, Z = 2, Dc = 1.473 g cm–3, μ(Cu-Kα) = 2.328 mm–1, T = 173
K, colourless blocks, Agilent Xcalibur PX Ultra A diffractometer; 10425 independent
measured reflections (Rint = 0.0459), F2 refinement,36,37 R1(obs) = 0.0465, wR2(all) = 0.1214,
7550 independent observed absorption-corrected reflections [|Fo| > 4σ(|Fo|), completeness to
θfull(67.7°) = 98.3%], 568 parameters. CCDC 1984670. See ESI for further discussion about
this X-ray crystal structure.
Crystal data for 4: [C45H53N8O8P2Zn2](BF4)·0.5(C7H8)·8(H2O)·1.5(CH4O), M = 1351.70,
triclinic, P-1 (no. 2), a = 12.3489(3), b = 13.7028(5), c = 18.1556(8) Å, α = 90.414(3), β =
93.300(3), γ = 100.305(3)°, V = 3017.13(18) Å3, Z = 2, Dc = 1.488 g cm–3, μ(Cu-Kα) = 2.229
mm–1, T = 173 K, colourless tabular needles, Agilent Xcalibur PX Ultra A diffractometer;
11520 independent measured reflections (Rint = 0.0349), F2 refinement,36,37 R1(obs) = 0.0567,
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wR2(all) = 0.1796, 8585 independent observed absorption-corrected reflections [|Fo| >
4σ(|Fo|), completeness to θfull(67.7°) = 98.4%], 757 parameters. CCDC 1984671. See ESI for
further discussion about this X-ray crystal structure.
Acknowledgements
A.T.-H. acknowledges Mexico’s Consejo Nacional de Ciencia y Tecnología (CONACyT)
for his postdoctoral fellowship (grant number 298826). T.G.C. acknowledges UK’s
Engineering and Physical Sciences Research Council (EPSRC) for her PhD Studentship
(grant number EP/L016737/1).
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Graphical Abstract
DOI: 10.1039/D0DT00930J
Molecular recognition of bisphosphonate drugs by a di-zinc(II) receptor in solution and on
gold nanoparticles is reported.
HO
R
O
O
O
P
P
FAM
O
FAM
O
O
N
N
N
N
Zn
Zn
N
N
O
(CH2)2
N
S
S
S
S
AuNP