Fluorescent Arylphosphonic Acids: Synergic Interactions between Bone and the Fluorescent Core

Article abstract of DOI:10.1002/chem.202001613

Herein, we report the third generation of fluorescent probes (arylphosphonic acids) to target calcifications, particularly hydroxyapatite (HAP). In this study, we use highly conjugated porphyrin-based arylphosphonic acids and their diesters, namely 5,10,1

Full text of DOI:10.1002/chem.202001613

Accepted Article
Accepted Article
Title: Fluorescent arylphosphonic acids: synergic interactions between
bone and fluorescent core
Authors: Gündoğ Yücesan, Yunus Zorlu, Connor Brown, Claudia Keil,
Menaf M Ayhan, Hajo Haase, Richard B Thompson, and Imre
Lengyel
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To be cited as: Chem. Eur. J. 10.1002/chem.202001613
Link to VoR: https://doi.org/10.1002/chem.202001613
01/2020

10.1002/chem.202001613
Chemistry - A European Journal
RESEARCH ARTICLE
Fluorescent arylphosphonic acids: synergic interactions between
bone and fluorescent core.
Yunus Zorlu[a], Connor Brown[b], Claudia Keil[c], M. Menaf Ayhan[a], Hajo Haase[c],Richard B. Thompson[d],
Imre Lengyel[b], Gündoğ Yücesan*[c]
[a]
[b]
[c]
[d]
Dr. Yunus Zorlu, Dr. M. Menaf Ayhan,
Department of Chemistry, Faculty of Science
Gebze Technical University
41400, Gebze-Kocaeli
Dr. Connor Brown, Dr. Imre Lengyel
Wellcome-Wolfson Institute for Experimental Medicine,
School of Medicine, Dentistry and Biomedical Science,
Queen’s University Belfast, Belfast BT9 7BL, UK.
Dr. Gundog Yucesan, Dr. Claudia Keil, Prof. Dr. Hajo Haase
Technische Universität Berlin, Chair of Food Chemistry and Toxicology,
Straße des 17. Juni 135, 10623 Berlin, Germany
E-mail: yuecesan@tu-berlin.de
Dr. Richard B. Thompson
Department of Biochemistry and Molecular Biology
University of Maryland School of Medicine, Baltimore, Maryland 21201 USA
Supporting information for this article is given via a link at the end of the document.
Abstract: Herein, we report the third generation of fluorescent probes
(arylphosphonic acids) to target calcifications, particularly
hydroxyapatite (HAP). In this study, we use highly conjugated
porphyrin-based arylphosphonic acids and their diesters, namely
5,10,15,20-Tetrakis[m-(diethoxyphosphoryl)phenyl] porphyrin (m-
H8TPPA-OEt8), 5,10,15,20-Tetrakis [m-phenylphosphonic acid]
porphyrin (m-H8TPPA), in comparison with their positional isomers
5,10,15,20-Tetrakis[p-(diisopropoxyphosphoryl)phenyl] porphyrin (p-
H8TPPA-iPr8), 5,10,15,20-Tetrakis [p-phenylphosphonic acid]
porphyrin (p-H8TPPA) which have phosphonic acid units bonded to
sp2 carbons of the fluorescent core. The conjugation of the fluorescent
core is thus extended to the (HAP) via the sp2 bonded -PO3H2 units,
which generates increased fluorescence upon HAP binding. The
resulting fluorescent probes are highly sensitive towards the HAP in
rat bone sections. The designed probes are readily taken up by cells.
Due to the lower reported toxicity of (p-H8TPPA), these probes could
find applications in monitoring bone resorption or adsorption, or
imaging vascular or soft tissue calcifications for breast cancer
diagnosis etc.
can lead to uncontrolled access of extracellular materials or
prevent the of passive diffusion of molecules between cells and
their environment and likely cell death. [6] In addition, minerals
like HAP can facilitate the retention of molecules by providing a
binding surface for the pathological accumulation of molecules.
[7a] Detecting and monitoring increased calcification can be a
valuable early indication of breast cancer. [8] Conversely,
monitoring the decrease in mineralization can be useful for
assessing osteoporosis. [9] In addition, evidence is emerging that
HAP deposition may play a role in development and progression
of age-related macular degeneration in the retina and
neurodegeneration in the brain. [7] Therefore, monitoring
calcification in health and disease is critical for diagnosis,
monitoring progression and treatment.
There are several methods used to monitor calcification, some
applicable in vivo while others only work in vitro. [10, 11] A number
of radiological techniques have the potential to detect calcification
using radiography, [12, 13] fluoroscopy, [13] conventional
computed tomography (CT), [14] electron-beam tomography
(EBT), [15, 16] multi-detector CT, [17, 18] intravascular
ultrasound, [19, 20] magnetic resonance imaging (MRI), [21] and
transthoracic and transesophageal echocardiography. [22]
However, most current methods cannot resolve smaller than
millimeter sized mineralization and are usually invasive, costly
and not well tolerated. [23] In vitro, the resolution can be
significantly increased: The classic Von Kossa histochemical
method relies on silver precipitation at sites of phosphate
deposition [24], while Alizarin Red S, a colorimetric label, is widely
used to detect calcium deposition. [25] In combination these are
effective, inexpensive and widely used methods to detect calcium
Introduction
Dysregulation of calcium and phosphate homeostasis often leads
to the pathological deposition of minerals, such as calcium
carbonate (CC), calcium oxalate (CO), calcium phosphate (CP),
calcium pyrophosphate (CPP), and hydroxyapatite (HAP) in many
soft tissues, [1, 2] such as those in the brain, [3] eye, [4] kidney
[5] and skin. [2, 6] The pathological deposition of these minerals
causes these tissues to become inflexible or even brittle, which
1
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10.1002/chem.202001613
Chemistry - A European Journal
RESEARCH ARTICLE
phosphate mineralization in fixed cells in culture or postmortem
tissue sections, but they are not very sensitive and subcellular
resolution is limited. Fluorescent probes broadly offer better
sensitivity than colorimetric ones, but existing stains such as
Alizarin Red S, Xylenol Orange, or uranium have clear drawbacks
such as modest selectivity among mineral phases, pH
dependence, overlap of their fluorescence with tissue
autofluorescence. Frangioni and Kovar’s research groups
developed the second generation fluorescent probes coupling
HAP-binding moieties such as bisphosphonate or tetracycline
with red and near-infrared penta- or hepta-methine cyanine
fluorescent moieties; the longer wavelength excitation and
emission of these fluorophores substantially reduces scattering
and background fluorescence. [26-28] These probes were
sensitive, selective, and usable in vivo in animal models, but are
costly and by comparison with other probes that exhibit significant
or unique changes in fluorescence when binding to particular
targets such as DNSA (dansylamide) with carbonic
inasmuch as p-H8TPPA exhibited no toxicity for an intestinal cell
line at high concentrations. [34, 35] One of the unexplored
properties of phosphonic acid metal binding groups is their
potential role in imaging metal deposits in living systems. Our and
Demadis’ recent work, and Martell’s early work from the 1970s all
indicate that phosphonates exhibit high affinity towards alkali and
alkaline earth metal ions, as well as divalent transition metal ions.
[35-39] In addition, the pKa2 of phenylphosphonic acid (PhPO3H2)
is 7.44, giving PhPO32- a -2 charge under physiological pH. [40]
Therefore, the use of phosphonic acid metal binding unit(s) to
target biologically significant divalent metal ions offers highly
sensitive and pH independent metal probes working near
physiological pH. Bisphosphonates have been previously
attached to cyanine (Osteosense®) and fluorescein moieties with
long aliphatic hydrocarbon side chains to label target calcification
with green or near infrared fluorescence. [39, 41] The presence of
sp3 bonds in the aliphatic linker chain in these examples between
the phosphonic acid and the fluorescent core in these examples
merely used bisphosphonates as recognition moieties and
minimized any electronic interactions between the fluorescent
core and the phosphonic acid metal binding unit. Therefore, such
fluorescent labels provide little if any change in fluorescence
intensity upon HAP binding. [39, 41, 42, 43] By comparison, the
direct conjugation of phosphonic acid metal binding group with an
sp2 carbon atom of the fluorescent core extends the conjugation
of the fluorescent core up to the coordinated metal ion. Thus, it
offers the prospect of the metal ion binding the phosphonate and
affecting the fluorescence in useful ways. For instance, such
binding and recognition of a metal ion might cause useful changes
in fluorescence that might help discriminate metal-bound
fluorophores from unbound or non-specifically bound
fluorophores. Such fluorescent phosphonates having direct sp2
conjugation with the fluorescent core haven’t been reported in the
literature, perhaps due to synthetic difficulties and the high energy
requirement to form P-C bonds. Our recent research efforts with
arylphosphonic acid synthesis have generated novel strategies to
introduce phosphonic acid around aromatic scaffolds, via Suzuki
Cross Coupling or the Pd-catalyzed Arbuzov reaction. [44-47]
Scheme 1. The fluorescent probes used in this study A. 5,10,15,20-Tetrakis[p-
(diisopropoxyphosphoryl)phenyl] porphyrin (p-H8TPPA-iPr8) B. 5,10,15,20-
Tetrakis [p-phenylphosphonic acid] porphyrin (p-H8TPPA) C. 5,10,15,20-
Tetrakis[m-(diethoxyphosphoryl)phenyl] porphyrin (m-H8TPPA-OEt8) D.
5,10,15,20-Tetrakis [m-phenylphosphonic acid] porphyrin (m-H8TPPA).
For this study, as seen in Scheme 1, we have synthesized 4
fluorescent probes. Two of them incorporate phenylphosphonic
acid as the HAP binding unit with highly conjugated porphyrin
cores, namely 5,10,15,20-tetrakis[p-phenylphosphonic acid]
anhydrase, [29] the triphenyl methane dye Auramine O with LADH
(horse liver alcohol dehydrogenase) with, [30] or ethidium
bromide with DNA, [31] their fluorescence is insensitive to
whether they have bound to the (calcified) target or
nonspecifically to something else. To address this latter issue,
we sought to develop the third generation of fluorescent probes
of calcification bearing phosphonic acid metal binding units which
are bonded to one of the sp2 carbon atoms of the fluorescent
cores, with the goal of differentially perturbing the fluorescence of
the probe when bound to calcified substrates, and thereby
providing additional information. The use of such fluorophores
would be important with respect to generating synergic
interactions between the fluorescent core and the d orbitals of the
target divalent metal ions.
porphyrin
(p-H8TPPA)
and
5,10,15,20-tetrakis[m-
phenylphosphonic acid] porphyrin (m-H8TPPA) (Figure 1; please
see the supporting information for synthesis details). p-H8TPPA
has four para-positioned PPA units promoting direct conjugation
between the HAP and the fluorescent core. On the other hand, m-
H8TPPA has four meta-positioned PPA units creating a more
protective environment between the fluorescent porphyrin core
and the HAP. We then explored their potential for binding
hydroxyapatite. As a control group we used the isopropyldiester
and ethyldiester analogues of p-H8TPPA and m-H8TPPA, namely
p-H8TPPA-iPr8 and m-H8TPPA-OEt8 (See supporting information
In this sense, phosphonic acid metal binding group has recently
attracted significant attention in diverse scientific fields ranging
from material science to medicine [32, 33]. Our initial toxicity
studies with aromatic phosphonic acids and phosphonate metal-
organic solids were promising for their in vivo applications
2
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10.1002/chem.202001613
Chemistry - A European Journal
RESEARCH ARTICLE
for detailed synthesis). A previous synthesis of p-H8TPPA relied
on the Ni-catalyzed Arbuzov reaction. [48] Therefore, p-H8TPPA
that is synthesized using this route usually has Ni bound to the
nitrogens in the porphyrin core. In this study, we have used an
alternative method using Pd-catalyzed Arbuzov reaction to obtain
metal free p-H8TPPA. (please see the supporting information for
synthesis details, NMR, FT-IR spectra, and the crystallographic
tables).
Results and Discussion
To probe their HAP binding properties in a biological matrix, we
have used cross sectioned mouse ribs as an example for naturally
occurring HAP. The sections were incubated with p-H8TPPA, m-
H8TPPA and p-H8TPPA-iPr8 at concentrations ranging from 0.01
to 1.0 mg/mL in HEPES buffer for varying times at room
temperature, then imaged by fluorescence microscopy (details
below in the figure legends), to assess HAP binding capability.
Ribs incubated with 1 mg/mL p-H8TPPA showed a greater
fluorescence intensity than both 0.1 mg/mL and 0.01 mg/mL
(Supp. Fig. 17A-C, respectively), whilst still showing clear
fluorescence signal at the lowest concentration of 0.1 mg/mL used
here.
This indicates that the dye binding to HAP is concentration
dependent. When the mouse ribs were incubated with 1 mg/mL
p-H8TPPA in HEPES for differing times from 120 to 10 mins (Supp.
Fig. 18A-E), there was a clear decrease in fluorescence intensity
but even the 10 min short incubation yielded an appreciable
fluorescence compared to the negative control (Supp. Fig. 18E
compared to Supp. Fig. 18F). When ribs were incubated with p-
H8TPPA diluted to 1 mg/mL in different buffers, there was no clear
difference between the fluorescence intensity of HEPES, PBS,
TBS (Supp. Fig. 19A-C, respectively). However, when p-H8TPPA
was diluted in dH2O (Supp. Fig. 19D), there was no fluorescent
signal observed. Sections were re-imaged at several time points
after the original microscopy sessions to observe the stability of
labeling. There was a noticeable decrease in fluorescence
intensity in all of the solvents although signal diminished faster in
HEPES buffer than in PBS or TBS. (Supp. Fig. 20) At the two
week time point there was still a weak fluorescent signal observed
for p-H8TPPA diluted in PBS, but not for the other buffers (Supp.
Fig. 20N compared to Supp. Fig. 20M, O and P). No signal was
associated with incubating the ribs with the solvents as a negative
control (Supp. Fig. 20Q-T)
Figure 1. Mouse ribs incubated with either p-H8TPPA (λex em
/
595/613 nm;
exposure time – 200 ms) or m-H8TPPA (λex em 578/603 nm; exposure time –
/
100 ms) diluted to 1 mg/mL in HEPES (A-E or F-J, respectively) and counter-
stained with DAPI (λex/em 359/461 nm; exposure time – 200 or 100 ms,
respectively). Images were captured on the day of staining (A and F), as well as
after 4 days (B and G), 7 days (C and H) and 14 days (D and I). A negative
control of mouse ribs incubated with each buffer alone is also included (E and
J, respectively). Scalebar = 100 µm.
The phosphonic acid moieties in m-H8TPPA are more protected
compared to the para-positioned p-H8TPPA. When mouse ribs
were incubated with different concentrations of m-H8TPPA, (1 to
0.01 mg/mL in HEPES buffer) we found little to no difference in
the fluorescence intensities (Supp. Fig. 21A-C, respectively).
When the mouse ribs were incubated with 1 mg/mL m-H8TPPA in
HEPES for differing times, 10 to 120 mins, there were no
appreciable difference in fluorescence intensities at the different
incubation times (Supp. Fig. 22A-E, respectively) while the
fluorescent intensities were clearly distinguishable from HEPES
alone (Supp. Fig. 22F). Imaging of the sections were repeated two
weeks following the original microscopy and we found no
noticeable decrease in fluorescence intensity between ribs
incubated with 1 mg/mL m-H8TPPA in HEPES buffer and imaged
on the day of staining compared with images taken 4, 7 and 14
days later (Fig. 1F-I, respectively). This is a noticeable difference
compared to p-H8TTPA, with a less protected structure, where
there is a loss of fluorescent signal with increasing time after initial
staining (Fig. 1A-D, respectively). This could be associated with
the more protected HAP phosphonic acid interaction in the meta
position limiting the interactions with the surrounding molecules
buffers etc. Again, no signal was associated with incubating the
ribs with HEPES only as a negative control (Fig. 1E and I,
respectively).
3
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RESEARCH ARTICLE
As seen in Figure 2, the absorbance peak (Fig. 2A) of p-H8TTPA
in PBS, TBS and HEPES with the addition of HAP corresponds
with the absorbance of the dye in d-DMSO (Supp. Fig. 16). There
is a noticeable shift in the absorbance peak maximum when
diluted in dH2O, from 415 nm to 435 nm, which suggests a shift in
the fluorescent properties of the dye due to protonation. This is
also reflected in the maximal peak of the fluorescence intensity
(Fig. 2B), with a shift from λem 646-648 nm for PBS, TBS and
HEPES to λem 675 nm when diluted in dH2O. The fluorescence
intensity of p-H8TPPA is also substantially increased by 1.8-, 2.0-,
2.0-, and 1.6-fold upon HAP binding of the fluorescent probe p-
H8TPPA in PBS, TBS, HEPES and dH2O, respectively (Fig. 2B,
solid vs. dashed lines). Previously reported bisphosphonate
fluorophores had sp3 aliphatic carbons between the fluorescent
core and HAP binding, therefore, such systems merely functioned
as fluorescent labels with no change in emission due to HAP
binding since they lacked synergistic interaction between the HAP
and the fluorescent core. Phosphonic acids that have direct sp2
bonds to the fluorescent core could extend the conjugation of the
fluorescent core to the HAP and thereby could initiate changes in
the ground and excited states resulting in quenching or enhancing
the fluorescence emission. As seen in Figure 2 (B), upon binding
of HAP, p-H8TPPA produces increased fluorescence supporting
this hypothesis; p-H8TPPA is thus the first example of a single sp2
bonded phosphonic acid unit targeting HAP in the literature.
cellular uptake. In addition, albumin is a regular component of
culture media.
Figure 3. Fluorescence spectra of THP-1 monocytes cells incubated with p-
H8TPPA.
As seen in Figure 3, the fluorescence spectra of p-H8TPPA-
loaded THP-1 cells very much resembles the characteristics of
the sensor diluted in HEPES-buffer (see Fig. 1 A, F). Thus,
beyond the low toxicity p-H8TPPA provides two more positives -
cell permeability and cellular retention- desirable for imaging
applications. p-H8TPPA-iPr8 was much less efficient in labeling
the THP-1 cells (see Suppl. Fig 23). We hypothesize that the
phenylphosphonic acid porphyrins, being somewhat amphipathic,
were able to penetrate the cell membrane, whereas the more
nonpolar esters were not as efficient.
Conclusion
Herein, we report next generation fluorescent probes to target
calcifications namely p-H8TPPA, m-H8TPPA, p-H8TPPA-iPr8, m-
H8TPPA-OEt8, in which the phosphonic acid metal binding unit(s)
are exclusively bonded to sp2 carbon atoms of the fluorescent
core. Sp2 bonding of the metal binding unit to the fluorescent core
further extended the conjugation of the fluorescent core to the
HAP, leading to significant increase in fluorescence upon HAP
binding. We further used these fluorescent probes to target the
HAP in mouse ribs and observed their interaction with human
cells. Both m-H8TPPA and m-H8TPPA have shown HAP specific
binding in rat bone sections. The more protected metal binding
nature of the m-H8TPPA resulted in longer fluorescence period
compared to its positional isomer p-H8TPPA. As a control, we
have also synthesized phosphonatediesters of the synthesized
fluorescent probes, which showed no HAP binding. p-H8TPPA
can travel through the cellular membrane, whereas the presence
of bulky and hydrophobic isopropyl groups in p-H8TPPA-iPr8
hindered their passage through the cellular membrane. This is the
first study utilizing sp2 bonded phosphonic acids with the
fluorescent cores to target HAP mineralizations. We have shown
that compact fluorescent probes with phosphonic acid metal
binding group(s) may be used to monitor microcalcifications,
calcifications. In addition, their easy acceptance into the cellular
matrix indicate that they could be used to target organelle specific
calcifications such as mitochondrial calcifications. The reported
low toxicity of p-H8TPPA and the new synthetic methods indicate
that they can be used in targeting wide range of calcifications in
vivo. We are currently developing our library to target organelle
specific calcifications.
Figure 2. The absorbance (A) and fluorescence (B) spectra of 0.01 mg/mL p-
H8TPPA in the presence (dashed lines) and absence (solid lines) of HAP .The
dye was diluted to 0.01 mg/mL in PBS (pH 7.4, green), TBS (pH 7.4, orange),
HEPES (pH 7.4, purple) and dH2O with absorbance and fluorescence spectra
obtained using the Flexstation 3 microplate reader.
Comparable experiments with mouse rib sections were
conducted using the isopropyldiester forms p-H8TPPA-iPr8 and
m-H8TPPA-OEt8 (See scheme 1a and 1c) as stains. We found a
complete lack of HAP-associated staining with these compounds
suggesting that the interaction of phosphonates with HAP was
reduced by the isopropylester groups on the phosphonates,
preventing binding and interaction of the fluorescent porphyrin
core with the HAP. We also noted that the presence of
hydrophobic isopropyl groups dramatically reduced their
solubilities in the aqueous.
Aiming to use phenylphosphonic acid functionalized porphyrins in
in vivo applications, the negative charge on the deprotonated
phosphonate might be a handicap by limiting cell permeability.
Optional demasking of phosphonate esters in metabolic
2-
surroundings might occur, recreating the metal binding PhPO3
.
This possibility led us to examine p-H8TPPA and p-H8TPPA-iPr8
suitability for in vivo cellular experiments by testing their
permeability on proliferating human THP-1 monocytes. The cells
were probed with either p-H8TPPA or p-H8TPPA-iPr8 in a
HEPES-based buffer containing 0.3% bovine serum albumin.
Albumin binds a wide variety of hydrophobic ligands under
physiological conditions, so we assumed an improved
solubility/accessibility for the isopropyl diester molecule for
4
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10.1002/chem.202001613
Chemistry - A European Journal
RESEARCH ARTICLE
Note: The detailed synthesis, FT-IR, MALDI-MS, UV-Vis, Fluorescent
Spectra, 1H-NMR, 13C-NMR, 31P-NMR, More detailed images of rat bone
sections are available in the Supporting Information section.
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Gündoğ Yücesan would like to thank the DFG for funding his
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10.1002/chem.202001613
Chemistry - A European Journal
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Entry for the Table of Contents
Arylphosphonic acids are highly selective for bone matrix and calcifications providing extended conjugation of the fluorescent core to
the bone matrix generating specific synergic interactions and turn up fluorescence upon HAP binding. The TOC picture depicts the
selectivity of p-H8TPPA for rat bone sections under physiological pH.
Institute and/or researcher Twitter usernames: @gundogyucesan @DrYunusZorlu @Drusen_Lengyel @TUBerlin
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