Synthesis and evaluation of the europiumIII and zinc II complexes as luminescent bioprobes in high content cell-imaging analysis

Article abstract of DOI:10.1016/j.jinorgbio.2011.08.023

Novel phenanthroline derivatives and their europium(III) and zinc(II) complexes have been prepared in up to 92%. In contrast to the stable zinc complexes, the europium compounds exhibit a strong luminescence in THF solution. However, quenching of the emis

Full text of DOI:10.1016/j.jinorgbio.2011.08.023

Journal of Inorganic Biochemistry 105 (2011) 1589–1595
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Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis and evaluation of the europiumIII and zincII complexes as luminescent
bioprobes in high content cell-imaging analysis
a
b
c
a,b
Natalia N. Sergeeva a,b, , Marion Donnier-Marechal , Gisela Vaz , Anthony M. Davies , Mathias O. Senge
⁎
a
SFI Tetrapyrrole Laboratory, School of Chemistry, Trinity College Dublin, Dublin 2, Ireland
Medicinal Chemistry, Institute of Molecular Medicine, Trinity Centre for Health Sciences, Trinity College Dublin, St James's Hospital, Dublin 8, Ireland
Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 8, Ireland
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 April 2011
Received in revised form 19 August 2011
Accepted 19 August 2011
Available online 14 September 2011
Novel phenanthroline derivatives and their europium(III) and zinc(II) complexes have been prepared in up to 92%.
In contrast to the stable zinc complexes, the europium compounds exhibit a strong luminescence in THF solution.
However, quenching of the emission is observed in DMSO indicating complete dissociation of the complexes back
to free ligands in this solvent. 1H NMR studies of the Eu(III)-complexes 5 and 6 also confirmed the existence of
different states depending on the solvent used. Moreover, it was found that compound 5 is stable in EtOH–PBS
solutions; here a strong signal in the emission spectra corresponding to the europium ion was detected. No spectral
changes were observed for the zinc(II) complexes, they were shown to be stable in the media. These metal com-
plexes can be used as fluorescence markers for the diagnosis of oesophageal squamous carcinoma (OE21) cells at
low concentrations. Cell images were acquired using the compounds 5, 7–9 as luminescent agents. The first images
were taken already after 20 min incubation time at a very low concentration range (0.7–1.6 μM).
© 2011 Elsevier Inc. All rights reserved.
Keywords:
Luminescent bioprobes
Lanthanides
Intracellular imaging
Photophysical analysis
Cancer
1. Introduction
only limited information is available on the real state of such metal com-
plexes in different media.
In past decades, molecular imaging technology has become a cen-
tral tool in biology, particularly in bioassays and cell imaging, cancer
research, and clinical trials in medicine [1–5]. The development of
novel luminescent probes for intracellular analysis, that are efficient
and kinetically stable, is one of the vital goals. Rare earth metal com-
plexes are now versatile targets in many areas of research, such as pho-
tochemistry, biomedicine and nanotechnology [6–13]. Special attention
has been given to the luminescence properties of lanthanide ions emitting
at a millisecond lifetime-scale and in the visible region for europium(III)
and terbium(III) complexes [14–17]. These remarkable photophysical
properties are key factors to make them molecules of choice for cellular
imaging and diagnostics [18–21]. A variety of the different Ln(III) com-
plexes have been synthesised, however a major problem is their instabil-
ity in aqueous media. The lanthanide ion and its neutral ligands are linked
via weak coordination bonds and generally exist in an equilibrium of “free
ligand”⇆“metal complex”, which can be shifted in a medium via solvent
exchange [22–23]. The metal complexes are prone to dissociation in the
presence of solvent molecules. Disadvantageously, in some cases this
can lead to an irreversible dissociation into the “free ligand” form in
water through completion of the first coordination sphere [22–25] of a
lanthanide ion with solvent molecules. This normally prevents the use
of Ln(III) complexes as luminescent markers for bioassays. Nevertheless,
Zinc compounds are known to be highly fluorescent and present
an alternative to lanthanide complexes. Thus, many photochemical
studies targeted at modelling the natural situation use more stable
zinc complexes. The propensity of zinc to act as an acceptor atom is
used in many supramolecular approaches and/or for the modulation
of chromophore properties via ligand binding. Due to their biological
relevance, technical importance and good stability, they have found
significant use as industrial pigments, electron transfer components,
for photochemical transformations and in photobiotechnology [26].
Here, we have studied the stability and photophysical efficiency of
europium and zinc complexes of phenanthroline derivatives. These mol-
ecules are strongly absorbing chromophores often used as antennas for
indirect excitation of metal ions, where direct excitation of a metal is inef-
ficient. Phenanthroline itself is known to inhibit metallopeptidases and its
various metal complexes have been prepared and tested as bioprobes
[27–37]. Here we present results based on the synthesis, behaviour indif-
ferent solvents monitored by NMR spectroscopy, photophysical analysis
and cellular imaging of novel metal complexes at low concentrations.
2. Experimental
2.1. Materials and methods
A 1 mM stock solution was prepared in THF, MeOH, DMSO and
EtOH for each compound. Dilutions were carried out directly before
starting the analyses. For cell stain imaging a stock solution prepared
⁎
Corresponding author at: SFI Tetrapyrrole Laboratory, School of Chemistry, Trinity
College Dublin, Dublin 2, Ireland. Tel.: +353 18968537; fax. +353 18968536.
E-mail address: sergeevn@tcd.ie (N.N. Sergeeva).
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.08.023

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N.N. Sergeeva et al. / Journal of Inorganic Biochemistry 105 (2011) 1589–1595
in EtOH was diluted with phosphate buffered saline (PBS) to different
concentrations. Buffer solutions prepared at the desired concentra-
tions were added directly to the cell culture. Total EtOH concentration
was lower than 0.1%. After adding compounds, cells were incubated
at 37 °C in 5% CO2 for 20 min to 24 h. Cells (human esophageal squa-
mous carcinoma OE21) were routinely cultured in RPMI 1640
(Hyclone, USA) with 10% inactivated foetal bovine serum (Hyclone,
USA) and 1% Penicillin/(Hyclone, USA). These cultures were grown
in sterile filtered top cell culture flasks (Nunc, Denmark). Cultures
were split 1:8 when 70–80% confluency was reached. Cells were
split using typsin 0.25% solution, with EDTA (Hyclone, USA) and
kept at 37 °C and 5% CO2 in a humidified atmosphere. Culture medi-
um was changed every 3 to 4 days until confluency. The cell lines
were plated at a concentration of 1.0×105 cells/mL into sterile 96-
well plates (Nunc, Denmark) and left to attach overnight.
2.4.2. 2-{4-[1′-(2′-Trimethylsilyl)ethynyl]phenyl}imidazo[4,5-f]-1,
10-phenanthroline 3
Yellow solid (1.24 g, 66%). Mp 258 °C; δH (400 MHz, DMSO-d6)
9.06 (m, 2H, Hphenan), 8.92 (d, J=6.9 Hz, 2H, Hphenan), 8.29 (d,
J=8.4 Hz, 2H, HPh), 7.86 (dd, J=8.0 Hz, J=4.3 Hz, 2H, Hphenan),
7.70 (d, 2H, J=8.4 Hz, HPh), 0.27 (s, 9H, TMS); δC (150.2 MHz,
DMSO-d6) 149.9, 148.3, 144.0, 132.6, 130.4, 129.9, 126.6, 123.3,
105.2, 96.5 (m); m/z (TOF MS) 393.152 (M+H. C24H21N4Si requires
393.153); UV–visible (UV–vis) (CH3OH): λmax (lgε) 283 (4.3), 335
(4.4).
2.4.3. 2-(4-Pyridinyl)imidazo[4,5-f]-1,10-phenanthroline [40] 4
Brown solid (1.07 g, 76%); δH (400 MHz, DMSO-d6) 9.20 (m, 2H,
H
phenan), 8.92 (m, 4H, Hphenan and HPy), 8.27 (m, 2H, HPy), 7.97 (m,
2H, Hphenan). UV–visible (UV–vis) (CH3OH): λmax (lgε) 273 (4.6),
322 (4.3), 387 (3.6).
2.2. General
2.5. Synthesis of the imidazo[4,5-f]-1,10-phenanthroline europium (III)
complexes 5–7
1H NMR spectra were recorded on a Bruker DPX 400 (400 MHz
and 600 MHz for 1H NMR). Chemical shifts are reported in ppm re-
ferred to TMS set at 0.00 ppm. All photophysical measurements
were performed in THF, MeOH, EtOH, DMSO and PBS. All emission
spectra were recorded on Fluorolog Horiba Jobin Yvon spectrometer.
High resolution mass-spectra (HRMS) were measured on MaldiQ-Tof
Premier Micromass and Micromass/Waters Corp. USA liquid chroma-
tography time-of-flight spectrometer equipped with ES source. UV–
visible (UV–vis) measurements were performed on Specord 250
Analytik Jena instrument. Melting points were acquired on Stuart
SMP10 melting point apparatus and are uncorrected. Thin layer chro-
matography (TLC) was performed on silica gel 60 F254 (Merck) pre-
coated aluminium sheets. Spectroscopy grade solvents were used in
all cases. All commercial chemicals and solvents were supplied by
Sigma-Aldrich and used without further purification. Abbreviations
“s” — singlet, “d” — doublet, “dd” — doublet of doublets, “m” — multi-
plet, “br” — broad, ES MS — electrospray mass-spectrometry, TOF MS
— time-of-flight mass-spectrometry, THF — tetrahydrofuran, MeOH —
methanol, EtOH — ethanol, DMSO — dimethylsulfoxide, PBS — phos-
phate buffered saline were used.
To an appropriate imidazo[4,5-f]-1,10-phenanthroline derivatives
2–4 (0.63 mmol) was dissolved in hot ethanol (100 mL) and an etha-
nolic solution of Eu(NO3)3•5H2O (90 mg, 0.21 mmol) was added. The
resulting mixture was heated at reflux until the starting material dis-
appeared (TLC-control, ca. 30 h). The yellow solid formed was fil-
tered, washed with ethanol and dried under vacuum to give the
desired product 5–7.
2.5.1. 2-(4-Bromophenyl)imidazo[4,5-f]-1,10-phenanthroline europium
(III) complex 5
Yellow solid (0.47 g, 58%). MpN300 °C; δH (400 MHz, THF-d8,
TMS) 13.19 (3H, br s, NH), 8.86 (6H, br m, Hphenan), 8.08 (16H, br
m, Hphenan and HPh), 6.50 (8H, br m, Hphenan); m/z (TOF MS)
1274.971 (M. C57H33Br3EuN12 requires 1274.976).
2.5.2. 2-{4-[1′-(2′-Trimethylsilyl)ethynyl]phenyl}imidazo[4,5-f]-1,10-
phenanthroline europium(III) complex 6
Yellow solid (0.77 g, 92%). MpN300 °C; δH (400 MHz, THF-d8, TMS)
13.00 (br s, 3H, NH), 8.75 (br m, 6H, Hphenan), 8.18 (br m, 10H, Hphenan
and HPh), 7.63 (m, 6H, HPh), 6.26 (br m, 8H, Hphenan), 0.20 (s, 27H,
TMS); m/z (TOF MS) 1302.292 (M-3Me+H2O. C69H53EuN12OSi3
requires 1302.299.
2.3. Synthesis of 1,10-phenanthroline-5,6-dione [38] 1
To a dry mixture of 1,10-phenanthroline (2.0 g, 0.01 mol) and KBr
(10.0 g, 0.08 mol), H2SO4 (40 mL) followed by HNO3 (20 mL) was
added dropwise at 0 °C. The resulting mixture was heated at 100 °C
until the bromine vapours disappeared. The solution was poured
carefully onto ice and slowly neutralised to pHN7 with Na2CO3 (satu-
rated solution and powder). The product was extracted with dichlor-
omethane and dried over Na2SO4. The solvents were evaporated to
give a yellow solid that was dried under vacuum (88–95%). 1H NMR
(400 MHz, CDCl3): δ 9.16 (dd, J=4.6 Hz, J=1.7 Hz, 2H); 8.54 (dd,
J=7.9 Hz, J=1.6 Hz, 2H); 7.62 (dd, J=7.9 Hz, J=4.7 Hz, 2H).
2.5.3. 2-(4-Pyridinyl)imidazo[4,5-f]-1,10-phenanthroline europium(III)
complex 7
Yellow solid (0.36 g, 55%). MpN300 °C; δH (400 MHz, D2O+
DMSO-d6, TMS) 7.82 (br m, 30H, Hphenan and HPh), 0.52 (br, 3H,
NH); m/z (ES) 968.17 (M-Py+H. C50H30EuN14 requires 968.21), m/z
(TOF MS) 906.18 (M-2Py+H2O). C44H27EuN13O requires 906.17,
1044.23 (M). C54H33EuN15 requires 1044.44, 1064.30 (M+H2O+
2H). C54H37EuN15O requires 1064.25.
2.4. Synthesis of imidazo[4,5-f]1,10-phenanthroline derivatives 2–4
2.6. Synthesis of imidazo[4,5-f]-1,10-phenanthroline zinc complexes
8–10
1,10-Phenanthrolin-5,6-dione 1 (1.0 g, 4.76 mmol) and ammoni-
um acetate (11.7 g, 0.15 mol) were dissolved in hot glacial acetic
acid (50 mL) at ca. 70 °C and an appropriate aldehyde (4.76 mmol),
dissolved in 20 mL of glacial acetic acid, was added. The resulting
mixture was heated at 70–80 °C for 2 h (TLC-control). Then the solu-
tion was allowed to cool to room temperature and was neutralised
with 1 N NaOH. The yellow compound was filtered, washed with
water and dried under vacuum.
To an appropriate imidazo[4,5-f]-1,10-phenanthroline derivatives
2 or 4 (0.54 mmol) dissolved in 10–20 mL of THF–MeOH (1:1, v/v)
was added a solution of ZnCl2 (0.27 mmol) in MeOH. A yellow precip-
itate was formed immediately after addition of the metal salt. The
mixture was stirred at room temperature for 24 h and filtered. The
solid residue was washed with ethanol and dried in vacuum.
2.6.1. 2-(4-Pyridinyl)imidazo[4,5-f]-1,10-phenanthroline zinc(II)
complex 8
Pale yellow solid (36 mg, 20%). MpN300 °C; δH (600 MHz, DMSO-
d6) 10.09 (s, 2H, NH), 9.16 (br, 8H, Hphenan), 8.96 (d, J=4.6 Hz, 4H,
2.4.1. 2-(4-Bromophenyl)imidazo[4,5-f]-1,10-phenanthroline 2
Yellow solid (1.43 g, 80%); analytical data were in accordance with
the literature [39].

N.N. Sergeeva et al. / Journal of Inorganic Biochemistry 105 (2011) 1589–1595
1591
Hphenan), 8.27 (br, 8H, HPy); δC (150.2 MHz, DMSO-d6) 162.1, 151.4,
144.1, 134.8, 133.3, 121.0; m/z (TOF MS ES+) 397.008 (M-L-Cl+H).
18H12N5ClZn requires 397.007; UV–visible (UV–vis) (CH3OH): λmax
C
(lgε) 277 (4.0), 319 (3.9).
2.6.2. 2-(4-Bromophenyl)imidazo[4,5-f]-1,10-phenanthroline zinc(II)
complex 9
Scheme 2. Synthesis of europium(III) complexes 5–7.
Pale yellow solid (152 mg, 69%). MpN300 °C. δH (600 MHz, DMSO-
d6) 14.22 (br, 2H, NH), 9.17 (br, 4H, Hphenan), 8.82 (m, 4H, Hphenan),
8.23 (d, 4H, HPh), 8.08 (dd, 4H, HPh), 7.84 (m, 4H, Hphenan); δC
(150.2 MHz, DMSO-d6) 150.8, 146.6 (m), 137.7, 132.1, 128.8, 125.6,
123.4; m/z (TOF MS) 812.968 (M+H). C38H23N8ZnBr2 requires
812.970; UV–visible (UV–vis) (CH3OH): λmax (lgε) 283 (4.8), 390
(4.8), 318 (4.7).
up to 69% yield (Scheme 3). Noteworthy, the different stoichiometric
salt-ligand ratios result only in the formation of the 2 to 1 complexes
8 and 9; neither 1:1 nor 3:1 complexes were observed, as reported in
some cases.
3.2. Photophysical behaviour of compounds 5–10
2.7. (2-(4-Bromophenyl)-N-hexyl)imidazo[4,5-f]-1,10-phenanthroline
zinc complex 10
Influence of the structural differences in complexes 5–10 on their
photophysical properties was investigated by comparing the spectro-
scopic profiles of the free ligands, zinc and europium compounds. The
complexes 5–10 exhibited absorption bands between 280 and
400 nm that strongly depended on the ligation state and type of
metal inserted. Slight shifts, intensity and shape differences were ob-
served in the absorption spectra for the free ligand 2 and its europium
5 and zinc 9 complexes (Fig. 1).
Emission spectra for this series of compounds were recorded at var-
ious irradiation energies. All three types of compounds exhibited an
emission band at 405 nm which is characteristic for the ligand's emis-
sion followed by an additional band corresponding to metal–ligand
transitions (Fig. 2).
The europium complex 5 showed the characteristic bands of lan-
thanide ion emission between 670 nm and 710 nm. In the case of
the zinc complex 9, a splitting of the band at 410 nm was observed
with the new maximum at 465 nm as the result of complex formation
(Table 1).
In addition to the emission profile requirements, fluorescence im-
aging of the biomaterials requires reasonable Stokes shifts. Indeed,
according to the results reported in Table 1, all compounds show
large Stokes shifts.
To a solution of 2-(4-bromophenyl)imidazo[4,5-f]-1,10-phenan-
throline 2 (230 mg, 0.61 mmol) in anhydrous DMF (10 mL), K2CO3
(170 mg, 1.21 mmol) was added under argon. The mixture was heated
at 60 °C for 2 h and 1-bromohexane (0.13 mL) was added. The reaction
was refluxed at 80 °C for 48 h and quenched with 10 mL of water. The
product was extracted with 3×20 mL of CHCl3, washed with water
and dried over Na2SO4. The solvents were evaporated to give the
crude product. To the residue (80 mg, 0.17 mmol) dissolved in 10 mL
of THF–MeOH (1:1, v/v) was added a solution of ZnCl2 (12 mg,
0.087 mmol) in MeOH. The reaction gave the complex 10 as a pale yel-
low solid (5 mg, 6%). Mp 260 °C. δH (400 MHz, DMSO-d6): 9.79 (d,
J=8.3 Hz, 2H, Hphenan), 9.54 (br, 2H, Hphenan), 9.23 (m, 4H, Hphenan),
8.47 (m, 2H, Hphenan), 8.32 (d, J=8.3 Hz, 4H, HPh), 8.14 (m, 2H, Hphenan),
7.89 (d, J=8.3 Hz, 4H, HPh), 5.92 (t, J=7.0 Hz, 4H, CH2), 2.09 (m, 4H,
CH2), 1.55 (m, 4H, CH2), 1.37 (m, 8H, CH2), 0.91 (t, J=7.0 Hz, 6H,
CH3). m/z (TOF MS) 981.154 (M+H. C50H47N8ZnBr2 requires
981.158); UV–visible (UV–vis) (CH3OH): λmax (lgε) 294 (4.2), 301
(4.2).
3. Results and discussion
3.1. Synthesis
3.3. Luminescence: solvents dependency and dissociation of the
europium(III) complexes
The neutral bidentate ligands 2–4 were synthesized using a sim-
ple two-step approach starting from commercially available 1,10-
phenanthroline. 1,10-Phenanthroline-5,6-dione 1 readily reacted with
various aldehydes to give compounds 2–4 in up to 80% yield (Scheme 1).
The metal complexes 5–10 were prepared using the derivatives 2–
4 and the corresponding metal salts in good yields. Formation of the
europium(III) complexes was achieved in ethanol in the presence of
europium(III) trinitrate with the ligands 2–4 in a stoichiometric
ratio of 1 to 3 yielding the compounds 5–7 in 58%, 92%, and 55%
yields, respectively (Scheme 2). The structure of the new europium
compounds have been confirmed by MS-spectroscopy and 1H NMR
(in THF) analysis.
A change in a photophysical profile of many optical materials such
as a “switch-on” and “-off” can be achieved simply by varying a sol-
vate medium through temporary quenching of the emission of one
or more components of the system. However, in some cases these
changes can be irreversible and can be used to detect a particular mol-
ecule or ion. We have analysed the behaviour of the phenanthrene-
based europium complexes 5–7 under different conditions. The effects
of the solvents can even be seen with the “naked eye”. Simple illumina-
tion of the different solutions of complex 5 with a standard laboratory
UV-lamp revealed the differences (Fig. 3).
A detailed analysis of compounds 5–7 was performed in various
solvents including THF, D2O, EtOH and DMSO. The presence of the
metal complexes was clearly verified for THF and D2O solutions.
Synthesis of the zinc(II) complexes was carried out using ZnCl2 in
THF–MeOH in a similar manner to produce the compounds 8–10 in
Scheme 1. Synthesis of 1,10-phenanthroline-5,6-dione 1 and derivatives 2–4.
Scheme 3. Synthesis of zinc(II) complexes 8–10.

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N.N. Sergeeva et al. / Journal of Inorganic Biochemistry 105 (2011) 1589–1595
Table 1
Photophysical parameters of 5 and 6 in THF and 8–10 in MeOH.
Entry
Absorption maxima, nm
Emission maxima, nma
5
279, 325
405; 591; 617; 646; 681
6
282; 339
405; 591; 617; 650; 681
8
9
10
277; 319
283; 290; 318
294; 301
400
560
495
a
Emission spectra recorded with 280 nm.
Fig. 1. Absorption spectra of compounds 2, 5 and 9 (10−5 M) in THF.
Fig. 3. Solvent dependency of solutions of 5 in THF, EtOH, D2O and DMSO (from left to
right) irradiated with UV-light (365 nm).
Here the typical emission signals corresponding to 5D0 → 7F0,1,2,3,4
transitions of the europium(III) ion in 5 and 6 were observed (Fig.4).
No lanthanide emission was found for 5 and 6 in DMSO. Here, only
the fluorescence corresponding to the ligand unit was detected. These
results indicate that the emission of the Eu(III) ion is either quenched
by solvent molecules entering the first solvation sphere or indicate
the complete dissociation of the complex back to a free ligand and
lanthanide salt with solvent molecules as new ligands (Scheme 4).
Detailed studies on solvent exchange mechanisms have been car-
ried out for the series of d- and f-metal complexes [22, 23]. The sol-
vent exchange rates differ greatly between harder and softer atom
donors of a solvent and different solvent exchange mechanisms (as-
sociative vs. dissociative activation) have been proposed [22, 23,
41]. Certainly, other factors such as different steric and electronic
characteristics of the system components, the presence of an exten-
sive π-moiety on the NN part of the ligand, an ease of the π-system
to interact with nonbonding electrons of the metal, and the flexibility
and stretching of the chelate bite mode have to be taken in account.
this, the effects of the different deuterated solvents were analysed by 1H
NMR spectroscopy. Fig. 5 gives an example of the different behaviours of
compound 5 in THF-d8, Methanol-d4 and DMSO-d6.
Clearly, europium complex 5 exists in its “metal complex”-form in
THF solution. Here, the phenanthroline hydrogen atoms in closest
proximity to the Eu(III)-centre are shifted up-field. This is in good
agreement with the fact that europium(III) complexes have a low
paramagnetic relaxation enhancement (T1 and T2 shortening effect)
and small paramagnetic shifts [22, 23, 41, 42]. The spectrum of com-
pound 5 in DMSO is identical to that of “free ligand” 2. Clearly, this is
the only form that exists in DMSO for compound 5. Similar results
were reported for europium(III) complexes with phosphine oxides
in DMSO-d6 [43]. However, complex 7 showed a notable stability in
comparison to 5 and 6 in a deuterated DMSO-D2O solvent mixture.
Interestingly, the mass spectrometric analysis of compounds 5 and
6 carried out with a DMSO solution did not give any molecular ion
corresponding to these complexes. Only the signal of the free ligand
was detected in all cases. These results were observed for the europi-
um complexes 5 and 6 and can be explained by a potential dissocia-
tion of the complexes through ligand exchange with solvent
molecules. No such changes were observed for the zinc complexes
8–10 in various solvents, including MeOH and THF.
3.4. NMR studies
As the luminescence studies indicated a possible decomposition of
the europium(III) complexes 5 and 6 in DMSO we employed NMR anal-
ysis to determine the existence of different states for these materials. For
Fig. 2. Comparison of the emission spectra of compound 2 and its europium 5 and zinc
9 complexes (10−5 M) at excitation 280 nm in THF.
Fig. 4. Emission spectra of complex 5 in different solvents at 335 nm excitation and
with a 420 nm filter.

N.N. Sergeeva et al. / Journal of Inorganic Biochemistry 105 (2011) 1589–1595
1593
Scheme 4. Dissociation of europium(III) complex in DMSO.
3.5. Aqueous solutions
The analysis of biological systems requires work under aqueous
conditions. Some luminescent materials can be reversibly quenched
by water via the formation of hydrogen bonds or directly by water
molecules. Thus, it is important to establish the real state of the com-
plex and its photophysical characteristics in aqueous solution before
the substance enters a cell. It is necessary to establish whether any
observed fluorescence quenching is the result of reduced emission
of the europium centre or is due to irreversible decomposition of
the metal complex.
Here, the emission spectrum of compound 5 in EtOH–PBS buffer at
physiological pH (7.4) is similar to that in THF. It clearly shows the
strong signals of the europium-ion bonded to the phenanthroline ligand
(Fig. 6).
The materials 5 and 7 were stable in the buffer solution for quite
some time (24–48 h); however, compound 6 does not exhibit any
lanthanide-corresponding emission under these conditions.
Fig. 6. Emission spectrum of complex 5 in EtOH–PBS at pH 7.4.
were taken after 20 min and 1 h incubation times at 405 nm irradia-
tion energy and detection at 535 nm (Fig. 7).
3.6. Cell imaging
The zinc complexes 8–10 were less fluorescent after 1 h incubation at
these concentrations, the fluorescent images were obtained for the zinc
compounds 8 (1.6 μM) and 9 (0.84 μM) after 2 and 6 h incubation, respec-
tively (Fig. 8). Interestingly, the fluorescence of 9 diminished with time,
and no intracellular fluorescence was observed for complex 10.
Overall, the europium compounds represent a good intensity after 1 h
incubation and are photostable over 6 h. In comparison to the europium
materials, the zinc complexes required longer incubation times and
show lower photostability. Furthermore, fluorescence decay occurred
over time as a result of photobleaching.
Oesophageal cancer is a common and aggressive form of cancer
and has shown a dramatic increase in Europe and North America in
the past few years [44–47]. Survival rates in Europe are only about
10% [48] and the incidence of this cancer, especially in the UK and
Eire, is rising [49]. Thus, we chose the oesophageal SCC cell line
OE21 (human oesophageal squamous carcinoma) for cell labelling
studies [50].
The determination of the lowest staining concentration was per-
formed in the 0.74 to 10 μM range. We have demonstrated that com-
plexes 5 and 7 are stable (up to 48 h) in aqueous solutions (EtOH–
PBS) and it still exhibits a strong luminescence (Fig. 6). The 1 mM
stock ethanolic solutions of the compounds 5 and 7 were diluted
with PBS buffer and an excess of crystals was filtered off to give con-
centrations of 0.74 μM for 5 and 8.2 μM for 7. The fluorescence images
4. Conclusion
We have demonstrated that the new zinc(II) and europium(III)
complexes 5–10 can be synthesized in good yield. The emission spectra
of compounds 5–7 show a strong signal corresponding to the Eu(III)-ion
Fig. 5. 1H NMR (400 MHz) of europium(III) complex 5 in different deuterated solvents.

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N.N. Sergeeva et al. / Journal of Inorganic Biochemistry 105 (2011) 1589–1595
Fig. 7. In cell images of complexes 5 (a) and 7 (b) in OE21-fixed cells after 1 h incubation (irradiation at 405 nm and detection at 535 nm).
Fig. 8. In cell images of the complexes 8 (a) and 9 (b) in EtOH–PBS pH 7.4 for OE21-fixed cells after 2 h incubation (irradiation at 405 nm and detection at 535 nm).
in solvents such as THF and D2O. However, quenching of the emission is
observed in DMSO. Moreover, NMR analysis has also confirmed that
DMSO-d6 solutions contain only the free ligand for compounds 5 and
6. Therefore, materials 5 and 6 are prone to dissociation in media such
as DMSO. Interestingly, complex 7 is still present in the D2O–DMSO-d6
mixture. Analysis of complexes 1–3 in EtOH–PBS aqueous buffer
revealed an intense emission of the lanthanide ion for 5 and 7; however,
6 exhibits a spectrum similar to the one in DMSO. No spectral changes
were observed for the zinc(II) complexes, they were shown to be stable
in the media. Fluorescence cell images were recorded between 20 min
to 6 h incubation times using complexes 5–9. The europium samples
show a good intensity after 1 h incubation and are photostable over
6 h. In contrast, the zinc complexes required longer incubation times
and are less photostable. Furthermore, a decrease in fluorescence over
time occurred as a result of photobleaching.
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