Bis(phosphonate)-building blocks modified with fluorescent dyes

Article abstract of DOI:10.1002/hc.21108

Preparation of two benzylic bis(phosphonic acids) modified with primary amine or carboxylic acid groups on the benzene ring is described. These compounds were prepared and characterized in the form of both bis(phosphonate) tetraesters and corresponding fr

Full text of DOI:10.1002/hc.21108

Heteroatom Chemistry
Volume 24, Number 5, 2013
Bis(phosphonate)-Building Blocks Modified
with Fluorescent Dyes
Toma´sˇ David,1 Jan Kotek,1 Vojteˇch Kub´ıcˇek,1 Zdeneˇk Tosˇner,2
Petr Hermann,1 and Ivan Lukesˇ1
1Department of Inorganic Chemistry, Faculty of Science, Charles University in Prague 128 40,
Prague 2, Czech Republic
2NMR Laboratory, Faculty of Science, Charles University in Prague 128 40, Prague 2, Czech
Republic
Received 1 March 2013; revised 26 June 2013
used model of bone tissue. Sorption ability of the
ABSTRACT:
Preparation
of
two
benzylic
prepared bis(phosphonate) compounds was similar
bis(phosphonic acids) modified with primary
amine or carboxylic acid groups on the benzene ring
is described. These compounds were prepared and
characterized in the form of both bis(phosphonate)
tetraesters and corresponding free acids. The phos-
phonic acid esters are suitable for further derivation,
mainly for conjugation through the amide bond.
Mild conversion of the bis(phosphonate) esters to
free acids using trimethylsilylbromide allowed to
work with functional groups sensitive to condi-
tions of acidic and/or alkaline hydrolysis. Three
bis(phosphonate)-containing fluorescent probes were
prepared from the building blocks, utilizing amide
and sulfamide bonds as spacers. Dyes containing the
dansyl group, rhodamine B, and fluorescein were
chosen due to their common availability and low
cost. The prepared bis(phosphonate)-building blocks
and modified fluorescent probes were used for ad-
sorption studies with hydroxyapatite, the commonly
ꢀC
to that of pamidronate.
2013 Wiley Periodicals,
Inc. Heteroatom Chem 24:413–425, 2013; View
this article online at wileyonlinelibrary.com. DOI
10.1002/hc.21108
INTRODUCTION
Geminal bis(phosphonates) are a widely studied
family of compounds as the group shows very high
affinity to hydroxyapatite (HA; Ca10(PO4)6(OH)2),
which is the main inorganic component of bones
[1–3]. This affinity is a basis for important ap-
plications in the treatment of diseases associated
with the disorder of calcium metabolism, includ-
ing osteoporosis, Paget’s disease, and cancer [1–4].
Owing to the affinity to the surface of other materi-
als, bis(phosphonates) are also used as corrosion in-
hibitors or as complexation agents in textile industry
or detergent formulations.
Correspondence to: Vojteˇch Kub´ıcˇek; e-mail: kubicek@
natur.cuni.cz.
Treatment of calcium metabolism diseases is not
the only medical application of bis(phosphonates).
Nowadays, geminal bis(phosphonates) are utilized
as potential vectors of other compounds to target
bone tissues [5–7]. Attachment of bis(phosphonate)
group changes significantly biodistribution and/or
pharmacokinetics of various pharmaceuticals, and
mostly leads to selective delivery of the drugs to
bone tissue. This concept is applied not only to
Contract grant sponsor: Long Term Research Plan of the Min-
istry of Education of the Czech Republic.
Contract grant number: MSM0021620857.
Contract grant sponsor: Grant Agency of the Czech Republic.
Contract grant number: P207/11/1437.
Contract grant sponsor: Ministry of Education of Czech
Republic.
Contract grant number: LD 13012.
Supporting Information is available in the online issue at
wileyonlinelibrary.com.
ꢀC
2013 Wiley Periodicals, Inc.
413

414 David et al.
the treatment of bone diseases but also to imaging
of bone-related diseases. Radiopharmaceuticals
based on 99mTc-bis(phosphonate) complexes used
for SPECT (single-photon emission computed to-
mography) imaging of bone metastases are clini-
cally important [7, 8]. Similar complexes with 186Re
and 188Re have been used for the bone-pain pal-
liation therapy [7]. Specific chelators bearing the
bis(phosphonate) group in a side chain have been
studied as bone-targeted transporters of other metal
ions in magnetic resonance imaging (Gd3+) [9, 10],
nuclear medicine methods SPECT (99mTc, 111In,
177Lu) [7, 11, 12], or positron emission tomogra-
phy (68Ga, 64Cu) [7, 13–15]. Fluorescent dyes [6] or
nanoparticles [16] containing the bis(phosphonate)
group have also been used for optical imaging of
calcified tissues.
mentary functional group [9, 10, 28–33]. The gen-
eral conjugation is illustrated in Fig. 1; the cou-
pling reaction should simplify the introduction of
the bis(phosphonate) group, decrease a number of
reaction steps, and reduce the time to synthesize the
conjugates.
Thus, the useful compound should be easily syn-
thesized in large scale in both ester and acid forms.
Therefore, we decided to synthesize and study two
bis(phosphonate)-building blocks containing amine
or carboxylate groups (compounds 3–7, Fig. 2) ful-
filling these requirements. The compounds were,
consequently, used to attach the bis(phosphonate)
group to fluorescent dye moieties - dansyl group,
rhodamine, and fluorescein (Fig. 2). The conjugates
with these common dyes were used for HA surface
modification.
Phosphonic acids are also known to exhibit a
high affinity for the surface of many inorganic ox-
ides and other inorganic materials [17–19]. The gem-
inal bis(phosphonate) group has been proved to have
much higher affinity to the metal oxide surfaces than
the simple phosphonate group [20] and is able to an-
chor other molecules to the surfaces with long-term
stability [21–25].
Diverse spectrum of compounds with unique
properties has been generated during studies on
bis(phosphonates) over past 40 years [26]. Deriva-
tization of the simplest bis(phosphonates) has been
usually employed for preparation of the more com-
plex molecules [27, 28]. However, for a number of
applications, the most important matter is just the
presence of the bis(phosphonate) group as it is solely
responsible for the desired properties as targeting
or surface modification. Thus, more easily available
bifunctional bis(phosphonates) as starting materi-
als, mainly the ester form, are sought as they could
simplify further modification of various molecules.
Such bis(phosphonate) “building blocks” contain-
ing a reactive functional group in the side chain
(e.g., in Fig. 1) might be attachable to a wide range
of substrates having the availability of a comple-
EXPERIMENTAL
Materials and Methods
The commercially available chemicals had synthetic
purity and were used as received. Dry solvents for
the synthesis were purchased from Sigma–Aldrich
(Prague, Czech Republic). Tetraethyl methylene-
bis(phosphonate) [34] and cationic piperazine
derivative of rhodamine B (III) [35] were pre-
pared according to the published procedures. 1H,
13C, and 2D NMR (H,H-COSY, H,C-HSQC, and
H,C-HMBC) spectra were recorded on a Bruker
Avance III 600 MHz spectrometer (Scientific In-
struments, Brno, Czech Republic) equipped with
the triple resonance cold probe. Chemical shifts
were referenced according to the literature [36].
The 31P NMR spectra were recorded on
a
Varian NMR system 300 MHz spectrometer
(Agilent, Santa Clara, CA, USA) equipped with an
AutoSwitchable probe, and 85% H3PO4 was used as
an external reference (δ = 0.00 ppm). The NMR spec-
tra were recorded at 25◦C. Chemical shifts (δ) are
given in ppm and the coupling constants (J) in Hz.
FIGURE 1 Schematic illustration of the conjugation reaction between a bis(phosphonate) building block with a general
substrate and examples of such building blocks. A is a functional group specifically reacting with a complementary functional
group B.
Heteroatom Chemistry DOI 10.1002/hc

Bis(phosphonate)-Building Blocks Modified with Fluorescent Dyes 415
FIGURE 2 Structures of the studied bis(phosphonate)-building blocks (3, 4, 6, 7) and the modified dyes (8–13).
Numbering schemes of bis(phosphonate) dye conju-
gates can be found in the Supporting Information
(Fig. S1). The ESI-MS spectra were recorded on a
Bruker Esquire 3000 spectrometer. High-resolution
MS spectra were measured on a Bruker APEX-Q
Fourier transform mass spectrometer. Fluorescence
spectra were recorded on a luminescence thermo
spectronic spectrometer AMINCO Bowman series 2
(Thermo Fisher Scientific, Pardubice, Czech Repub-
lic). UV–vis spectra were recorded on a Biochrom
WPA Lightwave II spectrophotometer (ChromSpec,
Prague, Czech Republic). HA was purchased from
Fluka (catalogue number 55496; Prague, Czech Re-
vent (50 mL) was added to the reaction mixture. The
resulting mixture was stirred at RT for additional
12 h. The reaction was quenched by addition of
EtOH (20 mL). The mixture was evaporated to dry-
ness. The crude product was dissolved in CHCl3
(150 mL) and extracted with water (3 × 150 mL).
The organic layer was evaporated to dryness, and the
resulting oil was extracted with water (3 × 25 mL).
The crude oily product was purified by column chro-
matography (SiO2). Fractions containing pure prod-
uct were combined, dried with anhydrous MgSO4,
and evaporated in vacuum to dryness. The product
was obtained as a viscous oil.
public). The specific surface area, 73 m2
g
−1, was
determined by N2 adsorption by means of a Quan-
tachrome Autosorb-6B apparatus (Quantachrome,
Odelzhausen, Germany). Surface analysis showed
the Ca:P ratio 5:4, as determined on an Omicron
ESCAProbeP apparatus (Omicron NanoTechnology,
Taunusstein, Germany). The ζ potential, –18 mV at
pH 7.5, was determined on a Brookhaven BI-Zeta
PALS apparatus (Brookhaven Instruments Corpora-
tion, Holtsville, NY, USA). For thin-layer chromatog-
raphy, Merck (Prague, Czech Republic) aluminum
foils with silica gel 60 F254 were used.
Tetraethyl
(phosphonate) (1). The reaction was per-
formed with 4-nitrobenzylbromide (4.75
g) in dry toluene with 90% purity after
extractions (determined by 31P NMR). The mo-
bile phase MeOH:MeCN:CHCl3 1:10:10 was used
for column chromatography. The product was
obtained as a yellow viscous oil. Yield: 5.03 g
(72%). NMR (CDCl3) 1H: δ 1.24 (CH3, m, 12H);
2-(4-Nitrophenyl)-ethane-1,1-bis
2
3
2.59 (CH P, tt, 1H, JHP = 24, JHH = 6); 3.27
3
3
(CH2 CH, td, 2H, JHP = 16, JHH = 6); 4.07
3
(CH2 O, m, 8H); 7.39 (CH
C
CH2, d, 2H, JHH
=
9); 8.08 (CH
C
N, d, 2H, 3JHH = 9); 13C{1H}: δ 16.4
Synthesis of Bis(phosphonate)-Building Blocks
(CH3, m); 31.4 (CH2 CH, m); 38.9 (CH P, t, 1JCP
=
General Procedure for Preparation of 4-Substituted
Benzyl-bis(phosphonates) (1, 5). Under an argon
atmosphere, a solution of CH2(PO3Et2)2 (9.51 g;
33 mmol) in dry solvent (50 mL; see below) was
slowly added, dropwise, to NaH (60% suspension
in mineral oil; 1.85 g, 46.3 mmol) suspended in dry
solvent (50 mL) and cooled to 0◦C. After liberation
of all hydrogen gas, the reaction mixture was left to
warm up to room temperature (RT). The solution of
the substituted benzylbromide (22 mmol) in dry sol-
133); 62.9 (CH2 O, m); 123.5 (CH
C
N, s); 130.0
(CH
C
CH2, s); 146.8 (C N, s); 147.5 (C CH2,
3
t, JCP = 8); 31P{1H}: δ 24.0 (s). ESI-MS m/z (–):
394.0 [M – Et]−; 422.0 [M – H]−; (+): 424.2 [M +
H]+; 446.1 [M + Na]+; 462.0 [M + K]+. TLC (SiO2,
MeOH:MeCN:CHCl3 1:10:10) Rf = 0.6.
Tetraethyl
2-[4-(Methoxycarbonyl)phenyl]-
ethane-1,1-bis(phosphonate) (5). The reaction was
performed with methyl 4-(bromomethyl)benzoate
Heteroatom Chemistry DOI 10.1002/hc

416 David et al.
(5.04 g) in dry THF with 87% purity after ex-
tractions (determined by 31P NMR). The mobile
phase MeCN:CHCl3 1:1 was used for SiO2 col-
umn chromatography. The product was obtained
as a light yellow viscous oil. Yield: 7.1 g (75%).
NMR (CDCl3) 1H: δ 1.24 (CH3 CH2, m, 12H);
with anhydrous MgSO4, and evaporated to dryness
in vacuum. The product was obtained as a light
yellow viscous oil. Yield: 4.07 g (59%). NMR (CDCl3)
1H: δ 1.22 (CH3, m, 12H); 2.63 (CH P, tt, 1H, 2JHP
=
3
3
24, JHH = 6); 3.27 (CH2 CH, td, 2H, JHP = 17,
3JHH = 6); 4.09 (CH2 O, m, 8H); 7.32 (CH
C CH2,
2
3
3
3
2.62 (CH P, tt, 1H, JHP = 24, JHH = 7); 3.25
d, 2H, JHH = 8); 7.94 (CH
C
CO, d, 2H, JHH
=
3
3
(CH2 CH, td, 2H, JHP = 17, JHH = 7); 3.88
8); 13C{1H}: δ 16.0 (CH3, m); 30.9 (CH2 CH, s);
1
(CH3 O, s, 3H); 4.09 (CH2 O, m, 8H); 7.32
38.2 (CH P, t, JCP = 130); 62.7 (CH2 O, m);
3
(CH
C
CH2, d, 2H, JHH = 8); 7.93 (CH
C
CO,
128.3 (C CO, s); 128.7 (CH C CH2, s);
d, 2H, JHH = 8); 13C{1H}: δ 16.5 (CH3, m);
129.7 (CH
C
CO, s); 144.7 (C CH2, t, JCP
=
3
3
1
31.5 (CH2 CH, s); 39.0 (CH P, t, JCP = 130); 52.3
8); 169.1 (CO, s); 31P{1H}: δ 22.6 (s). ESI-MS m/z (–):
392.7 [M – Et]−; 420.8 [M – H]−; (+): 422.9 [M +
H]+; 445.0 [M + Na]+; 460.9 [M + K]+. TLC (SiO2,
CH2Cl2:MeOH 10:1) Rf = 0.3.
(CH3 O, s) 62.8 (CH2 O, m); 128.6 (C CO, s);
129.1 (CH
C CH2, s); 129.8 (CH C CO, s); 145.3
3
(C CH2, t, JCP = 8); 167.2 (CO, s); 31P{1H}: δ 22.6
(s). ESI-MS m/z (–): 434.8 [M – H]−; (+): 437.0 [M +
H]+; 459.0 [M + Na]+; 473.0 [M + K]+. TLC (SiO2,
MeCN:CHCl3 1:1) Rf = 0.3.
General Procedure for Ester Hydrolysis (2, 4, 7).
A solution of particular bis(phosphonate) was dis-
solved in aqueous HCl (6 M; 15 mL) and heated at
90◦C (for reaction time see below). After cooling to
RT, the mixture was evaporated to dryness and three
times coevaporated with H2O (20 mL) and isolated
as described below.
Tetraethyl
2-(4-Aminophenyl)-ethane-1,1-bis
(phosphonate) (3). Under an argon atmosphere,
10% Pd/C (400 mg) was suspended in dry EtOH
(50 mL). The solution of 1 (4.05 g; 9.57 mmol) in dry
EtOH (100 mL) was added to the mixture, and H2
was continuously bubbled through the suspension at
RT overnight. The end of the reaction was detected
by TLC (a vanishing spot of the parent compound,
MeOH:MeCN:CHCl3 1:10:10). The catalyst was
filtered, and the filtrate was evaporated to dryness.
The product was obtained as a pale yellow viscous
oil. Yield: 3.54 g (94%). NMR (CDCl3) 1H: δ 1.24
2-(4-Nitrophenyl)-ethane-1,1-bis(phosphonic acid)
(2). The solution of
was heated for 24 h. After coevaporation
with H2O, the product was obtained as
1 (1.00 g; 2.36 mmol)
a
light yellow viscous oil. Slow crystallization
from aqueous solution afforded single crystals
suitable for X-ray diffraction analysis. Yield:
(CH3, m, 12H); 2.54 (CH P, tt, 1H, 2JHP = 24, 3JHH
=
0.70
g
(95%). Elemental analysis Calcd for
6); 3.10 (CH2 CH, td, 2H, 3JHP = 17, 3JHH = 6); 4.06
C8H11NO8P2·H2O (Mr = 329.1): C 29.5; H 3.7;
3
(CH2 O, m, 8H); 6.57 (CH
C
N, d, 2H, JHH
=
N 4.3; found: C 29.2; H 4.0; N 4.3 (%). NMR
3
9); 7.02 (CH
C
CH2, d, 2H, JHH = 9); 13C{1H}: δ
2
(DMSO-d6) 1H: δ 2.41 (CH P, tt, 1H, JPH = 23,
16.3 (CH3, m); 30.4 (CH2 CH, s); 39.3 (CH P, t,
3
3
3JHH = 6); 3.17 (CH2, dt, 2H, JPH = 16, JHH
1JCP = 132); 62.5 (CH2 O, m); 115.0 (CH
C
N, s);
3
= 6); 7.53 (CH
C
CH2, d, 2H, JHH = 9); 8.11
3
129.4 (C CH2, t, JCP = 7); 129.7 (CH
C CH2, s);
3
(CH
C
N, d, 2H, JHH = 9); 13C{1H}: δ 30.8 (CH2,
145.0 (C N, s); 31P{1H}: δ 23.2 (s). ESI-MS m/z (–):
392.1 [M – H]−. 394.2; (+): [M + H]+; 416.2 [M +
Na]+. TLC (SiO2, MeOH:MeCN:CHCl3 1:10:10) Rf =
0.4.
1
m); 39.8 (CH P, t, JPC = 126); 123.1 (CH
C N,
s); 130.1 (CH
C CH2, s); 145.9 (C N, s); 149.1
(C CH2, t, JPC = 7); 31P: δ 22.1 (dt, JPH = 23,
3JPH = 16). ESI-MS m/z (–): 291.9 [M – (H3O)]−;
309.9 [M – H]−; (+): 334.1 [M + Na]+; 356.1 [M
+ 2Na – H]+; 377.1 [M + 3Na – 2H]+. HRMS m/z
(–): 309.989 [M – H]−; 620.985 [2M – H]−; (+):
312.003 [M + H]+; 333.985 [M + Na]+; 349.959
[M + K]+; 622.999 [2M + H]+; 644.981 [2M + Na]+.
3
2
Tetraethyl
2-(4-Carboxyphenyl)-ethane-1,1-bis
(phosphonate) (6). To a solution of 5 (7.1 g;
16.3 mmol) in MeOH (200 mL), LiOH·H2O (3.42 g;
81.5 mmol) was added and the resulting suspen-
sion was stirred at RT for 12 days. The reaction
mixture was evaporated to dryness, the residue
was dissolved in water (150 mL), and aqueous HCl
(6 M) was slowly added to reach pH 3. The resulting
mixture was extracted with CHCl3 (3 × 150 mL). The
organic layers were combined and evaporated to
dryness. The crude product was purified by column
chromatography (SiO2; CH2Cl2:MeOH 10:1). Frac-
tions containing pure product were combined, dried
2-(4-Aminophenyl)-ethane-1,1-bis(phosphonic acid)
(4). The solution of 3 (715 mg; 1.81 mmol) was
heated for 24 h. After coevaporation with H2O, the
residue was redissolved in H2O (10 mL) and excess
of i-PrOH was added to cloudiness. The white pre-
cipitate formed was collected on glass frit and dried
over P2O5 under vacuum to give hydrochloride of the
Heteroatom Chemistry DOI 10.1002/hc

Bis(phosphonate)-Building Blocks Modified with Fluorescent Dyes 417
product as an off-white powder. Crystallization from
10% aqueous acetic acid afforded single crystals suit-
able for X-ray diffraction Yield: 401 mg (69%). Ele-
hydrous MgSO4 and evaporated to dryness in vac-
uum. The resulting oil was dissolved in benzene and
lyophilized to get the product as a dark yellow pow-
der (1.16 g). Yield: 68%. Elemental analysis Calcd
for C40H51N3P2O10S2 (Mr = 859.9): C, 55.8; H, 6.0; N,
4.9. Found: C, 55.1; H, 5.9; N, 4.8. NMR (CDCl3) 1H: δ
mental analysis Calcd for C8H13NO6P2·HCl (MR
=
317.6): C, 30.3; H, 4.4; N, 4.4. Found: C, 30.3; H,
1
4.6; N, 4.4. NMR (D2O/NaOD, pD = 10) H: δ 2.28
2
3
(CH P, tt, 1H, JPH = 21, JHH = 6); 3.16 (CH2,
1.26 (CH3 CH2, m, 12H); 2.54 (CH P, tt, 1H, 2JHP
=
3
3
dt, 2H, JHH = 6, JPH = 16); 6.93 (CH
C
N, d,
24, 3JHH = 6); 2.85 (CH3 N; s, 12H); 3.18 (CH2 CH,
3
3
3
3
2H, JHH = 8); 7.37 (CH
C
CH2, d, 2H, JHH = 8);
td, 2H, JHP = 16, JHH = 6); 4.07 (CH2 O, m, 8H);
13C{1H}: δ 30.8 (CH2, m); 41.8 (CH P, t, 1JPC = 111);
7.01 (2, d, 2H, JHH = 8); 7.08 (H6, m, 2H); 7.10
3
116.3 (CH
C
N, s); 129.9 (CH
C
CH2, s); 133.8
(H7, m, 2H); 7.12 (H3, m, 2H); 7,45 (H12, m, 2H);
(C CH2, m); 143.6 (C N, s); 31P: δ 22.1 (dt, JPH
=
7.75 (H8, d, 2H, 3JHH = 8); 8.26 (H11, d, 2H, 3JHH
=
2
21, 3JPH = 16). ESI-MS m/z (–): 261.9 [M – (H3O)]−;
279.9 [M – H]−; (+): 304.1 [M + Na]+; 226.1 [M +
2Na – H]+; 348.1 [M + 3Na – 2H]+. HRMS m/z (–):
280.015 [M – H]−; (+): 282.029 [M + H]+; 304.011
[M + Na]+; 319.985 [M + K]+; 563.051 [2M + H]+;
585.033 [2M + Na]+.
8); 8.56 (H13, d, 2H, JHH = 8); 13C{1H}: δ 16.3
(CH3 CH2, m); 30.8 (CH2 CH, s); 38.9 (CH P, t,
1JCP = 133); 45.6 (CH3 N, s); 62.8 (CH2 O, m);
115.3 (C6, s); 119.1 (C8, s); 122.9 (C12, s); 127.9 (C7,
s); 129.4 (C3, s); 129.5 (C14, s); 129.9 (C9, s); 131.6
(C1, s); 131.9 (C13, s); 132.1 (C2, s); 132.7 (C11, s);
3
3
133.9 (C10, s); 142.0 (C4, t, JCP = 7); 151.6 (C5,
2-(4-Carboxyphenyl)-ethane-1,1-bis(phosphonic
acid) (7). The solution of 5 (671 mg; 1.54 mmol)
was heated for 3 days. After co-evaporation with
H2O, product was re-dissolved in H2O (20 mL)
and filtered with charcoal. The filtrate was then
evaporated to dryness in vacuum and three times
coevaporated with H2O (20 mL). The solid product
was dried over P2O5 under vacuum to give a white
powder. Yield: 458 mg (96%). Elemental analysis
Calcd for C9H12O8P2·H2O (Mr = 328.2): C, 33.0; H,
s); 31P{1H}: δ 22.7 (s). ESI-MS m/z (+): 860.4 [M +
H]+; 882.4 [M + Na]+; 898.3 [M + K]+. TLC (SiO2,
MeOH:MeCN:CHCl3 1:10:10) Rf = 0.8.
Tetraethylester of Rhodamine–Bis(phosphonate)
Conjugate (10). Under an argon atmosphere, O-
(Benzotriazol-1-yl)-N,N,Nꢁ,Nꢁ-tetramethyluronium
tetrafluoroborate (TBTU) (740 mg; 2.30 mmol) was
added to a solution of rhodamine B hydrochloride
(1.10 g; 2.30 mmol) and triethylamine (640 μL;
4.60 mmol) in dry MeCN (25 mL). The mixture was
stirred at RT for 15 min. The solution of 3 (594 mg;
1.51 mmol) and triethylamine (320 μL; 2.30 mmol)
in dry MeCN (5 mL) was added to the rhodamine
derivative solution. The resulting mixture was
stirred at RT for 5 h. The mixture was evaporated
to dryness; the residue was dissolved in CHCl3
(50 mL) and extracted with water (5×50 mL). The
organic layer was evaporated to dryness. The crude
product was purified by column chromatography
(SiO2; MeOH:MeCN:CHCl3 1:10:10). The fractions
containing pure product were combined and evap-
orated to dryness in vacuum to get the product as
a purple viscous oil (807 mg). Yield: 65%. NMR
1
4.3. Found: C, 33.0; H, 4.0. NMR (DMSO-d6) H: δ
2.33 (CH P, tt, 1H, 2JHP = 24, 3JHH = 7); 3.11 (CH2,
3
3
td, 2H, JHP = 17, JHH = 7); 7.37 (CH
C CH2,
3
3
d, 2H, JHH = 8); 7.83 (CH
C
CO, d, 2H, JHH
=
8); 13C{1H}: δ 31.1 (CH2, s); 128.9 (C CO, s); 129.3
(CH
C CH2, s); 129.6 (CH C CO, s); 146.6
3
(C CH2, t, JCP = 8); 167.8 (CO, s); 31P: δ 20.3 (dt,
2JPH = 24; JPH = 17). ESI-MS m/z (–): 290.5 [M –
3
(H3O)]−; 308.5 [M – H]−. HRMS m/z (–): 308.994 [M
– H]−; 618.993 [2M – H]−; (+): 311.008 [M + H]+;
332.990 [M + Na]+; 348.964 [M + K]+; 621.009 [2M
+ H]+; 642.991 [2M + Na]+; 658.965 [2M + K]+.
Synthesis of Bis(phosphonate) Dyes
1
3
(CD3CN) H: δ 1.10 (CH3 CH2 N, t, 12H, JHH
=
Tetraethylester of Dansyl–Bis(phosphonate) Con-
jugate (8). To the stirred solution of 3 (1.08 g;
2.75 mmol) in dry MeCN (100 mL), dried K2CO3
(1.85 g; 13.4 mmol) was added. After 10 min, dan-
syl chloride (1.48 g; 5.50 mmol) was added to the
mixture. The resulting suspension was stirred at
RT for 5 days. Solids were filtered, and the fil-
trate was evaporated to dryness. The crude prod-
uct was purified by column chromatography (SiO2;
MeOH:MeCN:CHCl3 1:10:10). The fractions contain-
ing pure product were combined, dried with an-
7); 1.16 (CH3 CH2 O, m, 12H); 2.60 (CH P, tt,
2
3
1H, JHP = 24, JHH = 7); 3.04 (CH2 CH, td, 2H,
3JHP = 16, JHH = 7); 3.33 (CH2 N, q, 8H, JHH
=
=
3
3
4
7); 3.94 (CH2 O, m, 8H); 6.26 (H6, d, 2H, JHH
2); 6.39 (H10, dd, 2H, 3JHH = 9, 4JHH = 2); 6.63 (H9,
3
3
d, 2H, JHH = 9); 6.76 (H2, d, 2H, JHH = 9); 7.02
3
3
(H17, d, 1H, JHH = 7); 7.05 (H3, d, 2H, JHH = 9);
7.52 (H15, m, 1H); 7.54 (H16, m, 1H); 7.88 (H14,
d, 1H, JHH = 7); 13C{1H}: δ 12.7 (CH3 CH2 N, s);
3
2
16.6 (CH3 CH2 O, m); 31.5 (CH2 CH, t, JCP
=
1
5); 38,9 (CH P, t, JPC = 132); 44.9 (CH2 N, s);
Heteroatom Chemistry DOI 10.1002/hc

418 David et al.
63.1 (CH2 O, m); 66.7 (C11, s); 98.2 (C6, s); 106.8
(C8, s); 109.1 (C10, s); 123.7 (C14, s); 124.5 (C17, s);
127.7 (C2, s); 129.2 (C15, s); 129.8 (C9, s); 130.0 (C3,
s); 131.3 (C13, s); 134.0 (C16, s); 136.4 (C1, s); 138.9
uum, and the residue was worked up as described
below.
Dansyl–Bis(phosphonate) Conjugate, Free Acid
(9). TMSBr (1.20 mL; 9.1 mmol) was added to a
solution of 8 (400 mg; 465 μmol) in dry MeCN
(40 mL). The residue after evaporation was dis-
solved in dry MeCN (20 mL), and the result-
ing solution was quickly poured into a beaker
with H2O (200 mL). The formed precipitate was
collected on glass frit, washed with MeCN and
H2O, and dried over P2O5 under vacuum. The
product was obtained as a yellow powder. Yield:
347 mg (92%). Elemental analysis Calcd for
C32H35N3P2O10S2 (Mr = 747.7): C, 51.4; H, 4.7; N,
5.6. Found: C, 51.0; H, 4.7; N, 5.2. NMR (DMSO-
3
(C4, t, JCP = 7); 149.7 (C5, s); 153.7 (C7, s); 154.9
(C12, s); 168.3 (CO, s); 31P{1H}: δ 23.2 (s). ESI-MS
m/z (–): 788.2 [M – Et]−; 816.3 [M – H]−; (+): 818.4
[M + H]+; 840.4 [M + Na]+; 856.4 [M + K]+. TLC
(SiO2, MeOH:MeCN:CHCl3 1:10:10) Rf = 0.6.
Tetraethylester of Fluorescein–Bis(phosphonate)
Conjugate (12). Under an argon atmosphere, TBTU
(652 mg; 2.03 mmol) was added to a solution of 6
(660 mg; 1.56 mmol) and triethylamine (1.10 mL;
7.90 mmol) in dry MeCN (100 mL). The mix-
ture was stirred at RT for 15 min. The solu-
tion of 5-aminofluorescein (705 mg; 2.03 mmol) in
MeCN:THF (5:1; 120 mL) was then added to the re-
action mixture. The resulting solution was stirred at
RT for 12 h. The mixture was evaporated to dryness,
the residue was dissolved in CHCl3 (100 mL), and
the solution was extracted with water (5×100 mL).
The organic layer was evaporated to dryness. The
crude product was purified by column chromatog-
raphy (SiO2; acetone:toluene 8:1; Rf(12) = 0.7). The
fractions containing pure product were combined
and evaporated to dryness in vacuum to get the prod-
uct as a dark yellow viscous oil (880 mg). Yield: 75%.
NMR (CDCl3) 1H: δ 1.27 (CH3, m, 12H); 2.74 (CH P,
2
3
d6): 1H: δ 2.29 (CH P, tt, 1H, JHP = 22, JHH
=
5); 2.82 (CH3 N, s, 12H); 3.13 (CH2 CH, td, 2H,
3JHP = 16, 3JHH = 5); 6.89 (H2, d, 2H, 3JHH = 8); 7.11
3
(H7, m, 2H); 7.18 (H6, d, 2H, JHH = 7); 7.23 (H3,
3
3
d, 2H, JHH = 8); 7.53 (H8, d, 2H, JHH = 8); 7.57
(H12, m, 2H); 8.07 (H11, d, 2H, 3JHH = 7); 8.54 (H13,
d, 2H, 3JHH = 9); 13C{1H}: δ 30.8 (CH2 CH, m); 39.1
(CH P, t, 1JCP = 114); 45.1 (CH3 N, s); 115.5 (C6, s);
118.2 (C8, s); 123.5 (C12, s); 128.2 (C7, s); 128.5
(C14, s); 129.0 (C9, s); 129.4 (C3, s); 130.2 (C1, s);
131.3 (C2, s); 131.9 (C13, s); 132.6 (C11, s);
3
132.7 (C10, s); 143.6 (C4, t, JCP = 6); 151.0
(C5, s); 31P: δ 21.3 (dt, JPH = 22, JPH
=
2
3
tt, 1H, 2JHP = 19, 3JHH = 5); 3.26 (CH2, td, 2H, 3JHP
=
16). ESI-MS m/z (–): 728.0 [M – (H3O)]−; 746.0 [M –
H]−; (+): 792.1 [M + 2Na – H]+; 814.1 [M + 3Na –
2H]+. HRMS m/z (–): 728.106 [M – (H3O)]−; 746.117
[M – H]−; (+): 814.078 [M – 2H + 3Na]+.
16, 3JHH = 5); 4.12 (CH2 O, m, 8H); 6.55 (H10, d, 1H,
3JHH = 8); 6.63 (H9, d, 1H, 3JHH = 8); 6.73 (H6, s, 1H);
3
6.81 (H22 + H23, m, 2H); 6.89 (H17, d, 1H, JHH
=
3
8); 7.04 (H16, d, 1H, JHH = 8); 7.09 (H19, s, 1H);
7.29 (H14, d, 1H, 3JHH = 8); 7.41 (H3, d, 2H, 3JHH
=
Rhodamine–Bis(phosphonate) Conjugate, Free
Acid (11). TMSBr (1.90 mL; 14.6 mmol) was added
to a solution of 10 (597 mg; 730 μmol) in dry MeCN
(20 mL). The residue after evaporation was dissolved
in dry MeCN (10 mL), and MeOH (10 mL) was then
added. The resulting solution was evaporated to dry-
ness and three times coevaporated with MeOH. The
crude product was purified by column chromatog-
raphy (Amberlite CG50; H+-form; gradient elution
with H2O:MeOH from 100:0 to 0:100). The frac-
tions containing pure product were combined, evap-
orated to dryness in vacuum, and coevaporated sev-
eral times with MeOH. The resulting solid was dried
over P2O5 under vacuum to give the product as a
dark purple powder. Yield: 476 mg (71%). Elemental
analysis Calcd for C36H41N3O8P2·2H2O (Mr = 741.7):
8); 8.06 (H2, d, 2H, 3JHH = 8); 13C{1H}: δ 11.5 (CH3, d,
3JCP = 6); 26.6 (CH2 CH, t, 2JCP = 5); 33.8 (CH P, t,
1JPC = 132); 58.4 (CH2 O, m); 83.3 (C11, s);
102.8 (C6, s); 109.9 (C14, s); 110.2 (C8 + C19, s);
112.8 (C10, s); 117.2 (C21, s); 117.3 (C23, s); 123.4
(C16, s); 124.8 (C17, s); 127.4 (C1, s); 128.0 (C15, s);
129.0 (C22, s); 129.1 (C9, s); 129.2 (C3, s); 130.3 (C2,
s); 143.6 (C12, s); 145.7 (C4, t, 3JCP = 6); 146.6 (C13,
s); 151.9 (C20, s); 152.0 (C18, s); 152.3 (C7, s); 159.4
(C5, s); 164.4 (CO N, s); 169.7 (CO O, s); 31P{1H}:
δ 22.3 (s). ESI-MS m/z (–): 750.1 [M – H]−; (+): 752.2
[M + H]+; 774.2 [M + Na]+; 790.1 [M + K]+. TLC
(SiO2, acetone:toluene 8:1) Rf = 0.7.
General Procedure for Transesterification and
Subsequent Hydrolysis (9, 11, 13). To a stirred solu-
tion of particular bis(phosphonate) conjugate in dry
solvent, Bromotrimethylsilane (TMSBr) was added
(see below). The reaction mixture was stirred in dark
at RT for 24 h. Volatiles were then evaporated in vac-
C, 58.3; H, 6.1; N, 5.7. Found: C, 58.3; H, 5.8; N, 5.7.
3
NMR (DMSO-d6) 1H: δ 1.09 (CH3, t, 12H, JHH
=
2
3
7); 2.12 (CH P, tt, 1H, JHP = 23, JHH = 5); 2.95
(CH2 CH, tt, 2H, 3JHP = 17; 3JHH = 5); 3.31 (CH2 N,
3
4
q, 8H, JHH = 7); 6.29 (6, d, 2H, JHH = 2); 6.38
Heteroatom Chemistry DOI 10.1002/hc

Bis(phosphonate)-Building Blocks Modified with Fluorescent Dyes 419
3
4
Adsorption Experiments
(10, dd, 2H, JHH = 9, JHH = 2); 6.55 (H9, d, 2H,
3JHH = 9); 6.74 (H2, d, 2H, JHH = 9); 7.03 (H17, d,
3
Tetraethylester
of
Dansyl–Bis(phosphonate)
1H, 3JHH = 8); 7.05 (H3, d, 2H, 3JHH = 9); 7.52 (H15,
Conjugate (8). In
a
25-mL volumetric flask,
m, 1H); 7.54 (H16, m, 1H); 7.86 (H14, d, 1H, 3JHH
=
bis(phosphonate) (50 μmol) was dissolved in a
HEPES buffer solution (0.1 M; 25 mL; pH 7.5) to a
final bis(phosphonate) concentration of 2 mM. All
stock solutions were stored in dark at 8◦C.
7); 13C{1H}: δ 12.1 (CH3, s); 30.0 (CH2 CH, s); 48.3
(CH2 N, s); 66.2 (C11, s); 97.3 (C6, s); 105.6 (C8, s);
108.1 (C10, s); 122.7 (C14, s); 123.6 (C17, s); 125.4
(C2, s); 128.3 (C15, s); 128.4 (C9, s); 128.5 (C3, s);
129.4 (C13, s); 133.2 (C16, s); 134.6 (C1, s); 139.1 (C4,
Adsorption Experiments with Bis(phosphonate)-
Building Blocks. In ten 4-mL glass vials, HA (10 mg)
was suspended in the HEPES buffer solution (0.1 M;
pH 7.5) and the stock solution of the compound un-
der study (2, 4, and 7) was added to get a final volume
of 3.0 mL (the final total concentrations of the stud-
ied compounds in the samples were 40–600 μM). The
suspensions were gently shaken at 25◦C for 72 h and
3
t, JCP = 6); 148.2 (C5, s); 152.1 (C7, s); 153.6 (C12,
s); 166.7 (CO, s); 31P: δ 20.3 (dt, JPH = 20, JPH
=
2
3
16). ESI-MS m/z (–): 676.1 [M – Et]−; 686.1 [M –
(H3O)]−; 704.1 [M – H]−; (+): 706.3 [M + H]+; 728.3
[M + Na]+; 750.2 [M – H + 2Na]+. HRMS m/z (–):
648.167 [M + H – 2Et]−; 676.198 [M – Et]−; 686.219
[M – (H3O)]−; 704.230 [M – H]−; (+): 706.244 M +
H]+; 728.226 [M + Na]+; 750.208 [M – H + 2Na]+.
ꢀR
then filtered through a Millipore filter (Rotilabo -
syringe filter; PVDF; 0.22 μm). The amount of the
bis(phosphonate) remaining in the supernatant was
determined by UV–vis spectroscopy (λmax 2 = 295
nm, λmax 4 = 232 nm, and λmax 7 = 239 nm). The
experimental data were treated by a least-squares
fitting procedure using the Micromath Scientist pro-
gram, version 2.0 (Salt Lake City, Utah).
Fluorescein–Bis(phosphonate) Conjugate, Free
Acid (13). TMSBr (4 mL; 30.3 mmol) was added
to a solution of 12 (780 mg; 1.03 mmol) in a mix-
ture of dry MeCN (40 mL) and dry DMF (1 mL). The
residue after evaporation was dissolved in dry MeCN
(20 mL), and the resulting solution was quickly
poured into a beaker with water (200 mL). The
precipitate formed was collected on glass frit,
washed with MeCN and H2O, and redissolved in
MeOH. The resulting solution was evaporated to
dryness and three times coevaporated with MeOH.
The solid residue was dried over P2O5 under vac-
uum to give a product as a dark brown powder.
Yield: 502 mg (78%). Elemental analysis Calcd for
C29H23NO12P2·2H2O (Mr = 675.5): C, 51.6; H, 4.0; N,
2.1. Found: C, 51.8; H, 3.4; N, 2.3. NMR (DMSO-d6)
1H: δ 2.36 (CH P, tt, 1H, 2JHP = 23, 3JHH = 6); 3.17
Adsorption Experiments with Bis(phosphonate)
Dyes. In five 4-mL glass vials, HA (5–50 mg) was
suspended in the HEPES buffer solution (0.1 M;
pH 7.5) and the stock solution of the compound
under study (9, 11, and 13) was added to get a fi-
nal volume of 3.0 mL (the final total concentrations
of the studied compounds in the samples were 1.67
M). The suspensions were gently shaken in dark at
25◦C for 3 h and then filtered through a Millipore
ꢀR
filter (Rotilabo -syringe filter; PVDF; 0.22 μm). The
amount of the bis(phosphonate) remaining in the su-
pernatant was determined by UV–vis spectroscopy (λ
9 = 315, 325, and 335 nm; λ 11 = 230, 265, and 310
nm; and λ 13 = 235, 370, and 485 nm).
3
3
(CH2, td, 2H, JHP = 16, JHH = 6); 6.60 (H10, dd,
3
4
3
1H, JHH = 8, JHH = 2); 6.66 (H9, d, 1H, JHH = 8);
6.70 (H6, d, 1H, 4JHH = 2); 6.90 (H22, d, 1H, 3JHH
=
3
8); 6.99 (H17, d, 1H, JHH = 8); 7.06 (H16 + H23,
X-Ray Diffraction
4
m, 2H); 7.12 (H14, d, 1H, JHH = 1); 7.35 (H19, d,
4
3
1H, JHH = 2); 7.50 (H3, d, 2H, JHH = 8); 8.02 (H2,
Single crystals of 2·H2O and 4·H2O were obtained as
described above. The diffraction data were collected
at 150 K (Cryostream Cooler, Oxford Cryosystem)
using a Nonius Kappa CCD diffractometer and Mo
d, 2H, JHH = 8); 13C{1H}: δ 30.8 (CH2, t, JCP = 6);
40.0 (CH P, t, 1JPC = 132); 81.7 (C11, s); 102.1 (C6,
s); 107.5 (C14, s); 109.9 (C8, s); 110.3 (C19, s); 112.9
(C10, s); 117.6 (C21, s); 118.0 (C23, s); 122.7 (C16,
s); 124.3 (C17, s); 126.2 (C1, s); 127.1 (C15, s); 129.0
(C22, s); 129.1 (C9, s); 129.3 (C3, s); 129.7 (C2, s);
140.7 (C12, s); 147.1 (C4, t, 3JCP = 6); 148.9 (C13, s);
151.2 (C20, s); 151.6 (C18, s); 151.7 (C7, s); 159.4 (C5,
s); 164.2 (CO N, s); 169.0 (CO O, s); 31P: δ 20.7 (dt,
3
2
˚
Kα radiation (λ = 0.71073 A) and analyzed using the
HKL DENZO program package [37]. The structures
were solved by direct methods (SIR92) [38], and
refined by full-matrix least-squares techniques
(SHELXL97) [39]. All nonhydrogen atoms were
refined anisotropically. All hydrogen atoms were
located in a difference map of electron density; how-
ever, they were treated in theoretical (C H) or origi-
nal (O H, N H) positions with thermal parameters
2JPH = 23; JPH = 16). ESI-MS m/z (–): 637.9 [M –
3
H]−; (+): 640.1 [M + H]+; 662.1 [M + Na]+; 678.1
[M + K]+. HRMS m/z (–): 638.062 [M + H]−.
Heteroatom Chemistry DOI 10.1002/hc

420 David et al.
TABLE 1 Experimental Crystallographic Parameters of the
Studied Compounds
Compound
Parameter
2·H2O
4·H2O
Formula
Mr
C8H13NO9P2
329.13
C8H15NO7P2
299.15
Color, habit
Crystal system
Space group
Colorless, prism
Monoclinic
P21/n
12.9704(2)
6.8883(1)
15.6311(3)
113.5613(9)
1280.12(4)
4
Colorless, rod
Monoclinic
P21/c
8.6409(2)
23.2676(7)
11.9193(3)
95.1284(18)
2386.82(11)
8
˚
a (A)
˚
b (A)
FIGURE 3 Molecular structure of 2 found in the crystal
structure of 2·H2O.
˚
c (A)
β (◦)
3
˚
U, A
Z
are, generally, low yielding [27, 32, 41]. Their esters,
as it is common for 1-hydroxo-1,1-bis(phosphonate
esters), are prone to phosphonate–phosphate re-
arrangement [42]; however, coupling with the es-
ters can be done in organic solvents. As both
carboxylic acid and amine groups are the most
frequently utilized functional groups for fur-
ther modification, we decided to prepare suitable
methylene-bis(phosphonate)-building blocks, which
can be easily prepared in a gram scale, can be con-
jugated to other molecules and, thus, enable facile
bone targeting of different substrates.
Dcalc, g·cm−3
1.708
0.385
1.665
0.392
μ, mm−1
Total reflections
Observed reflections
(I > 2σ(I))
2928
2793
4704
3336
R; Rꢁ(I > 2σ(I))
wR; wRꢁ(I > 2σ(I))
0.0271; 0.0285
0.0732; 0.0743
0.0422; 0.0733
0.0936; 0.1079
Ueq(H) = 1.2 Ueq(X) as their free refinement led to
some unrealistic bond lengths. Crystallographic data
for the structures of 2·H2O and 4·H2O have been
deposited at the Cambridge Crystallographic Data
Centre, deposit number is CCDC-896879 for 2·H2O
and CCDC-896878 for 4·H2O. Table 1 presents
selected experimental crystallographic data.
Synthesis of the Building Blocks
Two building blocks, containing bis(phosphonic
acid) group and primary amine (4) or carboxylic
acid functional groups (7), were prepared accord-
ing to Scheme 1, and the identities of nitropre-
cursor 2 as well as that of final building block 4
were unambiguously determined by single-crystal X-
ray diffraction analysis (Figs. 3 and 4). The amine
derivative was described previously by Benedict [43];
however, the yield of the published synthesis was
only 8%. A tetrakis(P-methyl ester) analogue of 1 and
RESULTS AND DISCUSSION
The formation of an amide bond is the most com-
mon coupling reaction to form conjugates and var-
ious amide-coupling reagents have been developed
over the years [40]. However, coupling of simple
aminoalkyl-bis(phosphonates), for example, alen-
dronate, brings some problems. Reactions of free
zwitterions must be done in water at pH > 9 and
SCHEME 1 Reagents and conditions: (a) NaH, toluene, RT, 2 h, followed by 4-NO2-C6H4-CH2Br, toluene, RT, 4 h, 72%;
(b) aqueous HCl, 90◦C, 24 h, 95%; (c) H2, 10% Pd/C, atmospheric pressure, EtOH, RT, 94%; (d) aqueous HCl, 90◦C, 24 h,
69%; (e) NaH, THF, RT, 2 h, followed by 4-BrCH2-C6H4-CO2Me, toluene, RT, 16 h, 75%; (f) LiOH, MeOH, RT, 12 days, followed
by aqueous HCl, 59%; (g) aqueous HCl, 90◦C, 3 days, 97%.
Heteroatom Chemistry DOI 10.1002/hc

Bis(phosphonate)-Building Blocks Modified with Fluorescent Dyes 421
which is of sufficient purity for further couplings.
For their full characterization, they were purified by
column chromatography; it leads to the lower over-
all yields mentioned in the Experimental. Generally,
the phosphonate esters are cleaved by concentrated
aqueous acids or bases at nearly reflux tempera-
ture or the esters can be conveniently removed by
selective transesterification with trimethylbromosi-
lane under nonaqueous conditions [44], followed by
cleavage of the silylesters in water or alcohols.
FIGURE 4 Molecular structure of 4 found in the crystal struc-
ture of 4·H2O.
Synthesis of Dyes
As bis(phosphonate)-containing dyes are studied as
fluorescent probes for imaging of calcified tissues
and microcalcifications [6], conjugates with very
common fluorescent moieties were prepared. Three
conjugates were prepared from the building blocks
and commercially available dye precursors–dansyl
chloride, rhodamine B and 5-aminofluorescein
(Scheme 2). The reaction of amine-building block
4 with excess of dansyl-chloride in the presence of
anhydrous potassium carbonate afforded conjugate
8. Surprisingly, two bulky dansyl groups can be eas-
ily attached to one nitrogen atom by this procedure.
Hydrolysis in hydrochloric acid was not suitable
for preparation of free bis(phosphonate) due to the
instability of the sulfamide bond under acidic condi-
tions. Instead, transesterification with trimethylsilyl-
bromide in dry acetonitrile, followed by aqueous hy-
drolysis of the silyl groups was utilized. Rhodamine
3 as well as tetrakis(P-benzyl ester) analogue of 5 has
also been prepared but in only moderate yields [29].
Utilization of compounds 4 and 7 could be advanta-
geous as they are present in a form of the free acids
and thus they do not require consequent deesterifica-
tion. Both compounds are very well soluble in water
(except weakly acidic pH in the case of compound
4) and in DMSO. On the other hand, ethyl esters of
these compounds (3 and 6) have the true potential
of being useful starting material for further modifi-
cation. They can be prepared in two steps from eas-
ily available tetraethyl methylene-bis(phosphonate)
with moderate overall yields (68% and 44% for 3 and
6, respectively). Their synthesis is entirely scalable,
and these compounds can be obtained in approxi-
mately 90% purity without need of chromatography,
SCHEME 2 Reagents and conditions: (a) dansyl chloride, K2CO3, MeCN, RT, 5 days, 68%; (b) TMSBr, MeCN, RT, 24 h,
followed by H2O/MeCN, 92%; (c) rhodamine B hydrochloride, TBTU, TEA, MeCN, RT, 5 h, 65%; (d) TMSBr, MeCN, RT, 24 h,
followed by MeOH/MeCN, 71%; (e) 5-amino-fluorescein, TBTU, TEA, MeCN, THF, RT, 12 h, 75%; (f) TMSBr, MeCN, DMF, RT,
24 h, followed by H2O/MeCN, 78%.
Heteroatom Chemistry DOI 10.1002/hc

422 David et al.
FIGURE 5 Equilibrium between acidic (I) and alkaline (II) forms of rhodamine B and the structure of the cationic piperazine
derivative of rhodamine B (III) not undergoing the ring closure. The tautomeric states of the studied compounds were determined
according to chemical shifts of the bridging carbon atoms (denoted C*).
TABLE 3 Excitation and Emission Maxima of the Synthe-
derivative 11 was prepared by conjugation of build-
ing block 4 with rhodamine B hydrochloride using
TBTU followed by the above-mentioned transesteri-
fication of resulting esterified product 10. Similarly,
fluorescein derivative 13 was prepared from build-
ing block 6 and 5-amino-fluorescein.
sized Bis(phosphonated) Dyes (0.1 m HEPES buffer, pH 7.5)
Compound
Dye moiety
λmax(ex), nm
λmax(em), nm
9
11
13
Dansyl
Rhodamine
Fluorescein
299
523
558
495
541
586
Both rhodamine and fluorescein dyes are sen-
sitive toward ring-closing tautomerism generating
less fluorescent internal lactones or lactams, respec-
tively [45]. The equilibrium between the open form
of rhodamine B (I, acid form) and the closed form of
rhodamine B (II, alkaline form) is shown in Fig. 5.
Since common 1D NMR spectroscopy could not dis-
tinguish between these two forms, in-depth 2D NMR
measurements were performed to figure out the rela-
tionship between NMR parameters and the solution
structure of the prepared conjugates. We found that
chemical shift of one carbon atom (marked C* in
Fig. 5) is very sensitive to the tautomerism. Similar
analysis was then performed with compounds 10–
13 and also with compound III, whose secondary
amido group cannot undergo the ring-closing reac-
tion (Fig. 5). Results are summarized in Table 2.
It is clear that higher chemical shifts of C* atoms
in the open structures correspond to the aromatic
sp2-hybridized carbon atom. Likewise, lower chem-
ical shifts of C* carbons in the lactones and lac-
tames reflect the aliphatic sp3-hybridized carbon
atom. It is worth to notice that the nature of sol-
vent does not affect the chemical shift significantly.
Thus, bis(phosphonated) dyes 10–13 were prepared
in the closed form.
Florescence spectra of compounds 9, 11, and
13 were measured. The maxima of excitation and
emission wavelengths are summarized in Table 3.
As compounds 11 and 13 are present in the solution
in the lactam and lactone forms, respectively; their
fluorescence has approximately one order of magni-
tude lower intensity than that observed for reference
materials (the acidic form of rhodamine B and fluo-
rescein, respectively).
Adsorption Behavior of the Studied Compounds
There are several models characterizing adsorptions
onto surfaces [46]. The most common ones are the
Langmuir and Langmuir–Freudlich isotherms
TABLE 2 Chemical Shifts of the C*-Atoms in the Studied
X
(Kc)n
1 + (Kc)n
Compounds
=
Xm
Compound
Dye
Solvent
δ
13C* (ppm) Tautomer
where K is the affinity constant (dm3 mol−1), Xm the
maximum adsorption capacity (mol m−2), X the spe-
cific adsorbed amount (mol m−2), c the equilibrium
concentration in the solution (mol dm−3), and n is
a coefficient describing the adsorption energy distri-
bution (Langmuir, n = 1; Langmuir–Freundlich, 0 <
n < 1). The simpler Langmuir model does not take
into account the interaction between the adsorbed
Ia
Rhodamine DMSO-d6
Rhodamine DMSO-d6
Rhodamine CDCl3
Rhodamine CD3CN
Rhodamine DMSO-d6
Fluorescein CDCl3
159.1
85.3
155.8
66.7
66.4
84.3
81.8
Open
Lactone
Open
Lactame
Lactame
Lactone
Lactone
IIa
III
10
11
12
13
Fluorescein DMSO-d6
aCommercially available.
Heteroatom Chemistry DOI 10.1002/hc

Bis(phosphonate)-Building Blocks Modified with Fluorescent Dyes 423
FIGURE 7 Decrease of the supernatant concentration of
the dyes 9 (Dns ), 11 (Rhd •), and 13 (Flc ◦), with increasing
amount of HA in the suspension.
FIGURE
6 Adsorption isotherms of the prepared
bis(phosphonate)-building blocks 2 (NO2 ◦), 4 (NH2 ),
and 7 (COOH •) on HA (pH 7.4, 25◦C). The curves corre-
spond to the best fits obtained according to the Langmuir
model.
ture of the substituent on phenyl ring does not affect
adsorption properties significantly.
molecules, but results obtained with the fitting ac-
cording to the isotherm are usually satisfactory.
To characterize the adsorption behavior of the
bis(phosphonate)-building blocks, HA suspension in
water was utilized as a model of bone tissue. The
vials with various total concentrations of the stud-
ied compounds in solutions were incubated with the
constant amounts of HA. Adsorbed fractions of the
bis(phosphonates) were obtained indirectly as
the difference between their starting amount and
the amount remaining in solution (as determined
by UV–vis spectroscopy). The adsorption isotherms
of the bis(phosphonate)-building blocks bearing ni-
tro (2), amine (4), and carboxylic acid (7) groups are
depicted on Fig. 6.
The affinity constants, K, and maximum ad-
sorption capacities, Xm, obtained from the fitting
are summarized in Table 4. The results prove
strong adsorption of all studied compounds onto
HA. Comparison of the adsorption parameters with
bis(phosphonates) of the similar size (Table 4) sup-
ports the widely accepted fact that adsorption prop-
erties are determined mainly by the size of the
molecule. Similar values of both parameters found
for all three studied compounds indicate that the na-
The bis(phosphonate) dyes showed a slight shift
of the absorption maximum with the increasing con-
centration. Furthermore, substantial changes in the
UV–vis spectra over times were observed, apparently
due to photobleaching of the studied compounds.
This did not allow the precise quantification and
determination of the adsorption parameters and,
thus, sorption abilities of the title compounds were
demonstrated in a different manner. The vials with
the equal total concentration of the dyes were incu-
bated with various amounts of HA. A decrease in the
dye solution concentration with increasing amount
of HA is depicted in Fig. 7. The results indicate that
all compounds are adsorbed onto HA. Significant
difference in the adsorption of the fluorescein deriva-
tive 13 in comparison with other two derivatives is
probably the result of negatively charged phenolic
groups leading to a repulsion between molecules on
the surface. This is not the case of derivatives 9 and
11 containing tertiary amine groups, which could be
uncharged under conditions of the adsorption exper-
iments. Owing to the above-mentioned photobleach-
ing, the adsorption time was reduced to 3 h only.
Thus, the difference can also be ascribed to different
adsorption kinetics of the studied compounds.
TABLE 4 Adsorption Parameters for the Prepared Bis(phosphonate)-Building Blocks and Other Bis(phosphonates) of the
Similar Size on HA (pH 7.4, 25◦C)
Compound
2
4
7
PAMa
HEDPa
Mr, g·mol−1
Xm (× 10−5, mol·g−1
311.0
11.6 2.9
8.0 0.4
281.1
8.6 1.7
7.8 0.4
310.1
9.1 1.9
5.7 0.2
235.1
4.4
11.5
206.0
5.3
9.9
K (× 104, dm3·mol−1
)
)
aPAM = 3-aminopropyl-1,1-bis(phosphonic acid), HEDP = 1-hydroxyethyl-1,1-bis(phosphonic acid). Data are taken from the literature [47].
Heteroatom Chemistry DOI 10.1002/hc

424 David et al.
39, 396, (c) Ben-Haim, S.; Israel, O. Semin Nucl Med
2009, 39, 408.
[9] Kub´ıcˇek, V.; Rudovsky´, J.; Kotek, J.; Hermann, P.;
Elst, L. V.; Muller, R. N.; Kolar, Z. I.; Wolterbeek, H.
T.; Peters, J. A.; Lukesˇ, I. J Am Chem Soc 2005, 127,
16477.
[10] Vitha, T.; Kub´ıcˇek, V.; Kotek, J.; Hermann, P.; Elst, L.
V.; Muller, R. N.; Lukesˇ, I.; Peters, J. A. Dalton Trans
2009, 3204.
CONCLUSIONS
Two bifunctional bis(phosphonates), containing pri-
mary amine or carboxylic acid groups, were synthe-
sized in high overall yields. Both compounds were
prepared in the form of bis(phosphonate) ethylesters
as well as in the form of free acids. Owing to the
common availability of primary amine or carboxylic
acid functional groups in various substrates, the
synthesized building blocks can be easily attached
to various substrates via amide-coupling reactions.
Such derivatization was documented in the syn-
thesis of three fluorescent bis(phosphonate) dyes.
The conjugates were prepared by one-pot synthe-
sis from tetraethylester of the bis(phosphonates)
under nonaqueous conditions. In situ transesterifi-
cation with trimethylsilylbromide followed by aque-
ous hydrolysis afforded corresponding free acids
in high yields. NMR studies showed that the pre-
pared fluorescein and rhodamine bis(phosphonate)
derivatives are present in the lactone and lactame
forms, respectively. It results in less intensive fluo-
rescence compared with the starting dyes. All stud-
ied compounds show significant adsorption on HA;
their affinity to HA is slightly higher than that of
common bis(phosphonates) as pamindronate. The
results show that the presented bis(phosphonate)
derivatives could be considered as promising build-
ing blocks for modification of various substrates.
[11] Liu, W.; Hajibeigi, A.; Lin, M.; Rostollan, L. C.;
¨
Kova´cs, Z.; Oz, O. K.; Sun, X. Bioorg Med Chem Lett
2008, 18, 4789.
[12] Vitha, T.; Kub´ıcˇek, V.; Hermann, P.; Elst, L. V.;
Muller, R. N.; Kolar, Z. I.; Wolterbeek, H. T.; Bree-
man, W. A. P.; Lukesˇ, I.; Peters, J. A. J Med Chem
2008, 51, 677.
[13] (a) Fellner, M.; Baum, R. P.; Kub´ıcˇek, V.; Hermann,
P.; Lukesˇ, I.; Prasat, V.; Ro¨sch, F. Eur J Nucl Med Mol
Imaging 2010, 37, 834, (b) Fellner, M.; Biesalski, B.;
Bausbacher, N.; Kub´ıcˇek, V.; Hermann, P.; Ro¨sch, F.;
Thews, O. Nucl Med Biol 2012, 39, 993.
[14] Suzuji, K.; Satake, M.; Suwada, J.; Oshikiri, S.;
Ashino, H.; Dozono, H.; Hino, A.; Kasahara, H.; Mi-
namizawa, T. Nucl Med Biol 2011, 38, 1011.
[15] de Rosales, R. T. M.; Tavar, R.; Paul, R. L.; Jauregui-
Osoro, M.; Protti, A.; Glaria, A.; Gopal, V.; Szanda, I.;
Blower, P. J. Angew Chem, Int Ed 2011, 50, 5509.
[16] Ross, R. D.; Roeder, R. K. J Biomed Mater Res A
2011, 99, 58.
[17] Vioux, A.; Le Bideau, J.; Mutin, P. H.; Leclerq, D. Top
Curr Chem 2004, 232, 145.
[18] Mingalyov, P. G.; Lisichkin, G. V. Russ Chem Rev
2006, 75, 541.
[19] Mutin, P. H.; Guerrero, G.; Vioux, A. J Mater Chem
2005, 15, 3761.
ˇ
[20] Rehorˇ, I.; Kub´ıcˇek, V.; Kotek, J.; Hermann, P.;
Sza´kova´, J.; Lukesˇ, I. Eur J Inorg Chem 2011, 1981.
[21] Lalatonne, Y.; Paris, C.; Serfaty, J. M.; Weinmann, P.;
Lecouvey, M.; Motte, L. Chem Commun 2008, 2553.
[22] Benyettou, F.; Lalatonne, Y.; Sainte-Catherine, O.;
Monteil, M.; Motte, L. Int J Pharm 2009, 379, 324.
[23] Benyettou, F.; Lalatonne, Y.; Chebbi, I.; Di Benedetto,
M.; Serfaty, J.-M.; Lecouvey, M.; Motte, L. Phys Chem
Chem Phys 2011, 13, 10020.
ACKNOWLEDGMENTS
The work was conducted in the framework of the
TD1004, CM1006, and CM0802 COST Actions. We
thank Dr. Ivana C´ısarˇova´ (Charles University in
Prague) for performing X-ray measurements, and
the Laboratory of Molecular Structure Characteri-
zation (Academy of Science of the Czech Republic)
for performing HMRS measurements.
ˇ
[24] Rehorˇ, I.; Vil´ımova´, V.; Jendelova´, P.; Kub´ıcˇek, V.;
Jira´k, D.; Herynek, V.; Kapcalova´, M.; Kotek, J.;
ˇ
Cerny´, J.; Hermann, P.; Lukesˇ, I. J Med Chem 2011,
54, 5185.
[25] Ide, A.; Drisko, G. L.; Scales, N.; Luca, V.; Schiesser,
C. H.; Caruso, R. A. Langmuir 2011, 27, 12985.
[26] Russell, R. G. G. Bone 2011, 49, 2.
[27] (a) Zaheer, A.; Lenkinski, R. E.; Mahmood, A.; Jones,
A. G.; Cantley, L. C.; Frangioni, J. V. Nat Biotechnol
2001, 19, 1148, (b) Mizrahi, D. M.; Ziv-Polat, O.; Perl-
stein, B.; Gluz, E.; Margel, S. Eur J Med Chem 2011,
46, 5175, (c) Kowada, T.; Kikuta, J.; Kubo, A.; Ishii,
M.; Maeda, H.; Mizukami, S.; Kikuchi, K. J Am Chem
Soc 2011, 133, 17772.
[28] Wang, L.; Zhang, M.; Yang, Z.; Xu, B. Chem Commun
2006, 2795.
[29] (a) Gil, L.; Han, Y.; Opas, E. E.; Rodan, G. A.; Ruel, R.;
Seedor, J. G.; Tyler, P. C.; Young, R. N. Bioorg Med
Chem 1999, 7, 901, (b) Tanaka, K. S. E.; Hughton,
T. J.; Kang, T.; Dietrich, E.; Delorme, D.; Ferreira,
S. S.; Caron, L.; Viens, F.; Arhin, F. F.; Samiento, I.;
REFERENCES
[1] Fleisch, H. Bisphosphonates in Bone Disease; Aca-
demic Press: London, 2000.
[2] Fleisch, H. Endocr Rev 1998, 19, 80.
[3] Rogers, M. J.; Crockett, J. C.; Coxon, F. P.;
Mo¨nkko¨nen, J. Bone 2001, 49, 34.
[4] (a) Cle´zardin, P. Bone 2011, 48, 71, (b) Coleman, R.
E.; McCloskey, E. V. Bone 2011, 49, 71.
[5] Zhang, S.; Gangal, G.; Uludag, H. Chem Soc Rev
2007, 36, 507.
[6] Kub´ıcˇek, V.; Lukesˇ, I. Future Med Chem 2010, 2, 521.
[7] Palma, E.; Correia, J. D. G.; Campello, M. P. C.;
Santos, I. Mol BioSyst 2011, 7, 2950.
[8] (a) Brenner, A. I.; Koshy, J.; Morey, J.; Lin, C.; DiPoce,
J. Semin Nucl Med 2012, 42, 11, (b) Beheshti, M.;
Langsteger, W.; Fogelman, I. Semin Nucl Med 2009,
Heteroatom Chemistry DOI 10.1002/hc

Bis(phosphonate)-Building Blocks Modified with Fluorescent Dyes 425
Lehoux, D.; Fadhil, I.; Laquerre, K.; Liu, J.; Ostiguy,
V.; Poirier, H.; Moeck, G.; Parr, T. R., Jr.; Far, A. R.
Bioorg Med Chem 2008, 16, 9217.
[39] Sheldrick, G. M. SHELXL97. Program for Crystal
Structure Refinement from Diffraction Data; Univer-
sity of Go¨ttingen: Go¨ttingen, Germany, 1997.
[40] (a) El-Faham, A.; Albericio, F. Chem Rev 2011, 111,
6557, (b) Montalbetti, C. A. G. N.; Falque, V. Tetra-
hedron 2005, 61, 10827, (c) Han, S.-Y.; Kim, Y.-A.
Tetrahedron 2004, 60, 2447.
[30] Bansal, G.; Wright, J. E. I.; Zhang, S.; Zernicke, R. F.;
Uludag, H. J Biomed Mater Res 2005, 74, 618.
[31] Bansal, G.; Wright, J. E. I.; Kucharski, C.; Uludag, H.
Angew Chem, Int Ed 2005, 44, 3710.
[32] Bala, J. L. F.; Kashemirov, B. A.; McKenna, C. E.
Synth Commun 2010, 40, 3577.
[33] Yewle, J. N.; Puleo, D. A.; Bachas, L. G. Bioconjugate
Chem 2011, 22, 2496.
[41] Ehrick, R. S.; Capaccio, M.; Puleo, D. A.; Bachas, L.
G. Bioconjugate Chem 2008, 19, 315.
[42] Ruel, R.; Bouvier, J. P.; Young, R. N. J Org Chem
1995, 60, 5209.
[34] Kub´ıcˇek, V.; Kotek, J.; Hermann, P.; Lukesˇ, I. Eur J
Inorg Chem 2007, 333.
[35] Liu, W.; Xu, L.; Zhang, H.; You, J.; Zhang, X.; Sheng,
R.; Li, H.; Wu, S.; Wang, P. Org Biomol Chem 2009,
7, 660.
[36] Gottlieb, H.; Kotlyar, V.; Nudelman, A. J Org Chem
1997, 62, 7212.
[37] (a) Otwinovski, Z.; Minor, W. HKL DENZO and
Scalepack Program Package; Nonius BV: Delft, The
Netherlands, 1997, (b) Otwinovski, Z.; Minor, W.
Methods Enzymol 1997, 276, 307.
[43] Benedict, J. J.; Degenhardt, C. R.; Poser, J. W. US
Patent 4 830 847, 1987.
[44] McKenna, C. E.; Schmidhauser, J.: J Am Chem Soc,
1979, 17, 739.
[45] (a) Willwohl, H.; Wolfrum, J.; Gleiter, R. Laser Chem
1979, 10, 63, (b) Sueishi, Y.; Sugiyama, Y.; Ya-
mamoto, S.; Nishimura, N. J Phys Org Chem 1993, 6,
478.
[46] (a) Marczewski, A. W.; Jaroniec, M. Monatsh Chem
1983, 114, 711, (b) Jaroniec, M.; Derylo, A.; Mar-
czewski, A. W. Monatsh Chem 1983, 114, 393.
[47] Vitha, T.; Kub´ıcˇek, V.; Hermann, P.; Kolar, Z. I.;
Wolterbeek, H. T.; Peters, J. A.; Lukesˇ, I. Langmuir,
2008, 24, 1952.
[38] Altomare, A.; Cascarano, G.; Giacovazzo, C.;
Guagliardi, A.; Burla, M. C.; Polidori, G.; Camalli,
M. J Appl Crystallogr 1997, 27, 435.
Heteroatom Chemistry DOI 10.1002/hc