Fluorescent Arylphosphonic Acids: Synergic Interactions between Bone and the Fluorescent Core [original]

Fluorescent Imaging |Hot Paper|

Fluorescent Arylphosphonic Acids:Synergic Interactions between

Bone and the Fluorescent Core

Yunus Zorlu,[a] Connor Brown,[b] ClaudiaKeil,[c] M. Menaf Ayhan,[a] Hajo Haase,[c]

Richard B. Thompson,[d] Imre Lengyel,[b] and Gendog

˘Yecesan*[c]

Abstract: Herein, we report the third generation of fluo-

rescent probes (arylphosphonic acids) to target calcifica-

tions, particularly hydroxyapatite (HAP). In this study,we

use highly conjugated porphyrin-based arylphosphonic

acids and their diesters, namely 5,10,15,20-tetrakis[m-(di-

ethoxyphosphoryl)phenyl]porphyrin (m-H8TPPA-OEt8)and

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]por-

phyrin (p-H8TPPA-iPr8)and 5,10,15,20-tetrakis [p-phenyl-

phosphonic acid]porphyrin (p-H8TPPA), which have phos-

phonic acid units bonded to sp2carbon atoms of the fluo-

rescent core. The conjugation of the fluorescent core is

thus extended to the (HAP) through sp2-bonded @PO3H2

units, which generatesincreased fluorescenceupon HAP

binding. The resultingfluorescent probes are highly sensi-

tive towards the HAP in rat bone sections. The designed

probes are readily taken up by cells. Due to the lower re-

ported toxicity of (p-H8TPPA), these probes could find ap-

plications in monitoring bone resorption or adsorption, or

imaging vascular or soft tissue calcifications for breast

cancer diagnosis etc.

Dysregulation of calcium and phosphate homeostasisoften

leads to the pathological deposition of minerals, such as calci-

um 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 min-

erals causes these tissues to becomeinflexible or even brittle,

which can lead to uncontrolled access of extracellular materials

or preventthe passive diffusion of molecules between cells

and their environment, likelyleadingtocell death.[6] In addi-

tion, minerals like HAP can facilitate the retention of molecules

by providing abinding surface for the pathologicalaccumula-

tion of molecules.[7a] Detecting and monitoring increased calci-

fication can be avaluable early indication of breast cancer.[8]

Conversely,monitoring the decrease in mineralization can be

usefulfor assessing osteoporosis.[9] In addition, evidenceis

emerging that HAP deposition mayplay arole in the develop-

ment and progression of age-related maculardegeneration in

the retina and neurodegenerationinthe brain.[7] Therefore,

monitoring calcification in health anddisease is critical for di-

agnosis, monitoring progression and treatment.

There are severalmethods 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] convention-

al computed tomography (CT),[14] electron-beam tomography

(EBT),[15,16] multi-detector CT,[17,18] intravascular ultrasound,[19,20]

magnetic resonanceimaging (MRI),[21] and transthoracic and

transesophageal echocardiography.[22] However,most current

methodscannot resolve smaller than millimeter sized mineral-

izationand are usually invasive, costly and not well tolerat-

ed.[23] In vitro, the resolution can be significantly increased:The

classicVon Kossa histochemical method relies on silver precipi-

tation at sites of phosphate deposition,[24] while Alizarin Red S,

acolorimetric label, is widely used to detect calcium deposi-

tion.[25] In combination these are effective, inexpensive and

widely used methods to detect calcium phosphate mineraliza-

tion in fixed cells in culture or postmortem tissue sections, but

they are not very sensitive and subcellular resolution is limited.

[a] Dr.Y.Zorlu, Dr.M.M.Ayhan

Department of Chemistry,Faculty of Science

Gebze Technical University, 41400, Gebze-Kocaeli (Turkey)

[b] C. Brown, Dr.I.Lengyel

Wellcome-WolfsonInstitute for Experimental Medicine

SchoolofMedicine, Dentistry and Biomedical Science

Queen’s UniversityBelfast, Belfast BT9 7BL (UK)

[c] Dr.C.Keil, Prof. Dr.H.Haase, Dr.G.Yecesan

Technische Universit-tBerlin, Chair of Food Chemistry and Toxicology

Straße des 17. Juni 135, 10623 Berlin (Germany)

E-mail:yuecesan@tu-berlin.de

[d] Dr.R.B.Thompson

Department of Biochemistry and Molecular Biology

University of Maryland School of Medicine

Baltimore, Maryland 21201 (USA)

Supporting information and the ORCID identification number(s) for the

author(s) of this articlecan be found under:

https://doi.org/10.1002/chem.202001613. It contains detailed synthetic pro-

cedures, FTIR, MALDI-MS, UV/Vis, fluorescent spectra, 1HNMR, 13CNMR, and

31PNMR. In addition,more detailed imagesofrat bone sections are

included.

T2020 The Authors. Published by Wiley-VCH Verlag GmbH&Co. KGaA.

This is an open access article under the terms of the Creative Commons At-

tributionNon-Commercial NoDerivs License, which permitsuse and distri-

bution in any medium, provided the original work is properly cited, the use

is non-commercial and no modifications or adaptations are made.

Chem. Eur.J.2020,26,11129 –11134 T2020 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim11129

Chemistry—A European Journal

Communication

doi.org/10.1002/chem.202001613

Fluorescent probes broadly offer better sensitivity than colori-

metric ones, but existing stains such as Alizarin Red S, Xylenol

Orange, or uraniumhavecleardrawbacks such as modestse-

lectivity among mineral phases, pH dependence, overlap of

their fluorescencewith tissue autofluorescence. Frangioni and

Kovar’s research groupsdeveloped the second generation

fluorescent probescoupling HAP-binding moieties such as bis-

phosphonate or tetracycline with red and near-infrared penta-

or hepta-methine cyaninefluorescent moieties;the longer

wavelength excitation and emission of these fluorophores sub-

stantially reduces scatteringand background fluorescence.[26–28]

These probes were sensitive, selective, and usable in vivo in

animal models, but are costly and by comparison with other

probesthat exhibit significant or unique changes in fluores-

cence when bindingtoparticulartargets such as DNSA

(dansylamide) with carbonic anhydrase,[29] the triphenyl meth-

ane dye Auramine Owith LADH (horse liveralcohol dehydro-

genase) with,[30] or ethidium bromide with DNA,[31] their fluo-

rescenceisinsensitivetowhether they have bound to the (cal-

cified) target or nonspecifically to something else. To address

this latter issue, we soughttodevelop the third generation of

fluorescent probes of calcification bearing phosphonic acid

metal binding units which are bondedtoone of the sp2

carbon atoms of the fluorescent cores,with the goal of differ-

entiallyperturbing the fluorescenceofthe probe when bound

to calcified substrates, and thereby providing additional infor-

mation. The use of such fluorophores would be important with

respecttogenerating synergic interactions between the fluo-

rescent core and the dorbitals of the target divalent metal

ions.

In this sense, the phosphonic acid metal binding group has

recently attracted significant attention in diversescientific

fields ranging from material science to medicine.[32,33] Our initial

toxicitystudies with aromatic phosphonic acids and phospho-

nate metal-organic solids were promising for their in vivo ap-

plications inasmuch as p-H8TPPA exhibited no toxicityfor an

intestinal cell line at high concentrations.[34,35] One of the unex-

plored properties of phosphonic acid metal binding groups is

their potentialrole 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

towardsalkali and alkaline earth metal ions, as well as divalent

transition-metal ions.[35–39] In addition, the pKa2of phenylphos-

phonic acid (PhPO3H2)is7.44, giving PhPO32@a@2charge

under physiological pH.[40] Therefore, the use of phosphonic

acid metal-binding unit(s)totarget biologically significant diva-

lent metal ions offers highly sensitive and pH-independent

metal probesworkingnear physiological pH. Bisphosphonates

have been previously attached to cyanine(OsteosenseS)and

fluoresceinmoieties with long aliphatic hydrocarbon side

chains to label target calcification with green or near infrared

fluorescence.[39,41] The presence of sp3bonds in the aliphatic

linker chain in these examples between the phosphonic acid

and the fluorescent core in these examples merely used bis-

phosphonates as recognition moieties and minimized any elec-

tronic interactions between the fluorescent core and the phos-

phonic acid metal binding unit. Therefore, such fluorescent

labels provide little if any change in fluorescence intensity

upon HAP binding.[39,41–43] By comparison, the direct conjuga-

tion 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 prospectofthe metal-ion binding the phospho-

nate and affecting the fluorescenceinuseful ways. For in-

stance,such binding and recognition of ametal ion might

cause useful changes in fluorescence that might help discrimi-

nate metal-bound fluorophores from unbound or non-specifi-

cally boundfluorophores. Such fluorescent phosphonates con-

tainingdirect sp2conjugation with the fluorescent core have

not been reported in the literature, perhaps due to synthetic

difficulties and the high energy requirement to form P@C

bonds. Our recent research effortswitharylphosphonic acid

synthesis have generated novel strategies to introduce phos-

phonic acid aroundaromatic scaffolds, by Suzuki cross cou-

pling or the d-catalyzed Arbuzov reaction.[44–47]

For this study,asseen in Scheme1,wehave synthesized 4

fluorescent probes. Twoofthem incorporate phenylphosphon-

ic acid as the HAP binding unit with highlyconjugated porphy-

rin cores, namely 5,10,15,20-tetrakis[p-phenylphosphonic acid]-

porphyrin (p-H8TPPA)and 5,10,15,20-tetrakis[m-phenylphos-

phonic acid]porphyrin (m-H8TPPA)(Figure 1; please see the

Supporting Information for synthesis details). p-H8TPPA has

four para-positioned PPAunits promoting direct conjugation

between the HAP and the fluorescent core. On the other hand,

m-H8TPPA has four meta-positioned PPAunits creating amore

protectiveenvironmentbetween the fluorescent porphyrin

core and the HAP.Wethen explored their potentialfor binding

hydroxyapatite. As acontrolgroup we used the isopropyldiest-

er and ethyldiester analoguesofp-H8TPPA and m-H8TPPA,

namely p-H8TPPA-iPr8and m-H8TPPA-OEt8(see the Supporting

Scheme1.The fluorescentprobes used in this study:A)5,10,15,20-Tetra-

kis[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).

Chem.Eur.J.2020,26,11129 –11134 www.chemeurj.org T2020 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim11130

Chemistry—A European Journal

Communication

doi.org/10.1002/chem.202001613

Information for detailed synthesis).Aprevious synthesis of p-

H8TPPA relied on the Ni-catalyzed Arbuzov reaction.[48] There-

fore, 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 methodusing Pd-catalyzedArbu-

zov reaction to obtain metal free p-H8TPPA.(please see the

Supporting Information for synthesis details, NMR, FTIR spec-

tra, and the crystallographic tables).

To probe their HAP binding properties in abiological matrix,

we have used cross sectioned mouse ribs as an example for

naturallyoccurring HAP.The sections were incubated with p-

H8TPPA, m-H8TPPAand p-H8TPPA-iPr8at concentrations rang-

ing from 0.01 to 1.0 mgmL@1in HEPES buffer for varying times

at room temperature, then imaged by fluorescencemicroscopy

(detailsbelow in the figure legends), to assess HAP binding ca-

pability. Ribs incubated with 1mgmL@1p-H8TPPA showed a

greater fluorescence intensity than both 0.1 mgmL@1and

0.01 mgmL@1(Figure S17A–C, respectively), whilst still showing

clear fluorescencesignal at the lowestconcentration of

0.1 mgmL@1used here.

This indicates that the dye binding to HAP is concentration

dependent. When the mouse ribs were incubated with

1mgmL@1p-H8TPPA in HEPES for differing times from 120 to

10 mins (Figure S18A–E), there was aclear decrease in fluores-

cence intensity but even the 10 min short incubation yielded

an appreciable fluorescencecompared to the negative control

(Figure S18Ecompared to Figure S18F). When ribs were incu-

bated with p-H8TPPA diluted to 1mgmL@1in different buffers,

there was no clear differencebetween the fluorescenceinten-

sity of HEPES, PBS, TBS (Figure S19A–C, respectively). However,

when p-H8TPPA was diluted in dH2O(Figure S19D), there was

no fluorescent signal observed. Sections were re-imaged at

severaltime points after the originalmicroscopy sessions to

observe the stability of labeling. There was anoticeable de-

crease in fluorescenceintensity in all of the solvents although

signaldiminished faster in HEPES buffer than in PBS or TBS

(Figure S20). At the two week time point there was still aweak

fluorescent signal observed for p-H8TPPA diluted in PBS, but

not for the otherbuffers (Figure S20N compared to Fig-

ure S20M, Oand P). No signal was associated with incubating

the ribs with the solvents as anegative control (Figure S20Q-T)

The phosphonicacid moieties in m-H8TPPA are more pro-

tected compared to the para-positioned p-H8TPPA.When

mouse ribs wereincubated with different concentrations of m-

H8TPPA,(1to0.01 mgmL@1in HEPES buffer) we found little to

no difference in the fluorescence intensities (Figure S21A–C,

respectively). When the mouseribs wereincubated with

1mgmL@1m-H8TPPA in HEPES for differing times, 10 to 120

mins, there werenoappreciable difference in fluorescencein-

tensities at the differentincubation times (FigureS22A–E, re-

spectively) while the fluorescent intensities were clearly distin-

guishable from HEPES alone(FigureS22F). Imaging of the sec-

tions were repeated two weeks following the original micros-

copy and we found no noticeable decrease in fluorescencein-

tensity between ribs incubated with 1mgmL@1m-H8TPPA in

HEPES buffer and imaged on the day of stainingcompared

with images taken 4, 7and 14 days later (Figure 1F–I, respec-

tively).This is anoticeabledifference compared to p-H8TTPA,

with alessprotected structure, where there is aloss of fluores-

cent signal with increasing time after initial staining (Fig-

ure 1A–D, respectively). Thiscould be associated with the

more protected HAP phosphonic acid interaction in the meta

positionlimiting the interactions with the surrounding mole-

cules buffers etc. Again,nosignal wasassociatedwith incubat-

ing the ribs with HEPES only as anegative control (Figure 1E

and I, respectively).

As seen in Figure 2, the absorbance peak (Figure 2A)ofp-

H8TTPA in PBS, TBS and HEPES with the addition of HAP corre-

Figure 1. Mouse ribs incubated with either p-H8TPPA (lex/em 595/613 nm;ex-

posure time @200 ms)orm-H8TPPA (lex/em 578/603 nm;exposuretime

@100 ms) diluted to 1mgmL@1in HEPES(A–EorF–J, respectively) and coun-

ter-stained with DAPI (lex/em 359/461 nm;exposuretime: @200 or 100 ms,

respectively). Imageswere captured on the day of staining (A and F), as well

as after 4days (B and G), 7days (C and H), and 14 days(Dand I). Anegative

controlofmouse ribs incubated with each bufferalone is also included (E

and J, respectively). Scale bar=100 mm.

Chem. Eur.J.2020,26,11129–11134 www.chemeurj.org T2020 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim11131

Chemistry—A European Journal

Communication

doi.org/10.1002/chem.202001613

sponds with the absorbance of the dye in d-DMSO (Fig-

ure S16). There is anoticeable shift in the absorbance peak

maximum when diluted in dH2O, from 415 nm to 435 nm,

which suggests ashift in the fluorescent properties of the dye

due to protonation. This is also reflected in the maximal peak

of the fluorescenceintensity (Figure 2B), with ashift from lem

646–648nmfor PBS,TBS and HEPES to lem 675 nm when dilut-

ed in dH2O. The fluorescenceintensity of p-H8TPPA is also sub-

stantially 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 (Figure 2B,solidvs. dashed lines). Pre-

viously reported bisphosphonate fluorophores hadsp

3aliphat-

ic carbons between the fluorescent core andHAP binding,

therefore, such systems merelyfunctioned as fluorescent labels

with no change in emission due to HAP binding since they

lacked synergistic interaction between the HAP and the fluo-

rescentcore. Phosphonic acids that have direct sp2bonds to

the fluorescent core couldextend the conjugation of the fluo-

rescent core to the HAPand therebycould initiate changes in

the ground and excited states resulting in quenching or en-

hancing the fluorescenceemission. As seen in Figure 2(B),

upon binding of HAP, p-H8TPPA produces increased fluores-

cence supporting this hypothesis; p-H8TPPA is thus the first ex-

ample of asingle sp2bonded phosphonic acid unit targeting

HAP in the literature.

Comparable experimentswith mouserib sections were con-

ducted using the isopropyldiester forms p-H8TPPA-iPr8and m-

H8TPPA-OEt8(See Scheme 1a and 1c)asstains. We found a

complete lack of HAP-associated staining with these com-

pounds suggesting that the interaction of phosphonates with

HAP was reduced by the isopropylester groups on the phos-

phonates, preventing binding and interaction of the fluores-

cent porphyrin core with the HAP.Wealso noted that the pres-

ence of hydrophobic isopropyl groups dramatically reduced

their solubilities in the aqueous.

Aiming to use phenylphosphonicacid functionalized por-

phyrins in in vivo applications, the negative charge on the de-

protonated phosphonate might be ahandicap by limiting cell

permeability.Optional demasking of phosphonate esters in

metabolic surroundingsmight occur,recreating the metal

binding PhPO32@.This possibility led us to examine p-H8TPPA

and p-H8TPPA-iPr8suitability for in vivo cellular experiments

by testing their permeability on proliferating human THP-1

monocytes. The cells were probedwith either p-H8TPPA or p-

H8TPPA-iPr8in aHEPES-based buffer containing 0.3%bovine

serum albumin. Albumin binds awide variety of hydrophobic

ligandsunder physiological conditions, so we assumed an im-

provedsolubility/accessibility for the isopropyl diester mole-

cule for cellular uptake. In addition, albumin is aregular com-

ponentofculture media.

As seen in Figure 3, the fluorescencespectra of p-H8TPPA-

loaded THP-1 cells very much resembles the characteristics of

the sensordiluted in HEPES-buffer (see Figure 1A,F). Thus,

beyondthe low toxicity p-H8TPPA provides two more positives

-cell permeability and cellular retention- desirable for imaging

applications. p-H8TPPA-iPr8wasmuch less efficient in labeling

the THP-1 cells (see FigureS23). We hypothesize that the phe-

nylphosphonic acid porphyrins, being somewhat amphipathic,

were able to penetrate the cell membrane,whereas the more

nonpolaresters were not as efficient.

In conclusion, herein, we have reported next-generation

fluorescent probes to target calcifications, namely p-H8TPPA,

m-H8TPPA,p-H8TPPA-iPr8,and m-H8TPPA-OEt8,inwhich the

phosphonic acid metal binding unit(s)are exclusively bonded

to sp2carbon atoms of the fluorescent core. sp2bonding of

the metal binding unit to the fluorescent core furtherextend-

ed the conjugation of the fluorescent core to the HAP,leading

to significant increaseinfluorescence upon HAP binding. We

furtherused these fluorescent probestotarget the HAPin

mouse ribs and observed their interaction with human cells.

Both m-H8TPPA and m-H8TPPA have shown HAP specific bind-

ing in rat bone sections. The more protected metal binding

nature of the m-H8TPPA resulted in longerfluorescence period

compared to its positional isomer p-H8TPPA.Asacontrol, we

have also synthesized phosphonatediesters of the synthesized

fluorescent probes, which showed no HAP binding. p-H8TPPA

can travel throughthe cellular membrane,whereas the pres-

Figure 2. The absorbance (A) and fluorescence (B) spectra of 0.01 mgmL@1p-H8TPPA in the presence (dashed lines) and absence(solid lines)ofHAP.The dye

was diluted to 0.01 mgmL@1in PBS (pH 7.4,green), TBS (pH 7.4, orange), HEPES (pH 7.4, purple) and dH2Owith absorbance and fluorescencespectraobtained

using the Flexstation3microplate reader.

Figure 3. Fluorescence spectra of THP-1monocytes cells incubated with p-

H8TPPA.

Chem.Eur.J.2020,26,11129 –11134 www.chemeurj.org T2020 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim11132

Chemistry—A European Journal

Communication

doi.org/10.1002/chem.202001613

ence of bulky and hydrophobic isopropyl groupsinp-H8TPPA-

iPr8hindered their passage through the cellular membrane.

This is the first study utilizingsp

2-bonded phosphonicacids

with the fluorescent cores to target HAP mineralizations. We

have shown that compactfluorescent probeswith phosphonic

acid metal binding group(s)may be used to monitor microcal-

cifications and calcifications. In addition, their easy acceptance

into the cellular matrix indicatethat 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 tar-

geting wide range of calcifications in vivo. We are currently de-

velopingour library to target organelle specific calcifications.

Animal Experiments

Animal procedures were approved by Queen’sUniversity Belfast

Animal Welfare and Ethical Review Body (AWERB) and authorized

under the UK Animals (Scientific Procedures) Act 1986. Animal use

conformed to the standards of the ARVO Statement for the Use of

Animals in Ophthalmic and Vision Research and with European Di-

rective 210/63/EU.

Acknowledgements

G.Y.would like to thank the DFG for funding his work. C.N.B.

would like to thank to Department for EducationofNorthern

Ireland for PhD studenship and I.L. would like to thank to are-

search grant from the Belfast Association forthe Blind.

Conflict of interest

G.Y.and H.H. have pending patent protectingsome of the pre-

sented data.

Keywords: Alzheimer’s disease ·calcifications ·fluorescent

imaging ·ligand synthesis ·vascular calcifications

[1] A. Vallejo-Illarramendi, I. Toral-Ojeda, G. Aldanondo,A.LjpezdeMunain,

Expert.Rev.Mol. Med. 2014,16,E16.

[2] “Soft Tissue Calcification and Ossification”: A. H. Newberg in Imaging of

Arthritis and Metabolic Bone Disease (Ed.:B.N.Weissman), Elsevier,

2009,chap. 39, pp.681–696.

[3] E. M. Ramos, J. Oliveira, M. J. Sobrido,G.Cappola, PrimaryFamilial Brain

Calcification (Eds.:M.P.Adam,H.H.Ardinger,R.A.Pagon, S. E. Wallace,

L. J. H. Bean,K.Stephens, A. Amemiya) 2004,Apr 18 [Updated 2017

Aug 24].GeneReviewsS[Internet].Seattle (WA): University of Washing-

ton, Seattle;1993–2020.

[4] M. G. Pilgrim, I. Lengyel, A. Lanzirotti, M. Newville, S. Fearn, E. Emri, J. C.

Knowles, J. D. Messinger,R.W.Read, C. Guidry,C.A.Curcio, Invest. Oph-

thalmol. Vis. Sci. 2017,58,708–719.

[5] S. M. Moe, N. X. Chen, J. Am. Soc. Nephrol. 2008,19,213–216.

[6] a) G. M. Garcia,G.C.McCord, R. Kumar, Semin. Musculoskelet Radiol.

2003,7,187–193;b)M.E.Witte, A. M. Schumacher,C.F.Mahler,J.P.Be-

wersdorf, J. Lehmitz, A. Scheiter,P.S#nchez, P. R. Williams, O. Griesbeck,

R. Naumann, T. Misgeld, M. Kerschensteiner, Neuron 2019,101,615–

624.

[7] a) R. B. Thompson, V. Reffatto, J. G. Bundy, E. Kortvely,J.M.Flinn, A. Lan-

zirotti, E. A. Jones, D. S. McPhail, S. Fearn, K. Boldt, M. Ueffing, S. G. Ratu,

L. Pauleikhoff, A. C. Bird, I. Lengyel, Proc. Natl. Acad. Sci. USA 2015,112,

1565–1570;b)A.C.S.Tan, M. G. Pilgrim, S. Fearn, S. Bertazzo, E. Tsolaki,

A. P. Morrell, M. Li, J. D. Messinger,R.Doiz-Marco, J. Lei, M. G. Nittala,

S. R. Sadda, I. Lengyel, K. B. Freund, C. A. Curcio, Sci. Transl. Med. 2018,

10,eaat4544;c)T.C.von Erlach,S.Bertazzo, M. A. Wozniak, C.-M.Horejs,

S. A. Maynard,S.Attwood, B. K. Robinson, H. Autefage,C.Kallepitis, A.

del R&oHern#ndez, C. S. Chen,S.Goldoni, M. M. Stevens, Nat. Mater.

2018,17,237–242;d)V.Tadic, A. Westenberger,A.Domingo, D. Alvar-

ez-Fischer,C.Klein, M. Kasten, JAMA Neurol. 2015,72,460–467.

[8] J. J. Mordang, A. Gubern-M8rida, A. Bria, F. Tortorella,R.M.Mann,

M. J. M. Broeders, G. J. denHeeten, N. Karssemeijer, BreastCancer Res.

Treat. 2018,167,451–458.

[9] K. Ganesan,P.Bansal, A. Goyal, R. Douglas, Bisphosphonate,StatPearls

Publishing, 2020.

[10] A. Zaheer, M. Murshed, A. M. De Grand, T. G. Morgan, G. Karsenty,J.V.

Frangioni, Arterioscler.Thromb.Vasc. Biol. 2006,26,1132–1136.

[11] J. S. Lee, J. D. Morrisett, C.-H. Tung, Atherosclerosis 2012,224,340–347.

[12] V. Freire, T. P. Moser,M.Lepage-Saucier, Insights into Imaging 2018,9,

477–492.

[13] “Detection and Quantification of CoronaryCalcification”: R. Vliegenthart

in Coronary Radiology. Medical Radiology (Eds.: M. Oudkerk),Springer,

Berlin, Heidelberg, 2004,pp. 175–184.

[14] C. R. Becker,A.Knez, T. F. Jakobs, S. Aydemir,A.Becker,U.J.Schoepf,R.

Bruening, R. Haberl, M. F. Reiser, Eur.Radiol. 1999,9,620–624.

[15] R. Vliegenthart,B.Song, A. Hofman,J.C.M.Witteman, M. Oudkerk, Ra-

diology 2003,229,520–525.

[16] J. P. Shields, Jr., C. H. Mielke, T. H. Rockwood, R. A. Short, F. K. Viren, Am.

J. Card. Imaging 1995,9,62–66.

[17] L. Saba, G. Mallarin, Am. J. Neuroradiol. 2009,30,1445–1450.

[18] D. T. Boll, E. M. Merkle, E. K. Paulson, R. A. Mirza, T. R. Fleiter, Radiology

2008,249,119–126.

[19] G. S. Mintz, JACC-Cardiovasc.Imaging. 2015,8,461–471.

[20] N. B. Senguttuvan, S. Kumar,W.-S. Lee, S. Mishra, J. H. Cho, J. E. Kwon,

S. H. Hyeon, Y. S. Jeong, H. Won, S. Y. Shin, K. J. Lee, T. H. Kim, C. J. Kim,

S.-W.Kim, Plos One 2016,11,e0165885.

[21] A. Fatemi-Ardekani, C. Boylan,M.D.Noseworthy, Med. Phys. 2009,36,

5429–5436.

[22] S. Schwartzenberg, Y. Shapira, M. Vaturi, M. Nassar,A.Hamdan,I.Yedi-

dya, H. Ofek, S. Kazum, D. Monakier,R.Kornowski, A. Sagie, Eur.Heart J.

Cardiovasc. Imaging 2020,21,jez319.805.

[23] Y. Wang, M. T. Osborne, B. Tung, M. Li, Y. Li, J. Am. Heart Assoc. 2018,7,

e008564.

[24] J. Rungby,M.Kassem, E. F. Eriksen, G. Danscher, Histochem. J. 1993,25,

446–451.

[25] A. Bensimon-Brito, J. Cardeira, G. Dion&sio, A. Huysseune, M. L. Cancela,

P. E. Witten, BMC Dev.Biol. 2016,16,2.

[26] “Red and Near-Infrared Fluorometry”: R. B. Thompson in Topics in Fluo-

rescenceSpectroscopy Vol. 4: Probe Design and Chemical Sensing (Eds.:

J. R. Lakowicz), 1994,New York, Plenum Press, pp. 151–181.

[27] G. Hong, A. Antaris, H. Dai, Nat. Biomed.Eng. 2017,1,0010.

[28] J. L. Kovar,X.Xu, D. Draney,A.Cupp, M. A. Simpson, D. M. Olive, Anal.

Biochem. 2011,416,167–173.

[29] R. F. Chen, J. C. Kernohan, J. Biol. Chem. 1967,242,5813–5823.

[30] R. H. Conrad, J. R. Heitz, L. Brand, Biochemistry 1970,9,1540–1546.

[31] J.-B. LePecq, C.Paoletti, J. Mol. Biol. 1967,27,87–106.

[32] G. Yecesan, Y. Zorlu, M. Stricker,J.Beckmann, Coord.Chem. Rev. 2018,

369,105–122.

[33] R. vonMoos, L. Costa, E. Gonzalez-Suarez,E.Terpos, D. Niepel, J.-J.

Body, Cancer Treat. Rev. 2019,76,57–67.

[34] A. Bulut, M. Maares, K. Atak, Y. Zorlu, B. C¸ os¸ut, J. Zubieta, J. Beckmann,

H. Haase, G. Yecesan, CrystEngComm 2018,20,2152–2158.

[35] M. Maares, M. M. Ayhan, K. B. Yu,A.O.Yazaydin,K.Harmandar,H.

Haase,J.Beckmann, Y. Zorlu, G. Yecesan, Chem.Eur.J.2019,25,11214–

11217.

[36] R. J. Motekaitis, I. Murase, A. E. Martell, Inorg. Chem. 1976,15,2303–

2306.

[37] T. K. Hurst, D. Wang, R. B. Thompson, C. A. Fierke, Biochim. Biophys. Acta

Proteins Proteomics 2010,1804,393–403.

[38] I. R. Salcedo, R. M. P. Colodrero, M. Bazaga-Garca, A. Vasileiou, M. Papa-

daki, P. Olivera-Pastor, A. Infantes-Molina, E. R. Losilla, G. Mezei, A.

Cabeza, K. D. Demadis, CrystEngComm 2018,20,7648–7658.

Chem. Eur.J.2020,26,11129 –11134 www.chemeurj.org T2020 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim11133

Chemistry—A European Journal

Communication

doi.org/10.1002/chem.202001613

[39] H. Hyun, H. Wada, K. Bao, J. Gravier,Y.Yadav,M.Laramie, M. Henary,

J. V. Frangioni, H. S. Choi, Angew.Chem.Int. Ed. 2014,53,10668–10672;

Angew.Chem. 2014,126,10844–10848.

[40] K. Nagarajan, K. P. Shelly,R.R.Perkins, R. Stewart, Can. J. Chem. 1987,

65,1729–1733.

[41] A. M. Sim, N. A. Rashdan,L.Cui, A. J. Moss, F. Nudelman,M.R.Dweck,

V. E. MacRae, A. N. Hulme, Sci. Rep. 2018,8,17360.

[42] K. R. Bhushan,E.Tanaka, J. V. Frangioni, Angew.Chem. Int. Ed. 2007,46,

7969–7971; Angew.Chem. 2007,119,8115–8117.

[43] S. Sun, K. M. Błaz

˙ewska, A. P. Kadina, B. A. Kashemirov,X.Duan, J. T. Trif-

fitt, J. E. Dunford,R.Graham, G. Russell, F. H. Ebetino, A. J. Roelofs, F. P.

Coxon, M. W. Lundy,C.E.McKenna, Bioconjugate Chem. 2016,27,329–

340.

[44] A. Schetrumpf, A. Duthie,E.Lork, G. Yecesan, J. Beckmann, Z. Anorg.

Allg. Chem. 2018,644,1134–1142.

[45] A. Schetrumpf, E. Kirpi, A. Bulut, F. L. Morel, M. Ranocchiari, E. Lork,Y.

Zorlu, S. Grabowsky,G.Yecesan, J. Beckmann, Cryst. Growth Des. 2015,

15,4925–4931.

[46] A. Bulut, Y. Zorlu, M. Wçrle, S. Pas¸a,H.Kurt, J. Zubieta, J. Beckmann, G.

Yecesan, Eur.J.Inorg. Chem. 2016,3506–3512.

[47] A. Bulut, Y. Zorlu, M. Wçrle, A. C¸ etinkaya, H. Kurt, B. Tam, A. :zgerYa-

zaydin,J.Beckmann,G.Yecesan, ChemistrySelect 2017,2,7050–7053.

[48] T. Rhauderwiek, H. Zhao, P. Hirschle, M. Dçblinger, B. Bueken, H. Re-

insch, D. De Vos, S. Wuttke, U. Kolb, N. Stock, Chem. Sci. 2018,9,5467–

5478.

Manuscript received:April 3, 2020

Accepted manuscript online:April 15, 2020

Version of record online:July 28, 2020

Chem. Eur.J.2020,26,11129 –11134 www.chemeurj.org T2020 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim11134

Chemistry—A European Journal

Communication

doi.org/10.1002/chem.202001613