Design of novel, water soluble and highly luminescent europium labels with potential to enhance immunoassay sensitivities

Article abstract of DOI:10.3390/molecules22101807

To meet the continual demands of more-sensitive immunoassays, the synthesis of novel luminescent Eu(III) chelate labels having similar substituted 4-(phenylethynyl)pyridine chromophores in three different chelate structure classes are reported. Significan

Full text of DOI:10.3390/molecules22101807

molecules
Article
Design of Novel, Water Soluble and Highly
Luminescent Europium Labels with Potential to
Enhance Immunoassay Sensitivities
Henri Sund 1, Kaj Blomberg 1, Niko Meltola 1,2 and Harri Takalo 1,
*
1
Radiometer Turku Oy, Biolinja 12, 20750 Turku, Finland; henri.sund@radiometer.fi (H.S.);
kaj.blomberg@radiometer.fi (K.B.); niko.meltola@arcdia.com (N.M.)
Present affiliation ArcDia International Oy Ltd., Lemminkäisenkatu 32, 20520 Turku, Finland
Correspondence: harri.takalo@radiometer.fi or harrijuha.takalo@gmail.com; Tel.: +358-40-508-7104
2
*
Received: 29 September 2017; Accepted: 19 October 2017; Published: 24 October 2017
Abstract: To meet the continual demands of more-sensitive immunoassays, the synthesis of novel
luminescent Eu(III) chelate labels having similar substituted 4-(phenylethynyl)pyridine chromophores
in three different chelate structure classes are reported. Significantly enhanced luminescence intensities
were obtained, evidently caused by the intra-ligand charge transfer (ILCT) mediated sensitization,
but the alternative ligands triplet state process cannot be ruled out. Based on the present study, even
quite small changes on the chelate structure, and, especially, on the substituents’ donor/acceptor
strength on both ends of 4-(phenylethynyl)pyridine subunits have an unpredictable effect on the
luminescence. The highest observed brightness was 16,400 M−1 cm−1 in solution and 69,500 M−1
cm−1 on dry surface, being 3.4 and 8.7 fold higher compared to the reference chelate. The new label
chelates provide solutions for improved assay sensitivity up-to tenfold from the present concepts.
Keywords: europium chelate; label; (phenylethynyl)pyridine chromophore; luminescence; intra-ligand
charge transfer
1. Introduction
Time-resolved fluorometry (TRF) employing long-lifetime emitting luminescent lanthanide
chelates has been applied in many specific binding assays, such as immunoassays, DNA hybridization
assays, receptor-binding assays, enzymatic assays, bio-imaging such as immunocytochemical,
immunohisto-chemical assays or cell based assays to measure the wanted analyte at very low
concentration [1–3]. Lanthanide chelates have special properties offering proper alternative markers
in bio-affinity assays: (i) the difference between the excitation and emission wavelengths is
large; (ii) normally, they have long emission life-time compared to the background, and, thus,
the time-resolved fluorescence (TR-FIA) measurement techniques together with narrow emission
lines eliminate the fluorescence background almost completely; (iii) the concentration quenching is
small and enables using wide dynamic assay range without additional dilutions, and (iv) several
different sample matrixes e.g., whole blood, serum and plasma can be used for the assays. Applications
based on TRF have been commercialized in the fields of clinical diagnostics and drug discovery [1].
For TRF applications, an optimal label has to fulfill several strict requirements. First, the label has
to be photo-chemically stable both in the ground and excited states, and it has to be kinetically and
chemically stable. The excitation wavelength has to be as high as possible, preferable over 300 nm. It has
to have efficient cation emission i.e., intense brightness (excitation coefficient x quantum yield, εΦ).
The lanthanide chelate has to have a high absorptivity and efficient energy transfer from the excited
level of the so-called antenna ligand (i.e., chromophore) to the proper emitting level of the lanthanide
ion. Luminescence quenching by water molecules should be prevented to enable a long luminescence
Molecules 2017, 22, 1807; doi:10.3390/molecules22101807
www.mdpi.com/journal/molecules

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decay time, but, at the same time, the chelate has to have good water solubility. For the purpose of
labeling, it should have a reactive group to allow covalent attachment to a bio-specific binding reactant,
and the affinity and nonspecific binding properties of the labeled biomolecules have to be retained.
It is challenging to prepare a chelate-label molecule that fulfills all requirements, and, therefore, certain
compromises are generally made in the development of suitable labels. As a consequence hereof,
a number of attempts (see e.g., [4]) have been made to tune the photo-physical properties of the chelate
labels suitable for TRF applications, and only a few viable chelate labels are in commercial use.
Scheme 1. The studied label structures numbered 1–8.
Since the discovery in the mid-1980s and the later publication of the phenylethynylpyridine
antenna for Eu(III) chelates [
in literature, inter alia three [
macro-cyclic ligands [ 16 19]. The nine-dentate Eu(III) label (1
5
,
6
6
], several new chelates utilizing the chromophore have been described
10], seven [ 11 13] and nine-dentate acyclic [ 14 15] as well as
in Scheme 1) has been applied already
–
6–
8,
–
8, ,
8,
–
almost for 15 years in TRF immunoassays [14,20–24] especially in near-patient testing such as critical
care and emergency situations. The technology has offered quick, easy-to-use, high quality and

Molecules 2017, 22, 1807
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quantitative point-of-care testing platform called as AQT90 FLEX (Radiometer Medical ApS). The aim of
the present work is to enhance the brightness and to red-shift the excitation wavelength of the present
label
For that purpose, we prepared three different nine-dentate chelate label designs i.e., with two and three
individual chromophore moieties in acyclic and macrocyclic ligand formats (chelates in Scheme 1).
1 to be able to use a UV LED based excitation [25] with significantly improved assay sensitivity.
2–8
The objective was to study the effect of electron-releasing substituents (OCH2COO−) in mesomeric
para- and orto-position of phenylethynylpyridine moiety together with electron-withdrawing (COO−)
as well as releasing (CH2) groups at the other end of the chromophoric moiety in those three different
ligand groups. The novel chelates were tested after coupled with taurine (2a–8a) and protein (2b–8b)
being comparable to the derivative 1a,b of the used reference chelate 1.
2. Results
2.1. Syntheses
The syntheses of two different terminal acetylenes 14 and 15 used in the study started from
4-bromobenzene-1,3-diol and benzene-1,3,5-triol, which were transformed to the corresponding
intermediates 11 and
9 with the reaction of ethyl bromoacetate in dry MeCN and using K2CO3
as the needed base (Scheme 2). For the synthesis of compound 9, careful control of reaction conditions
is required to achieve a reasonable yield; otherwise, a dramatic drop in yield together with purification
problems will be the result. Direct iodination of the compound
9 with I2 and NaHCO3 in H2O/CHCl3
mixture afforded poor results, whereas the reaction with N-chlorosuccimide and NaI in acetic
acid described by Yamamoto et al. [26] produced the iodo derivative 10 in quantitative yield.
As an alternative route to the compound 10, a reaction of 2-iodo-benzene-1,3,5-triol [27] with
ethyl bromoacetate was entirely unsuccessful. Both halides 10 and 11 were conjugated with
trimethysilylacetylene using the Sonogashira reaction in the presence of a catalytic amount of
Pd(II) catalyst and CuI under argon. Microwave heating at 100 ◦C for 30–45 min in diethyl amine
(DEA) and dimethylformamide (DMF) gave appropriate yields of products. In the reaction with 11,
an improved yield was obtained by using PPh3 in the mixture. We also tested using a corresponding
bromo derivative to the compound 10—nicely obtained by the reaction of 9 with N-bromosuccimide,
but, unfortunately, without success. Finally, the deprotection with tetrabutylammonium fluoride gave
the wanted phenylacetylenes 14 and 15 with quantitative yields.
The second Sonogashira reaction between the acetylenes 14 and 15 and the dibromo intermediate
16 [14] gave ligand esters 17 and 18, respectively. The esters 17 and 18 were hydrolyzed with KOH in
EtOH–H2O followed by the forming of chelates 19 and 20 with EuCl3 in slightly acidic conditions and
precipitating the excess europium as Eu(OH)3 by adjusting the pH to 8.5. The chelates 19 and 20 were
purified by the semi-preparative HPLC.
The activation of the amino group of the chelates by transformation to the corresponding
isothiocyanato group in the final labeling reagents
in H2O/CHCl3.
2 and 3 was performed with thiophosgene
The synthesis of the acyclic labeling regents (
from the protection of aminophenyl acetylene with trifluoroacetic acid anhydride to get compound 21
After the Sonagashira coupling reaction with the 6-bromo-2,6-di-hydroxy-methylpy-ridine 22 28],
diol 23 was transformed with PBr3 into bis(bromomethyl) derivative 24. This product was conjugated
4 and 5 in Scheme 3) with three chromophores started
.
[
with two pyridine subunits 26 in the dry MeCN in the presence of K2CO3 to get the key intermediate 27
.
The needed building block 26 was prepared from 4-bromo-6-bromomethyl-2-carboxyethylpyridine
25 [14] using five-fold excess of glycine ethyl ester hydrochloride in dry MeCN and di-isopropylethylamine.
After the triplet bond coupling reaction of the two bromo groups in the compound 27 with the
acetylenes 14 and 15, the target ester ligands 28 and 29 were hydrolyzed, transformed to the
corresponding Eu(III) chelates 30 and 31 including the HPLC purifications, and, finally, activated to
the target labeling chelates 4 and 5 as described above with the chelates 2 and 3.

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OH
OH
Br
O
COOEt
O
i)
HO
OH
OH
EtOOC
O
COOEt
9
ii)
iv)
O
COOEt
O
O
COOEt
O
O
COOEt
O
iii)
v)
COOEt
R1
COOEt
EtOOC
O
COOEt
Br
I
12: R1 = H
13: R1 = OCH2COOEt
10
11
SiMe3
vi)
COOEt
EtOOC
O
EtOOC
COOEt
O
O
COOEt
O
NH2
NH2
O
O
R1
COOEt
R1
R1
Br
Br
14 and 15
N
N
N
N
N
N
vii)
N
N
N
N
COOEt EtOOC
COOEt EtOOC
COOEt EtOOC
COOEt EtOOC
17 and 18
16
viii)
COO-Na+
+Na-OOC
+Na-OOC
COO-Na+
COO-Na+
+Na-OOC
+Na-OOC
COO-Na+
O
O
O
O
NH2
NCS
O
O
O
O
R1
R1
R1
R1
ix)
N
N
N
N
N
N
Eu3+
-OOC
Eu3+
-OOC
N
N
-OOC
N
N
-OOC
COO-
COO-
COO-
COO-
Na+
Na+
19: R1 = H
20: R1 = OCH2COO-Na+
2 and 3
Scheme 2. Synthesis of compounds
2 and 3. Reactions: (i) and (iv) BrCH2COOEt, K2CO3, MeCN;
(ii) NCS, NaI, CH3COOH; (iii) and (v) trimethylsilylacetylene (TMSA), Pd(II), CuI, DEA, DMF; with the
compound 11 PPh3 was used as an additional catalyst; (vi) TBAF, CH2Cl2; (vii) Pd(II), CuI, TEA, THF;
(viii) KOH, EtOH, H2O, EuCl3; (ix) CSCl2, NaHCO3, CHCl3, H2O.
The synthesis of the unsymmetrical acyclic analogue to the labeling reagent
5
i.e., the Eu(III)
28
chelate in Scheme 4 was more complicated. First, the 6-bromo-2,6-dibromomethylpyridine 32
6
[ ]
was transformed to diester 33 by the reaction with two ethyl glysinates, and, after conjugation to
one equivalent of the compound 25, the triester 34 was obtained with a reasonable yield. It is worth
mentioning that, although in the last step only one main product spot is seen on TLC, besides
the wanted product 34, the TLC spot actually also contains a product in which two equivalents
of the compound 25 have reacted with the compound 33. With a careful column chromatography,

Molecules 2017, 22, 1807
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the two products can be separated from each other. The secondary amino in 33 was coupled to the
bromomethyl group of 37 in almost quantitative yield. The coupling reaction between acetylene 21 and
4-bromo-6-hydroxymethyl-2-carboxyethylpyridine 35 [14] followed by the bromination of the hydroxyl
group in 36 with PBr3 gave the compound 37. After a similar Sonagashira reaction as described above,
the ligand ester 39 was formed followed by the hydrolysis, the formation of Eu(III) chelate 40, the HPLC
purification and activation to the labeling reagent 6.
Scheme 3. Synthesis of compounds 4 and 5. Reactions: (i) TFAA; (ii) Pd(II), CuI, triethylamine (TEA),
tetrahydrofuran (THF); (iii) PBr3, CHCl3; (iv) NH2CH2COOEt, N,N-diisopropylethylamine (DIPEA),
MeCN; (v) K2CO3, MeCN; (vi) Pd(II), CuI, TEA, DMF; (vii) KOH, EtOH, H2O, EuCl3; (viii) CSCl2,
NaHCO3, CHCl3, H2O.

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Scheme 4. Synthesis of compound 6. Reactions: (i) NH2CH2COOEt, DIPEA, MeCN; (ii) and (v) K2CO3,
MeCN; (iii) and (vi) Pd(II), CuI, TEA, THF; (iv) PBr3, CHCl3; (vii) KOH, EtOH, H2O, EuCl3; (viii) CSCl2,
NaHCO3, CHCl3, H2O.

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Scheme 5. Synthesis of compounds
7 and 8. Reactions: (i) Pd(II), CuI, TEA, THF; (ii) PBr3, CHCl3;
(iii) K2CO3, MeCN; (iv) TFA; (v) DIPEA, MeCN; (vi) KOH, EtOH, H2O, EuCl3, citric acid; (vii) CSCl2,
NaHCO3, CHCl3, H2O.
The synthesis of macrocyclic Eu(III) chelates
7 and 8 is presented in Scheme 5. After the
organometallic coupling of the acetylenes 14 and 15 with the 4-bromo-6-hydroxymethyl-2-carboxy-
ethylpyridine 35 to get the conjugates 41 and 42, and the bromination of the hydroxyl group with PBr3,
the needed chromophores 43 and 44 were isolated to be used for coupling with two amino functions
of the macrocyclic intermediate 46. The diBoc-protected 1,4,7-triazacyclononane derivative 45 formed

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from 37 and di-tert-butyl 1,4,7-triazacyclononane-1,4-dicarboxylate (diBocTACN) was deprotected
with CF3COOH to get the compound 46. The two phenylethynylpyridine subunits 43 and 44 were
coupled with 46 in dry MeCN and DIPEA with high yields. It is notable that, according to mass spectra
analysis of the crude reaction mixture, a mass corresponding of an over conjugated product was found
i.e., the reactivity of bromide in 43 and 44 is high enough to be able to form ternary substituted amino
function, although steric hindrance could be assumed significant enough to prevent such a reaction.
The synthesis strategy to prepare the chromophores before conjugation to the 1,4,7-triazacyclononane
(TACN) was chosen as the organometallic coupling reaction was not efficient when TACN was present,
probably due to possible complex formations between Cu(I)/Pd(II) and the azacrown, which lead to
incomplete reaction.
The ethyl ester groups of 47 and 48 were saponified with KOH in EtOH–H2O mixture; however,
the conventional Eu(III) chelate formation in slightly acidic solution at room temperature lead to
incomplete chelate formation according to HPLC and MS analyses. After multiple tests, the reasonable
complex formation of 49 and 50 was achieved within several days incubation at 95 ◦C and pH 9.5 in
aqueous citric solution to prevent Eu(OH)3 precipitation. The possible reason for the slow complexation
could be the high negative charge of the two chromophores causing repulsion and slow rotation of
the chromophores to reach the final complex i.e., high rigidity of the ligand and/or a possible strong
encapsulation of potassium ion in the TACN cavity during the earlier phase, as has been seen with
e.g., crown ethers and cryptands. After the HPLC purification of chelates 49 and 50, the amino groups
were activated with thiophosgene to receive the labeling reagents 7 and 8.
2.2. Photophysical Properties
The reference chelate (
→1a 8a) and protein (→1b
the absorption maxima (λabs), the excitation maxima (λexc), decay-times (τ), the corresponding molar
absorption coefficients ( ), brightness (εΦ) and quantum yield ( ) in tris-saline-azide (TRIS) buffer
1) and prepared labeling reagents (2–8) were conjugated to taurine
(
–
–
8b) followed by purifications. The photochemical properties i.e.,
ε
Φ
(TRIS 50 mM, NaCl 0.9%, pH 7.75) are presented in Table 1. The table includes also the obtained
enhanced signals from dried troponin I immunoassay wells, compared with the corresponding signal
of the reference chelate with a known effect of drying [20].
Table 1. Absorption maxima (λabs) and excitation maxima (
absorption coefficients ( ), brightness (εΦ) and quantum yields (
the enhanced brightness on dried surface.
λ
exc), luminescence decay times (
τ), molar
ε
Φ
) in TRIS buffer (pH 7.75) as well as
−1
−1
−1
−1
−1
εΦ 1 (M cm
)
Chelate λabs (nm)
λ
(nm)
τ (ms)
ε (M cm
)
εΦ (M−1 cm
)
Φ (%)
exc
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
320
325
343
344
351
349
343
348
349
351
352
350
348
352
354
356
325
1.01
1.01
0.96
0.97
0.88
0.90
0.84
0.80
0.70
0.69
0.77
0.69
0.58
0.51
0.48
0.42
1
55,000
55,000
57,000
55,000
58,000
68,000
85,000
77,000
64,000
78,000
80,000
75,000
89,000
99,000
77,000
104,000
4800
4800
8500
5700
4400
4900
14,900
16,100
16,400
11,400
15,400
9700
600
9
9
15
10
8
-
325
340
340
346
345
343
343
343
344
342
345
341
346
345
348
8000
-
16,100
-
7
20,200
18
21
26
15
19
13
0.7
0.5
0.9
1.0
-
24,100
-
34,400
-
37,200
-
40,200
-
500
700
1000
69,500
Enhanced brightness after drying.

Molecules 2017, 22, 1807
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The excitation wavelengths of the new chelates are ca. 15–20 nm red-shifted compared to
the reference chelate, and are between 340–350 nm due to the additional electron-releasing ether
functions at para- and meta-positions of the chromophores. The excitation maxima fit perfectly to
a recently published new optical system based on UV light emitting diode excitation at 340 nm [25].
The third ether substituent (e.g., chelate
5
compared to
4) does not have any notable effect on the
excitation wavelength, except with the chelate
2
(ca. 345 nm) compared to the corresponding complex
3
(ca. 350 nm). Generally, the increase of the chromophores decreases the H2O solubility as well as
increases the formation of aggregates during the bio-molecule labeling process and non-specific binding
properties for the labelled proteins. Aggregations will produce purification problems and reduce
the yield of labelled material. Moreover, increased non-specific binding of the labelled biomolecule
can enhance the assay background, and, thus, reduces the assay sensitivity. In the present study,
the additional COO−Na+ groups, together with the ether substituents offer high solubility in H2O,
reduce the nonspecific binding of the labelled biomolecule, and the affinity properties of the used
biomolecules are almost unchanged despite the increased negative charge of the labels. However,
such groups close to each other might cause nonspecific binging to positive groups present in the assay,
and, thus, interference with assay components might be obtained.
The absorptivities between the two chromophore chelates 1a
55,000 to 58,000 M−1 cm−1. As it could be assumed, the third chromophore increases the absorptivities.
The measured values from 64,000 (5a) to 89,000 M−1 cm−1
7a) express the higher value for
–3a are close to each other from
(
the 2,4-substituted taurine chelate designs compared to the corresponding 2,4,6-substitued ones.
The coupling of chelate to the protein changes the polarity of the chelates environment and seems
to enhance absorptivity with all new chelates except 4b and 6b, and the change is strongest with the
TACN based label 8b (104,000 vs. 77,000 M−1 cm−1).
The luminescence decay-times of the new chelates with two chromophores
shorter compared to , and are more reduced with the acyclic chelates with three chromophores
being lowest with TACN derivatives and . Moreover, the life-times with 2,4,6-trisubstituted
chelate designs are shorter compared to the corresponding 2,4-disubstitued analogues ( vs.
vs. and vs. ). Moreover, the chromophore with an electron donating NH(C=S)NH group
2 and 3 are slightly
1
4
–
6
7
8
3
2,
5
4
8
7
can have its own influence on the luminescence lifetime. This phenomenon of luminescence lifetime
reduction is rather surprising as all chelate structures are nine-dentate and the chromophores are
similar. Generally, the sensitization of lanthanide luminescence occurs through the ligands’ triplet
excited state. However, lately, an alternative route through an intra-ligand charge transfer (ILCT)
state has been demonstrated [10,29]. Ligands, which contain a conjugated π-electron skeleton,
such 4-(phenylethynyl)pyridine moiety, substituted by an electron-donating substituent together with
an electron accepting substituent at the other end of the antenna, can be excited through the ligands’s
CT-state. Such antenna chromophore can present a dipolar (push-pull) geometry. The lanthanide ion
acting as a Lewis acid increases the acceptor strength of this push–pull feature and lowers the energy
of the relaxed CT excited state. When the CT state is low enough, the excited energy can transfer
back from the excited state of lanthanide ion, which is observed as a reduced luminescence life-time.
Thus, the shortened luminescence life-time can be assumed to reflect the energy transfer through the
ILCT together with the original triplet state mediating route. It is known that the relaxation in the
CT excitation path is strongly correlated with the donor/acceptor pair, which supports the shorter
luminescence life-time of the chelates having chromophores with only one CH2 group and one electron
accepting COO− group in the pyridines (
with two CH2 groups. Thus, the chelates
7
7
and
and
8
8
) over the other chelates having chromophores also
have chromophores with lower CT states, whereas
the others have subunits with CT state(s) at a higher energy level, which minimized both the energy
transfer trough ILCT and the energy backflow. Similarly, the complexes with two chromophores
1
–
3
have only electron donating CH2 groups in pyridine and show the longest lifetimes. Moreover,
reference chelate has only one electron donating substituent in the phenyl ring compared to others,
and, thus, the longest decay time is observed. As a consequence, the increase of luminescence lifetime
1

Molecules 2017, 22, 1807
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in order 1 > 2 > 3 > 4 > 5 = 6 > 7 > 8 can be attributed to the push–pull feature of the complex
chromophores. Further evidence for the energy back-transfer phenomenon due to the low lying ILCT
state indicates the luminescence lifetime measurements of chelates 6b (0.55 ms) and 8b (0.42 ms) in
D2O, which rule out the possible lifetime reduction caused by water molecules.
The luminescence effectiveness of the chelates is affected by the molar absorption coefficient (
and the quantum yield ( ), and it can be illustrated by the product εΦ, called brightness. From the
chelates with two chromophores 2a gives almost two fold higher signal, whereas is about at the
same level compared to reference . The acyclic chelates show notable enhanced luminescence
intensities (9700–16,400 M−1 cm−1), being 2.0–3.1 times higher compared to the chelate
. In addition,
the quantum yields (13–26%) are significantly increased. A remarkable drop of brightness and quantum
yield is seen with the chelates and . The reason behind the obtained luminescence intensities cannot
ε)
Φ
3
1
4–6
1
7
8
be explained by differences between triplet state energy levels as those can be assumed to be near
to each other. Earlier triplet state energy level measurements from several different chelate designs
having the same (phenylethynyl)pyridine subunit including three, seven and nine-dentate chelates
−1
have given the triplet state energy values in a range of 21,600–21,830 cm
[8]. Therefore, the observed
effect on the luminescence intensity should be attributed to the excited energy transfer through the
low lying ILCT states, although the triplet mediated process cannot be entirely ruled out. If the ILCT
state is too low, the signal is reduced in aqueous solutions. Further evidence of the energy transfer
through the ILCT state is seen from the brightness measurements on the surface dried assay wells
after an immunoreaction (Table 1) compared to the corresponding wet measurements. The signal
increase of 60% (i.e., 1.7 fold) for the chelate 1 has been demonstrated in literature [20]. It is known that
with lanthanide chelates, which have low-lying CT state, the signal is highly dependent on solvent
or environment polarity [29]. The enhanced luminescence on the dried surface are without doubt
partly caused by the loss of H2O molecules causing the signal reduction due to the IR overtones of
OH bonds in aqueous solution but also by the polarity change. The signal enhancement between
wet and dry formats is ca. 2.8–4.1, 1.5–3.8 and 69–80 folds for the two chromophores containing
chelates
When compared to the dry signal level of chelate
obtained. More evidence of the CT route and its significance on the luminescence is clearly seen from
Figure 2. A quite clear correlation is seen between the decay constants (kchel = 1/ ) in TRIS buffer and
2
and
3, the acyclic chelates
4
–6
and the TACN chelates
7 and 8, respectively (see also Figure 1).
1, brightness improvement from 2.0 to 8.7 fold is
τ
the measured enhanced brightness of the different chelates (1b–8b) after the drying.
70,000
60,000
50,000
40,000
30,000
20,000
10,000
0
1b
2b
3b
4b
5b
6b
7b
8b
Figure 1. Brightness of the chelates 1b
–8b in 0.05 M TRIS buffer (dark grey) and on a dried surface
(light grey).

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70,000
60,000
50,000
40,000
30,000
20,000
10,000
0
0.5
1
1.5
2
2.5
kchel (1/ms)
Figure 2. Measured enhanced brightness of chelates 1b–8b on the dried surface vs. decay constant
(kchel = 1/τ) measured in 0.05 M TRIS buffer.
Finally, the dependency of luminescence on the measurement temperature between three chelates
6b and 8b in Figure 3 shows increasing luminescence quenching in order 8b 6b 1b being
1b
,
>
>
accordance with above observations, and is in agreement with the behavior of ILCT transition i.e.,
hypsochromic shift upon decreasing the temperature obtained earlier with phenylethynylpyri-dine
Eu(III) chelates [29].
Figure 3. Relative luminescence intensity of chelate 1b (square), 6b (triangle) and 8b (circle) vs.
measurement temperature.
The luminescence intensities after drying are really remarkable e.g., when compared to the
brightness of the well-known dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA®)

Molecules 2017, 22, 1807
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−1
−1
enhancement solution (26,320 M cm in [30]) used in many sensitive bio-affinity assays. Therefore,
it should be possible to design highly sensitive TR-FIA assays by using the novel labeling chelates.
Actually, preliminary troponin I assays performed have given three, five and seven folds improved
assay sensitivities by using e.g., chelate labels 3, 6 and 8, respectively.
3. Materials and Methods
3.1. Materials
All commercially available solvents and reagents were used without further purifications. MeCN,
THF and TEA were dried with molecular sieves and K2CO3 was dried overnight at 140 ◦C before use.
The organic intermediates were purified by silica gel 60 (Merck, Darmstadt, Germany) column
chromatography. The low fluorescence MaxiSorp single wells made of irradiated polystyrene were
purchased from Nunc (Thermo Fisher Scientific, Waltham, MA, USA). The monoclonal anti-troponin
I detection antibodies were manufactured by International Point of Care Inc., Toronto, ON, Canada.
The troponin I capture antibody and antigen were purchased from HyTest Ltd. (Turku, Finland).
3.2. Syntheses
The general synthetic pathways used for the Eu(III) Chelates 2–8 are shown in Schemes 2–5.
The more detailed synthetic procedures and the results of the spectroscopic product characterization
are presented in the Supporting information. Microwave assisted syntheses were performed with
a Initiator+ microwave synthesizer from Biotage (Uppsala, Sweden).
The Eu(III) chelates were analysed and purified by using a reversed phase HPLC (2996 Photodiode
Array Detector, 600 Controller, Delta 600 Fraction Collector III; Waters, Milford, MA, USA) with a RP-18
column. The solvents were A: triethylammonium acetate buffer (20 mM, pH 7) and B: 50% acetonitrile
in triethylammonium acetate buffer (20 mM, pH 7). The gradient was started from 5% of solvent B and
the amount of solvent B was linearly raised to 100% within 30 min.
The 1H and 13C-NMR spectra were recorded on an AVANCE 500 DRX (Bruker, Billerica, MA, USA).
The chemical shifts are given in ppm from the internal tetramethylsilane. Mass spectra were recorded
on Applied Biosystems QSTAR® XL ESI-TOF (electrospray ionization-time-of-flight mass analyzer)
instrument (Thermo Fisher Scientific) using α-cyano-4-cinnamic acid matrix. UV-Vis spectra were
recorded on an Ultrospec® 3300 pro (GE Healthcare Life Sciences, Chicago, IL, USA) or a UV-1800
spectrophotometer (Shimadzu, Kyoto, Japan).
Eu(III) Chelate (1a). A mixture of Eu(III) chelate
1 (2.5 µmol) and taurine (3.1 mg, 25 µmol) in aqueous
50 mM Na2CO3 buffer (0.125 mL, pH 9.8) was stirred overnight at RT (room temperature). The product
was purified by HPLC. Rf(HPLC): 15.1 min. UV: 320 nm.
Triethyl 2,20,200-[benzene-1,3,5-triyltris(oxy)]triacetate (
added within 2 h to a mixture of benzene-1,3,5-triol (5.04 g, 40 mmol) and dry K2CO3 (18.2 g, 132 mmol)
9). Ethyl bromoacetate (14.6 mL, 132 mmol) was
◦
◦
in dry MeCN (300 mL) at 50 C under argon. After stirring for ca. 20 h at 50 C, the mixture was
filtered, the solid material washed with MeCN and the filtrate evaporated to dryness. The product
was purified by silica gel column chromatography using MeOH/CH2Cl2 (first 0:100, then 1:99) as
1
an eluent. Yield: 10.8 g (70%). H-NMR [DMSO-d6 (dimethyl sulfoxide),
δ
ppm]: 6.13 (3H, s), 4.74
ppm): 169.02, 159.84,
95.12, 65.27, 61.10, 14.51. MS(ESI-TOF) calculated for C18H24O9 [M + H]+: 385,15, found: 385.04.
(6H, s), 4.17 (6 H, q, J = 7.1 Hz), 1.22 (9H, t, J = 7.1 Hz). 13C-NMR (DMSO-d6,
δ
Triethyl 2,20,200-[(2-iodobenzene-1,3,5-triyl)tris(oxy)]triacetate (10). The compound
(8.36 g, 22 mmol)
9
in CH3COOH (40 mL) was added to a mixture of N-chlorosuccinimide (2.95 g, 22 mmol) and NaI
(3.30 g, 22 mol) in CH3COOH (65 mL). After stirring for 1.5 h at RT, the mixture was evaporated to
dryness. The residue was dissolved in EtOAc (ethyl acetate, 320 mL) and neutralized with saturated
NaHCO3 solution. The phases were separated, the aqueous phase was extracted with EtOAc (80 mL)
and the combined organic phases were dried with Na2SO4. Yield: 11,2 g (100%). 1H-NMR (CDCl3,

Molecules 2017, 22, 1807
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δ
ppm): 6.10 (2H, s), 4.65 (4H, s), 4.55 (4H, s), 4.27 (2H, q, J = 7.15 Hz), 4.26 (4H, q, J = 7.15 Hz),
1.31 (3H, t, J = 7.15 Hz), 1.30 (6H, t, J = 7.15 Hz). 13C-NMR (CDCl3,
δ ppm): 168.14, 168.01, 159.89,
158.65, 94.34, 66.61, 65.61, 61.52, 61.43, 14.05. MS(ESI-TOF) calculated for C18H23IO9 [M + H]+: 511.05,
found: 510.93.
0
Diethyl 2,2 -[(4-bromo-1,3-phenylene)bis(oxy)]diacetate (11). A mixture of 4-bromobenxene-1,3-diol (3.18 g,
16.8 mmol), dry K2CO3 (5,12 g, 37,0 mmol) and ethyl bromoacetate (4.11 mL, 37.0 mmol) in dry MeCN
(60 mL) was stirred for 24 h at 50 ◦C. The mixture was filtered, the solid material washed with MeCN
and the filtrate evaporated to dryness. The product was purified by silica gel column chromatography
1
using MeOH/CH2Cl2 (first 0:100, then 1:99) as an eluent. Yield: 5.97 g (98%). H-NMR (DMSO-d6,
δ
ppm): 7.46, (1H, d, J = 8.8 Hz), 6.65 (1H, d, J = 2.7 Hz), 6.51 (1H, dd, J = 2.7 and 8.8 Hz), 4.91 (2H, s),
4.79 (2H, s), 4.18 (2H, q, J = 7.1 Hz), 4.16 (2H, q, J = 7.1 Hz), 1.22 (3 H, t, J = 7.1 Hz), 1.21 (3 H, t,
J = 7.1 Hz). 13C-NMR (DMSO-d6,
δ ppm): 168.65, 158.62, 155.08, 133.55, 108.65, 102.64, 102.09, 65.81,
65.44, 61.21, 61.15, 14.49. MS(ESI-TOF) calculated for C14H17BrO6 [M + H]+: 361.03 and 363.03 found:
360.89 and 362.92.
General Procedure for the Synthesis of Compounds 12 and 13. A mixture of compound 10 or 11 (5.33 mmol),
bis(triphenylphosphine)palladium(II) chloride (186 mg, 0.27 mmol), CuI (51 mg, 0.27 mmol),
triphenylphosphine (142 mg, 0.54 mmol; only for compound 12) in diethylamine (10 mL) and dry DMF
(5 mL) was de-aerated with argon. After addition of trimethylsilylacetylene (1.17 mL, 8.34 mmol),
◦
the mixture was stirred for 45 or 30 min for compound 10 and 11, respectively, at 100 C using
microwave heating. After evaporation to dryness, the residue was dissolved in CH2Cl2 (50 mL),
washed with H2O (2 × 25 mL), dried with Na2SO4 and purified with column chromatography.
Diethyl 2,20-{{4-[(trimethylsilyl)ethynyl]-1,3-phenylene}bis(oxy)}diacetate (12). The product was purified
by silica gel column chromatography using EtOAc/petroleum ether (20:80) as an eluent. Yield: 94%.
1H-NMR (DMSO-d6,
δ
ppm): 7.31 (1H, d, J = 8.8 Hz), 6.52 (1 H, d, J = 2.4 Hz), 6.61 (1H, dd, J = 2.4
and 8.8 Hz), 4.85 (2H, s), 4.81 (2H, s), 4.18 (2H, q, J = 7.1 Hz), 4.17 (2H, q, J = 7.1 Hz), 1.23 (3H, t,
J = 7.1 Hz), 1.21( 3H, t, J = 7.1 Hz), 0.22 (9H, s). 13C-NMR (DMSO-d6,
ppm): 168.73, 160.08, 159.70,
δ
134.94, 107.33, 105.18, 102.16, 100.66, 97.00, 65.66, 65.30, 61.17, 14.51, 14.49, 0.57. MS(ESI-TOF) calculated
for C19H22O6Si [M + H]+: 379.16, found 379.39.
Triethyl 2,20,200-{{2-[(trimethylsilyl)ethynyl]benzene-1,3,5-triyl}tris(oxy)}triacetate (13). The product was
purified by silica gel column chromatography using EtOAc/petroleum ether (first 20:80, then 30:70)
1
as an eluent. Yield: 70%. H-NMR (CDCl3,
δ
ppm): 6.10 (2H, s), 4.67 (4H, s), 4.55 (2H, s), 4.27 (2H, g,
J = 7.15 Hz), 4.26 (4H, q, J = 7.15 Hz), 1.30 (6H, t, J = 7.15 Hz), 1.29 (3H, t, J = 7.15 Hz), 0.26 (9H, s).
13C-NMR (DMSO-d6,
δ ppm): 168.60, 168.56, 160.98, 159.66, 101.34, 98.17, 95.29, 93.43, 65.77, 65.31,
61.06, 61.04, 14.41, 14.38, 0.56. MS(ESI-TOF) calculated for C23H32O9Si [M + H]+: 481.19, found: 481.99.
General Procedure for the Synthesis of Compounds 14 and 15. A mixture of compound 12 or 13 (10.4 mmol)
and tetrabutylammonium fluoride (3.25 g, 12.4 mmol) in CH2Cl2 (200 mL) stirred for 1.5 h at RT under
argon. Mixture was washed with aqueous 10% citric acid solution (200 mL), H2O (200 mL), died with
Na2SO4 and used for the next step without further purification as no impurities were found with the
TLC analysis.
1
Diethyl 2,20-{[4-(ethynyl)-1,3-phenylene]bis(oxy)}diacetate (14). Yield: 100%. H-NMR (DMSO-d6,
δ ppm):
7.34 (1H, d, J = 8.8 Hz), 6.53 (1H, d, J = 2.4 Hz), 6.52 (1H, dd, J = 2.4 and 8.8 Hz), 4.89 (2H, s), 4.81 (2H, s),
4.17 (2H, q, J = 7.1 Hz), 4.16 (2H, q, J = 7.1 Hz), 4.10 (1H, s), 1.22 (3H, t, J = 7.1 Hz), 1.21 (3H, t, J = 7.1 Hz)
.
13C-NMR (DMSO-d6,
δ ppm): 168.84, 168.83, 160.29, 159.59, 134.90, 107.29, 104.62, 100.49, 83.70, 80.35,
65.40, 65.29, 61.17, 14.49. MS(ESI-TOF) calculated for C16H18O6 [M + H]+: 307.12, found: 307.01.
1
Triethyl 2,20,200-{[2-(ethynyl]benzene-1,3,5-triyl]tris(oxy)}triacetate (15). Yield: 100%. H-NMR (CDCl3,
δ
ppm): 6.06 (2H, s), 4.69 (4H, s), 4.55 (2H, s), 4.27 (2H, q, J = 7.15 Hz), 4.26 (4H, q, J = 7.15 Hz),
3.50 (1H, s), 1.30 (3H, t, J = 7.15 Hz), 1.29 (6H, t, J = 7.15 Hz). 13C-NMR (CDCl3,
δ
ppm): 168.23, 168.10,

Molecules 2017, 22, 1807
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161.48, 159.39, 95.25, 94.18, 85.18, 66.42, 65.49, 61.64, 61.49, 14.14. MS(ESI-TOF) calculated for C20H24O9
[M + H]+: 409.15, found: 409.20.
General Procedure for the Synthesis of Ligand Esters 17 and 18. A mixture of compound 16 (0.22 g, 0.25 mmol; [31])
and compound 14 or 15 (0.60 mmol) in dry TEA (2 mL) and THF (4 mL) was de-aerated with argon.
After an addition of bis(triphenylphosphine)palladium(II) dichloride (11 mg, 16
µmol) and CuI
(6 mg, 32
µ
mol), the mixture was stirred overnight at 55 ◦C and evaporated to dryness. The residue
was dissolved in CH2Cl2 (30 mL), washed with H2O (2
column chromatography.
×
15 mL), dried with Na2SO4 and purified by
Ligand Ester 17. The product was purified by silica gel column chromatography using TEA/EtOAc/
petroleum ether (20:40:40) as an eluent. Yield: 48%. 1H-NMR (DMSO-d6,
ppm): 7.47 (2 H, d, J = 1.1 Hz),
δ
7.45 (2H, d, J = 8.6 Hz), 7.43 (2H, d, J = 1.1 Hz), 6.79 (2H, d, J = 8.4 Hz), 6.61 (2H, d, J = 2.3 Hz), 6.55
(2H, dd, J = 8.4 and 2.3 Hz), 6.44 (2H, d, J = 8.4 Hz), 4.91 (4H, s), 4.83 (4H, s), 4.75 (2H, s), 4.17 (4H, q,
J = 7.1 Hz), 4.17 (4H, q, J = 7.1 Hz), 4.05 (4H, q, J = 7.1 Hz), 3.94 (4H, s), 3.82 (4H, s), 3.56 (8H, s), 2.65
(4H, s), 1.23 (6H, t, J = 7.1 Hz), 1.20 (6H, t, J = 7.1 Hz), 1.16 (12H, t, J = 7.1 Hz). 13C-NMR (DMSO-d6,
δ
ppm): 171.14, 168.77, 168.702, 160.28, 160.14, 159.58, 159.21, 146.96, 134.86, 129.34, 129.28, 129.18,
127.50, 122.84, 122.50, 114.42, 107.61, 104.26, 100.64, 90.62, 90.38, 65.65, 65.35, 61.21, 61.19, 59.73, 59.60,
56.67, 54.98, 32.61, 14.50, 14.49, 14.46. MS(ESI-TOF) calculated for C70H84N6O20 [M + 2H]+: 1329.59,
found: 1330.66.
Ligand Ester 18. The product purified by silica gel column chromatography using MeOH/CH2Cl2 (5:95)
1
as an eluent. Yield: 73%. H-NMR (CDCl3,
δ
ppm): 7.56 (2H, s), 7.51 (2H, s), 6.88 (2H, d, J = 8.3 Hz)
,
6.56 (2H, d, J = 8.3 Hz), 5.98 (4H, s), 4.70 (8H, s), 4.52 (4H, s), 4.28 (4H, q, J = 7.1 Hz), 4.26 (8H, q,
J = 7.1 Hz), 4.15 (8H, q, J = 7.1 Hz), 4.04 (4H, s), 3.87 (4H, s), 3.62 (8H, s), 2.80–2.70 (4H, m), 1.65 (2H, bs),
1.31 (6H, t, J = 7.1 Hz), 1.25 (12H, t, J = 7.1 Hz), 1.24 (12H, t. J = 7.1 Hz). 13C-NMR (CDCl3,
δ ppm):
171.14, 168.45, 168.14, 160.91, 159.58, 159.52, 157.81, 144.59, 133.17, 130.33, 129.52, 123.52, 122.58, 115.26,
96.52, 95.52, 93.46, 85.52, 66.04, 65.29, 61.54, 61.32, 60.49, 60.35, 59.92, 56.02, 54.88, 32.58, 14.24, 14.16,
14.15. MS(ESI-TOF) calculated for C78H96N6O26 [M + 2H]+: 1533.65, found: 1532.96.
General Procedure for the Synthesis of Eu(III) Chelates 19 and 20. Compound 17 or 18 (52 µmol) in 0.5 M
KOH in EtOH (5.4 mL) was stirred for 30 min at RT, H2O (2.7 mL) was added and the mixture was
further stirred at RT for 2 h. After evaporation of EtOH and an additional stirring for 2 h at RT,
the pH was adjusted to 6.5 by addition of 6 M HCl. EuCl3 (21 mg, 57 µmol) in water (0.5 mL) was
added within 5 min and the pH was maintained at 6.0–6.5 with suitable additions of solid NaHCO3.
After stirring overnight at RT, the pH was adjusted to 8.5 with 1 M NaOH. The precipitate was removed
by centrifugation and the supernatant evaporated to dryness. The product was purified by HPLC.
Eu(III) Chelate 19. Yield: 88%. Rf(HPLC): 14.8 min. UV: 342 nm.
Eu(III) Chelate 20. Yield: 92%. Rf(HPLC): 12.3 min. UV: 346 nm.
General Procedure for the Synthesis of Eu(III) Chelates
chelate 19 or 20 (55 mol) was added within 5 min to a mixture of CSCl2 (30
(37 mg, 0.44 mmol) in CHCl3 (1.3 mL). After stirring for 45 min at RT, the two phases were separated and
the aqueous phase was washed with CHCl3 (3 1.3 mL). The product was precipitated with acetone
(ca. 45 mL), isolated by centrifugation, washed with acetone (2 10 mL) and dried overnight in vacuum
2
and
3. An aqueous solution (1.3 mL) of the Eu(III)
µ
µL, 0.39 mmol) and NaHCO3
×
×
desiccator. The products were used as such for next phase or for labeling the antibodies.
Eu(III) Chelate 2. Rf(HPLC): 18.4 min. UV: 346 nm.
Eu(III) Chelate 3. Rf(HPLC): 14.6 min. UV: 350 nm.
General Procedure for the Synthesis of Eu(III) Chelates 2a and 3a. A mixture of Eu(III) chelate
2 or 3
(2.5
µmol) and taurine (3.1 mg, 25 µmol) was stirred in aqueous 50 mM Na2CO3 buffer (0.125 mL,
pH 9.8) for overnight at RT. The product was purified by HPLC.

Molecules 2017, 22, 1807
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Eu(III) Chelate 2a. Rf(HPLC): 15.1 min. UV: 340 nm.
Eu(III) Chelate 3a. Rf(HPLC): 12.5min. UV: 351 nm.
N-(4-Ethynylphenyl)-2,2,2-trifluoroacetamide (21). 4-Ethynylaniline (1.17 g, 10 mmol) was added in small
portions to a ice-cold trifluoroacetic anhydride (5.6 mL, 40 mmol). After stirring for 2.5 h at RT,
cold H2O was added, the mixture filtrated and the product washed with cold H2O. Yield: 2.12 g (98%).
1H-NMR (CDCl3,
13C-NMR (CDCl3,
δ ppm): 8.08 (1H, bs), 7.55 (2H, d, J = 8.8 Hz), 7.51 (2 H, d, J = 8.8 Hz), 3.1 (1H, s).
δ
ppm): 154.72, 135.40, 133.23, 129.92, 120.25, 120.01, 82.67, 77.99. MS(ESI-TOF)
calculated for C10H6F3NO [M + H]+: 213.04, found 213.05.
N-{4-{[2,6-Bis(hydroxymethyl)pyridin-4-yl]ethynyl}phenyl}-2,2,2-trifluoroacetamide (23) A mixture of the
compound 21 (0.55 g, 2.58 mmol) and 4-bromo-2,6-dihydroxymethylpyridine (22; [28]) (0.47 g,
2.15 mmol) in dry TEA (5 mL) and THF (10 mL) was de-aerated with argon. After addition of
bis(triphenylphosphine)palladium(II) chloride (30 mg, 43
was stirred for 19 h at 55 C. After evaporation to dryness, the residue was treated with a cold mixture
µ
mol) and CuI (16 mg, 86
µ
mol), the mixture
◦
of CH2Cl2 (40 mL) and H2O (20 mL), filtered and the product washed with cold H2O (10 mL) and
1
CH2Cl2 (10 mL). Yield: 0.56 g (75%). H-NMR (DMSO-d6,
δ
ppm): 11.5 (1H, bs), 7.79 (2H, d, J = 8.8 Hz),
ppm): 162.25,
7.68 (2H, d, J = 8.8 Hz), 7.41 (2H, s), 5.49 (2H, bs), 4.56 (4H, s). 13C-NMR (DMSO-d6,
δ
155.53, 155.23, 154.94, 154.64. 138.01, 133.12, 131.39, 121.52, 120.00, 119.58, 118.63, 117.29, 114.99, 92.93,
87.98, 64.39. MS(ESI-TOF) calculated for C17H13F3N2O3 [M + H]+: calculated 351.10, found 351.96.
N-{4-{[2,6-bis(bromomethyl)pyridin-4-yl]ethynyl}phenyl}-2,2,2-trifluoroacetamide (24). PBr3 (0.35 mL, 3.67 mmol)
was added in a suspension of the compound 23 (0.86 g, 2.45 mmol) in CHCl3 (100 mL). After stirring
for 20 h at 60 ◦C, the mixture was neutralized with 5% NaHCO3 (50 mL). The aqueous phase was
extracted with CHCl3 (50 mL) and the combined organic phases were dried with Na2SO4, filtered
and evaporated to dryness. The product 24 (1.09 g, 93%) was used for the next step without further
1
purifications. H-NMR (CDCl3,
δ
ppm): 7.93 (1H, s), 7.63 (2H, d, J = 8.7 Hz), 7.59 (2H, d, J = 8.7 Hz),
ppm): 157.02, 155.16, 154.85, 154.55, 154.23, 134.92, 133.36,
7.47 (2H, s), 4.53 (4H, s). 13C-NMR (CDCl3,
δ
133.11, 124.55, 120.26, 119.73, 118.98, 116.68, 114.38, 112.10, 93.70, 86.77, 32.98. MS(ESI-TOF) calculated
for C17H11Br2F3N2O [M + H]+: 474.93, 476.93, 478.92, found 475.38, 477.44, 479.45.
Ethyl-2-{[4-bromo-6-(carboxyethyl)pyridine-2-yl]methylenenitrilo}acetate (26). A mixture of 4-bromo-6-
bromomethyl-2-carboxyethylpyridine (25; [14]) (1.66 g, 5.15 mmol), glycine ethyl ester hydrochloride
(3.60 g, 25.8 mmol) and di-isopropylethylamine (9.1 mL) in dry MeCN (70 mL) was stirred
overnight at RT. The mixture was evaporated to dryness and the product was purified by silica gel
column chromatography using MeOH/CH2Cl2 (3:97) as an eluent. Yield 1.55 g (87%). 1H-NMR:
(CDCl3,
δ ppm): 8.14 (1H, d, J = 1.65 Hz), 7.85 (1H, d, J = 1.60 Hz), 4.47 (2H, q, J = 7.12 Hz),
4.20 (2H, q, J = 7.13 Hz), 4.05 (2H, s), 3.47 (2H, s), 2.26 (1H, s), 1.43 (3H, t, J = 7.13 Hz), 1.28 (3H, t,
J = 7.13 Hz). δ ppm): 172.41, 164.08, 162.23, 148.76, 133.81, 128.53, 126.29, 62.07,
13C-NMR (DMSO-d6,
60.47, 53.65, 50.24, 14.59, 14.54. MS(ESI-TOF) calculated for C13H17BrN2O4 [M + H]+: 345.05, 347.05,
found 344.97, 346.97.
Synthesis of Compound 27. A mixture of compounds 24 (0.44 g, 0.94 mmol), 26 (0.65 g, 1.89 mmol),
dry K2CO3 (0.52 g, 3.76 mmol) was stirred in dry MeCN (30 mL) for 27 h at 70 ◦C. The mixture was
filtrated and the filtrate evaporated to dryness, and the product was purified by silica gel column
1
chromatography using TEA/EtOAc/petroleum ether (1:69:30) as an eluent. Yield: 66%. H-NMR
(CDCl3,
7.60 (2H, d, J = 8.73 Hz), 7.34 (2H, s), 4.44 (4H, q, J = 7.08 Hz), 4.18 (4 H, q, J = 7.15 Hz); 4.10 (4H, s), 3.98
(4 H, s), 3.50 (4 H, s), 1.40 (6H, t, J = 7.08 Hz), 1.28 (6H, t, J = 7.15 Hz). 13C-NMR (CDCl3,
ppm): 171.02,
δ ppm): 8.53 (1H, s), 8.14 (2H, d, J = 1.80 Hz), 8.11 (2H, d, J = 1.80 Hz), 7.68 (2 H, d, J = 8.73 Hz),
δ
164.11, 161.85, 158.48, 155.23, 154.92, 154.62, 154.32, 148.52, 136.02, 134.37, 132.98, 132.16, 129.28, 128.35,
127.01, 123.43, 120.28, 119.04, 116.74, 114.45, 112.15, 92.79, 87.68, 62.23, 60.68, 59.82, 59.64, 55.41, 14.24.
MS(ESI-TOF) calculated for C43H43Br2F3N6O9 [M + H]+: 1003.15, 1005.15, 1007.15, found 1003.71,
1005.48, 1007.58.

Molecules 2017, 22, 1807
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General Procedure for the Synthesis of Ligand Esters 28 and 29. A mixture of compound 27 (0.120 g,
0.148 mmol), compound 14 or 15 (0.355 mmol) in dry TEA (1 mL) and THF (2 mL) was de-aerated with
argon. After an addition of bis(triphenylphosphine)palladium(II) dichloride (10 mg, 14
µmol) and CuI
(6 mg, 28
µ
mol), the mixture was stirred overnight at 55 ◦C and evaporated to dryness. The residue
was dissolved in CH2Cl2 (30 mL), washed with H2O (2
silica gel column chromatography.
×
15 mL), dried with Na2SO4 and purified by
Ligand Ester 28. The product purified by silica gel column chromatography using 10% EtOH/CH2Cl2 as
1
an eluent. Yield: 69%. H-NMR (DMSO-d6,
δ
ppm): 11.44 (1H, s), 7.85 (2H, s), 7.77 (2H, s), 7.69 (2H, d, J
= 8.1 Hz), 7.57 (2H, d, J = 6.7 Hz), 7.45 (2H, d, J = 8.1 Hz), 7.43 (2H, s), 6.60 (2H, s), 6.53 (2H, d, J = 6.7 Hz)
,
4.91 (4H, s), 4.82 (4 H, s), 4.30 (4H, q, J = 6.7 Hz), 4.17 (8H, q, J = 7.1 Hz), 4.06 (4H, q, J = 6.9 Hz), 4.00
(4 H, s), 3.95 (4 H, s), 3.53 (4 H, s), 1.30 (6H, t, J = 6.7 Hz), 1.22 (6H, t. J = 7.1 Hz), 1.20 (6H, t, J =
7.1 Hz), 1.18 (6H, t, J = 6.9 Hz). 13C-NMR (DMSO-d6,
δ ppm): 171.19, 168.67, 168.62, 164.58, 160.62,
160.32, 159.04, 155.47, 155.18, 154.89, 154.59, 147.74, 137.93, 134.95, 133.48, 132.98, 130.67, 129.50, 127.50,
124.78, 123.44, 121.34, 119.46, 118.50, 117.26, 114.96, 112.67, 107.69, 103.73, 100.64, 92.13, 89.65, 87.58,
65.70, 65.34, 61.84, 61.20, 60.48, 59.64, 56.50, 55.71, 55.38, 14.51, 14.46. MS(ESI-TOF) calculated for
C75H77F3N6O21 [M + H]+: 1455.52, found 1455.36.
Ligand Ester 29. The product was purified by silica gel column chromatography using TEA/EtOAc/
1
petroleum ether (1:69:30) as an eluent. Yield: 57%. H-NMR (CDCl3,
δ
ppm): 9.12 (1H, s), 8.09 (2H, d,
J = 1.25 Hz), 8.06 (2H, d, J = 1.25 Hz), 7.57 (2 H, s), 7.34 (2H, d, J = 8.80 Hz), 7.30 (2H, d, J = 8.80 Hz),
5.97 (4H, s), 4.63 (8H, s), 4.60 (4H, s), 4.45 (4H, q, J = 7.12 Hz), 4.30 (4H, q, J = 7.12 Hz), 4,21 (8H, q,
J = 7.12 Hz), 4.17 (4H, q, J = 7.12), 4.10 (4H, s), 4.02 (4H, s), 3.52 (4H, s), 1.42 (6H, t, J = 7.12 Hz),
1.33 (6H, t, J = 7.12 Hz), 1.29 (6H, J = 7.12 Hz), 1.25 (12H, t, J = 7.12 Hz). 13C-NMR (CDCl3,
δ ppm):
171.13, 168.69, 168.69, 165.07, 161.08, 160.53, 159.98, 159.03, 154.95, 154.66, 147.65, 136.35, 133.99, 132.56,
132.17, 128.47, 127.64, 125.43, 123.12, 119.95, 116.90, 114.59, 96.39, 94.76, 93.71, 92.65, 87.75, 87.21, 66.12,
65.22, 61.91, 61.89, 61.51, 60.57, 60.10, 59.94, 55.11, 14.32, 14.27, 14.13. MS(ESI-TOF) calculated for
C83H89F3N6O27 [M + H]+: 1659.58, found 1659.81.
General Procedure for the Synthesis of Eu(III) Chelates 30 and 31. Compound 28 or 29 (90 µmol) was
stirred in 0.5 M KOH in EtOH (13 mL) for 30 min at RT. H2O (10 mL) was added and the mixture was
further stirred at RT for 3 h. After evaporation of EtOH and an additional overnight stirring at RT,
the pH was adjusted to 6.5 by addition of 6 M HCl. EuCl3 (33 mg, 90 µmol) in water (0.5 mL) was
added within 5 min and the pH was maintained at 6.0–6.5 with suitable additions of solid NaHCO3.
After stirring overnight at RT, the pH was adjusted to 8.5 with 1 M NaOH. The precipitate was removed
by centrifugation and the supernatant evaporated to dryness. The product was purified by HPLC.
Eu(III) Chelate 30. Yield: 100%. Rf(HPLC): 14.2 min. UV: 340 nm.
Eu(III) Chelate 31. Yield: 100%. Rf(HPLC): 14.5 min. UV: 359 nm.
General Procedure for the Synthesis of Eu(III) Chelates
4
and
5. An aqueous solution (2.7 mL) of the Eu(III)
chelate 30 or 31 (0.10 mmol) was added within 5 min to a mixture of CSCl2 (53
µL, 0.70 mmol) and
NaHCO3 (67 mg, 0.80 mmol) in CHCl3 (2.7 mL). After stirring for 30 min at RT, the two phases were
separated and the aqueous phase was washed with CHCl3 (3 × 3 mL). The product was precipitated
with acetone (ca. 45 mL), isolated by centrifugation, washed with acetone (2
overnight in vacuum desiccator.
×
10 mL) and dried
Eu(III) Chelate 4. Rf(HPLC): 18.4 min. UV: 346 nm.
Eu(III) Chelate 5. Rf(HPLC): 20.9 min. UV: 325 (sh), 340 and 362 (sh) nm.
General Procedure for the Synthesis of Eu(III) Chelates 4a and 5a. A mixture of Eu(III) chelate
(6.3 mol) and taurine (8 mg, 63 mol) was stirred in aqueous 50 mM Na2CO3 buffer (0.64 mL, pH 9.8)
and DMF (0.64 mL) for overnight at RT. The product was purified by HPLC.
4 or 5
µ
µ

Molecules 2017, 22, 1807
17 of 23
Eu(III) Chelate 4a. Rf(HPLC): 16.7 min. UV: 340 nm.
Eu(III) Chelate 5a. Rf(HPLC): 14.3 min. UV: 349 nm.
Diethyl 2,20-{[(4-bromopyridine-2,6-diyl)bis(methylene)]bis(azanediyl)}diacetate (33). A mixture of 4-bromo-
2,6-dibromomethylpyridine (32, [28]) (3.50 g, 10 mmol), glycine ethyl ester hydrochloride (14.0 g,
0.10 mol) and di-isopropylethylamine (35 mL) in dry MeCN (130 mL) was stirred overnight at RT.
After evaporation to dryness, the residue was dissolved in CH2Cl2 (100 mL), washed with H2O
(3
using TEA/EtOAc/petroleum ether (first 10:20:70, then 15:30:55) as an eluent. Yield 2.48 g (64%).
1H-NMR (CDCl3,
ppm): 7.43 (2H, s), 4.20 (4H, q, J = 7.2 Hz), 3.91 (4H, s), 3.46 (4H, s), 2.25–2.15
×
50 mL), dried with Na2SO4, and the product was purified by silica gel column chromatography
δ
(2H, bs), 1.28 (6H, t, J = 7.2 Hz). 13C-NMR (CDCl3,
δ ppm): 172.00, 160.40, 133.83, 60.75, 54.00, 50.34,
14.18. MS(ESI-TOF) calculated for C15H22BrN3O4 [M + H]+: 388.09 and 390.09, found 388.75 and 390.75.
Ethyl 4-bromo-6-{{{{4-bromo-6-{[(2-ethoxy-2-oxoethyl)amino]methyl}pyridin-2-yl}methyl}(2-ethoxy-2-oxo-ethyl)
amino}methyl}picolinate (34). 4-Bromo-6-bromomethyl-2-carboxyethylpyridine (25, [14]) (2.06 g, 6.39 mmol)
was added in small portions to a mixture of the compound 33 (2.49 h, 6.39 mmol), dry K2CO3
(3.53 g, 25.6 mmol) in dry MeCN (215 mL) within 2 h at RT. After stirring for 22 h at RT, the mixture
was filtrated and the filtrate evaporated to dryness. The product was purified by silica gel column
chromatography using TEA/EtOAc/petroleum ether (first 10:25:65) as an eluent. Yield 2.03 g (50%).
1H-NMR (CDCl3,
δ ppm): 8.12 (1H, d, J = 1.7 Hz), 8.07 (1H, d, J = 1.7 Hz), 7.55 (1H, d, J = 1.5 Hz),
7.43 (1H, d, J = 1.5 Hz), 4.46 (2H. q, J = 7.2 Hz), 4.19 (4H, q, J = 7.2 Hz), 4.09 (2H, s), 3.95 (2H, s), 3.90
(2H, s), 2.3–2.1 (1H, bs), 1.43 (3H, t, J = 7.2 Hz), 1.29 (3H, t, J = 7.2 Hz), 1.29 (3H, t, J = 7.2 Hz). 13C-NMR
(CDCl3,
δ ppm): 171.98, 170.81, 164.03, 161.57, 160.41, 159.70, 148.54, 134.26, 133.90, 129.13, 126.95,
124.60, 123.77, 62.16, 60.75, 60.63, 59.61, 59.48, 55.38, 54.00, 50.36, 14.19, 14.17, 14.14. MS(ESI-TOF)
calculated for C24H30Br2N4O6 [M + H]+: 629.06, 631.05 and 633.06, found 629.54, 631.56 and 633.54.
Ethyl 6-(hydroxymethyl)-4-{[4-(2,2,2-trifluoroacetamido)phenyl]ethynyl}picolinate (36). A mixture of the
compound 21 (0.34 g, 1.60 mmol) and 35 (0.47 g, 2.15 mmol; [14]) in dry TEA (5 mL) and THF (10 mL)
was de-aerated with argon. After addition of bis(triphenylphosphine)palladium(II) chloride (19 mg,
27 µmol) and CuI (10 mg, 53 µ
mol), the mixture was stirred for 24 h at 55 ◦C. After evaporation to
dryness, the product was purified by silica gel column chromatography using EtOH/CH2Cl2/TEA
1
(10:89:1) as an eluent. Yield: 0.49 g (94%). H-NMR (CDCl3,
δ ppm): 8.49 (1H, s), 8.08 (1H, s), 7.66
(2H, d, J = 8.7 Hz), 7.60 (1H, s), 7.59 (2H, d, J = 8.7 Hz), 4.85 (2H, s), 4.49 (2 H, q, J = 7.1 Hz), 1.43 (3H, t,
J = 7.1 Hz). 13C-NMR (CDCl3,
δ
ppm): 164.55, 160.42, 155.24, 154.94, 154.64, 154.34, 147.42, 136.24,
133.10, 132.99, 125.58, 125.27, 120.30, 119.36, 118.94, 116.65, 114.35, 112.06, 94.24, 87.57, 64.30, 62.10,
14.18. MS(ESI-TOF) calculated for C19H15F3N2O4 [M + H]+ 393.18; found 394.16.
Ethyl 6-(bromomethyl)-4-{[4-(2,2,2-trifluoroacetamido)phenyl]ethynyl}picolinate (37). A mixture of compound 36
(2.10 g, 5.35 mmol) and PBr3 (0.75 mL, 8.03 mmol) in dry CHCl3 (180 mL) was stirred for 18 h at
◦
55 C, neutralized with aqueous 5% NaHCO3 (150 mL), the aqueous phase was extracted with CHCl3
(2 × 50 mL) and the combined organic phases were dried with Na2SO4. The product was purified
by silica gel column chromatography using EtOH/CH2Cl2 (10:90) as an eluent. Yield: 2.01 g (84%).
1H-NMR (CDCl3,
δ
ppm): 8.25 (1H, s), 8.01 (1H, d, J = 1.1 Hz), 7.75 (1H, d, J = 1.1 Hz); 7.68 (2H, d,
J = 8.7 Hz), 7.65 (2H, d, J = 8.7 Hz); 4.62 (2H, s), 4.50 (2H, q, J = 7.1 Hz), 1.45 (3H, t, J = 7.1 Hz). 13C-NMR
(CDCl3, ppm): 164.32, 157.68, 155.16, 154.85, 154.55, 154.25, 148.06, 136.21, 133.59, 133.06, 128.36,
δ
126.09, 120.26, 119.32, 118.92, 116.62, 114.32, 112.03, 94.61, 86.29, 62.25, 32.62, 14.20. MS(ESI-TOF)
calculated for C19H14BrF3N2O3 [M + H]+ 455.02 and 457.02; found 455.78 and 457.73.
Compound 38. A mixture of compound 34 (2.02 g, 3.20 mmol), compound 37 (1.46 g, 3.20 mmol),
dry K2CO3 (1.77 g, 12.8 mmol) in dry MeCN (70 mL) was stirred for 18 h at RT under argon. The mixture
was filtrated and the filtrate evaporated to dryness. The product was purified by silica gel column
1
chromatography using EtOH/CH2Cl2 (first 5:95, then 10:90) as an eluent. Yield: 3.03 g (94%). H-NMR
(DMSO-d6, δ ppm): 11.46 (1H, s), 7.97, (1H, s), 7.90 (1H, s), 7.82–7.77 (4H, m), 7.65 (2H, d, J = 8.4 Hz),

Molecules 2017, 22, 1807
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7.58 (1H, s), 7.54 (1H, s), 4.35 (2H, q, J = 7.1 Hz), 4.32 (2H, q, J = 7.2 Hz), 4.07 (2H, q, J = 7.1 Hz), 4.06
(2H, q, J = 7.1 Hz), 4.01 (2H, s), 3.98 (2H, s), 3.90 (2 H, s), 3.89 (2H, s), 3.57 (2H, s), 3.53 (2H, s), 1.34
(3H, t, H = 7.1Hz),1.31 (3H, t, J = 7.2 Hz), 1.20 (3H, t, J = 7.1 Hz), 1.18 (3H, t, J = 7.1 Hz). 13C-NMR
(DMSO-d6, δ ppm): 170.71, 170.69, 170.68, 170.56, 164.00, 163.38, 161.62, 160.41, 159.98, 159.81, 154.94,
154.64, 154.36, 154.03, 148.04, 147.33, 137.57, 135.94, 133.09, 132.78, 132.66, 132.62, 131.45, 128.42, 125.84,
120.81, 120.61, 118.07, 116.68, 114.39, 112.06, 93.81, 86.24, 61.43, 61.28, 61.14, 58.89, 58.19, 58,13, 58.94,
58.88, 55.32, 55.21, 14.07, 14.01, 13.96, 13.94. MS(ESI-TOF) calculated for C43H43Br2F3N6O9 [M + H]+
1003.22, 1005.22 and 1007.22; found 1003.59, 1005.48 and 1007.40.
Ligand Ester 39. A mixture of compound 38 (0.23 g, 0.23 mmol), compound 15 (0.22 g, 0.55 mmol)
in dry TEA (2 mL) and THF (4 mL) was de-aerated with argon. After an addition of
bis(triphenyl-phosphine)palladium(II) dichloride (10 mg, 14
µmol) and CuI (6 mg, 28 µmol),
the mixture was stirred overnight at 55 ◦C and evaporated to dryness. The product was purified by
silica gel column chromatography using EtOH/CH2Cl2/TEA (10:89/1) as an eluent. Yield: 0.31 g
1
(57%). H-NMR (DMSO-d6,
δ ppm): 11.43 (1H, s), 7.90 (1H, s), 7.87 (1H, s), 7.83 (1H, s), 7.77 (1H, s),
7.71 (2H, d, J = 8.6 Hz), 7.60 (2H, d, J = 8.6 Hz), 7.38 (1H, s), 7.37 (1H, s), 4.87 (4H, s), 4.83 (4H, s), 4.82
(2H, s), 4.79 (2H, s), 4.32 (2H, q, J = 7.1 Hz), 4.31 (2H, q, J = 7.1 Hz), 4.18 (4H, q, J = 7.1 Hz), 4.17 (2H,
s), 4.16 (4H, q, J = 7.1 Hz), 4.13 (4H, q, J = 7.1 Hz), 4.06 (2H, q, J = 7.2 Hz), 4.05 (2H, q, J = 7.2 Hz),
4.04 (2H, s), 3.96 (2H, s), 3.95 (2H, s), 3.54 (2H, s), 3.52 (2H, s), 1.31 (3H, t, J = 7.1 Hz), 1.30 (3H, t, J = 7.1
Hz), 1.22 (6H, t, J = 7.1 Hz), 1.20 (6H, t, J = 7.1 Hz), 1.18 (6H, t, J = 7.1 Hz), 1.17 (3H, t, J = 7.2 Hz), 1.16
(3H, t, J = 7.2 Hz). δ ppm): 171.27, 171.21, 168.60, 164.73, 164.62, 162.78, 161.07,
13C-NMR (DMSO-d6,
160.92, 160.66, 160.55, 158.80, 158.68, 155.53, 155.25, 154.96, 154.65, 147.81, 138.43, 133.45, 133.10, 132.74,
132.13, 127.72, 127.21, 124.93, 124.63, 123.14, 123.08, 121.35, 119.62, 118.07, 117.33, 115.04, 112.74, 94.65,
94.43, 94.29, 93.93, 93.88, 93.44, 93.42, 89.09, 87.53, 86,80, 65.83, 65.76, 65.46, 65.44, 61.82, 61.75, 61.19,
61.17, 61.14, 60.40, 60.35, 59.59, 59.53, 59.29, 59.25, 55.37, 55.02, 14.50, 14.48, 14.47, 14.46, 14.45, 14.44,
14.42. MS(ESI-TOF) calculated for C83H89F3N6O27 [M + H]+ 1659.58; found 1659.34.
Eu(III) Chelates 40. The compound 39 (0.28 g, 0.169 mmol) in 0.5 M KOH in EtOH (18.2 mL) was
stirred for 30 min at RT, H2O (10 mL) was added and the mixture was further stirred at RT for 7 h.
After evaporation of EtOH and an additional overnight stirring at RT, the pH was adjusted to 6.5 by
addition of 6 M HCl. EuCl3 (68 mg, 0.186 µmol) in water (1.0 mL) was added within 5 min and the pH
was maintained at 6.0–6.5 with suitable additions of solid NaHCO3. After stirring overnight at RT,
the pH was adjusted to 8.5 with 1 M NaOH. The precipitate was removed by centrifugation and the
supernatant evaporated to dryness. The product was purified by HPLC. Yield: 0.20 g (56%). Rf(HPLC):
15.2 min. UV: 358 nm.
Eu(III) Chelate
within 5 min to a mixture of CSCl2 (64
(2.25 mL). After stirring for 15 min at RT, the two phases were separated and the aqueous phase was
washed with CHCl3 (3 2.25 mL). The product was precipitated with acetone (ca. 45 mL), isolated by
centrifugation, washed with acetone (2 10 mL) and dried overnight in vacuum desiccator over silica
gel. Yield: 0.12 g. Rf(HPLC): 21.9 min. UV: 342 nm.
6
. An aqueous solution (2.25 mL) of the Eu(III) chelate 40 (0.13 g, 61
µmol) was added
µL, 0.85 mmol) and NaHCO3 (82 mg, 0.97 mmol) in CHCl3
×
×
Eu(III) Chelate 6a. A mixture of Eu(III) chelate (25 mg 11.7 µmol) and taurine (15 mg, 0.117 mmol) in
6
aqueous 50 mM Na2CO3 buffer (0.88 mL, pH 9.8) was stirred for overnight at RT. The product was
purified by HPLC. Rf(HPLC): 14.9 min. UV: 352 nm.
General Procedure for the Synthesis of Compounds 41 and 42. A mixture of compound 14 or 15 (1.00 mmol),
and compound 35 (0.22 g, 0.84 mmol; [28]) in dry TEA (5 mL) and THF (10 mL) was de-aerated with
argon. After an addition of bis(triphenylphosphine)palladium(II) dichloride (10 mg, 14
(6 mg, 28
mol), the mixture was stirred overnight at 55 ◦C and evaporated to dryness. The residue
was dissolved in CH2Cl2 (30 mL) washed with H2O (3 10 mL), dried with Na2SO4 and evaporated
to dryness. The product was purified by silica gel column chromatography.
µmol) and CuI
µ
×

Molecules 2017, 22, 1807
19 of 23
0
Diethyl 2,2 -{{4-[(2-(ethoxycarbonyl)-6-(hydroxymethyl)pyridin-4-yl]ethynyl)-1,3-phenylene}bis-(oxy)}-di-acetate
(
41). The product was purified by silica gel column chromatography using MeOH/CH2Cl2 (5:95) as
1
an eluent. Yield: 80%. H-NMR (CDCl3,
δ ppm): 8.08 (2H, s), 7.60 (2H, s), 7.45 (1H, d, J = 8.5 Hz),
6.50 (1H, dd, J = 2.0 and 8.5 Hz), 6.45 (1H, d, J = 2.0 Hz), 4.85 (2H, s), 4.71 (2H, s), 4.63 (2H, s), 4.47
(2H, q, J = 7.1 Hz), 4.30 (2H, q, J = 7.1 Hz), 4.29 (2H, q, J = 7.1 Hz), 1.44 (3H, t, J = 7.1 Hz), 1.32 (3H, t,
J = 7.1 Hz), 1.31 (3H, t, J = 7.1 Hz). 13C-NMR (CDCl3,
δ ppm): 168.11, 168.00, 164.63, 161.71, 160.08,
159.95, 147.27, 134.81, 127.02, 125.50, 106.42, 105.38, 100.99, 91.30, 89.67, 65.91, 65.34, 64.32, 62.24, 61.93,
61.54, 14.20, 14.19, 14.07. MS(ESI-TOF) calculated for C25H27NO9[M + H]+ 486.18; found 486.46.
0
00
Triethyl 2,2 ,2 -{{2-{[2-(ethoxycarbonyl)-6-(hydroxymethyl)pyridin-4-yl]ethynyl)}benzene-1,3,5-triyl}tris-(oxy)}
triacetate (42). The product was purified by silica gel column chromatography using EtOAc/petroleum
1
ether/TEA (from 69:30:1 to 89:10:1) as an eluent. Yield: 76%. H-NMR (CDCl3,
δ ppm): 8.11 (1H, d,
J = 0.5 Hz), 7.64 (1H, d, J = 0.5 Hz), 6.09 (2H, s), 4.84 (2 H, s), 4.70 (4H, s), 4.58 (2H, s), 4.47 (2H, q,
J = 7.1 Hz), 4.29 (4H, q, J = 7.1 Hz), 4.28 (2 H, q, J = 7.1 Hz), 3.45 (1 H, bs), 1.44 (3H, t, J = 7.1 Hz), 1.32
(3H, t, J = 7.1 Hz), 1.31 (6H, t, J = 7.1 Hz). 13C-NMR (CDCl3,
δ ppm): 167.97, 167.89, 164.70, 161.04,
160.22, 158.87, 147.21, 134.13, 125.42, 125.17, 96.17, 94.27, 93.80, 87.78, 66.21, 65.42, 64.29, 61.86, 61.61,
61.49, 14.20, 14.08, 14.06. MS(ESI-TOF) calculated for C29H33NO12[M + H]+ 588.56; found 589.03.
General Procedure for the Synthesis of Compounds 43 and 44. A mixture of compound 41 or 42 (0.613 mmol)
and PBr3 (86
µL, 0.919 mmol) in dry CHCl3 (20 mL) was stirred for 2.5 h at RT, neutralized with
aqueous 5% NaHCO3 (20 mL), the aqueous phase was extracted with CHCl3 (2
the combined organic phases were dried with Na2SO4. The product was purified by silica gel
column chromatography.
×
20 mL) and
Diethyl 2,20-{{4-{[2-(bromomethyl)-6-(ethoxycarbonyl)pyridin-4-yl]ethynyl}-1,3-phenylene}bis-(oxy)}diacetate
(
43). The product was purified by silica gel column chromatography using EtOH/CH2Cl2 (10:90) as
1
an eluent. Yield: 89%. H-NMR (CDCl3,
δ ppm): 8.10 (1H, d, J = 1.2 Hz), 7.75 (1H, d, J = 1.2 Hz), 7.46
(1H, d, J = 8.5 Hz), 6.50 (1H, dd, J = 2.3 and 8.5 Hz), 6.46 (1H, d, J = 2.3 Hz), 4.72 (2H, s), 4.63 (2H, s), 4.60
(2H, s), 4.49 (2H, q, J = 7.1 Hz), 4.30 (2H, q, J = 7.1 Hz), 4.29 (2H, q, J = 7.1 Hz), 1.45 (3H, t, J = 7.1 Hz),
1.32 (3H, t, J = 7.1 Hz), 1.31 (3H, t, J = 7.1 Hz). 13C-NMR (CDCl3,
δ ppm): 168.10, 167.97, 164.43, 160.05,
160.03, 157.41, 147.95, 134.88, 127.65, 126.00, 106.42, 105.28, 100.97, 91.97, 89.34, 65.92, 65.35, 62.40, 62.09,
61.54, 61.49, 32.88, 14.22, 14.09, 14.07. MS(ESI-TOF) calculated for C25H26BrNO8[M + H]+ 548.09 and
550.09; found 548.83 and 550.80.
Triethyl 2,20,200-{{2-{[2-(bromomethyl)-6-(ethoxycarbonyl)pyridin-4-yl]ethynyl}benzene-1,3,5-triyl}-tris(oxy)}-
triacetate (44). The product was purified by silica gel column chromatography using EtOH/CH2Cl2
1
(10:90) as an eluent using EtOH/CH2Cl2 (10:90) as an eluent. Yield: 82%. H-NMR (CDCl3, δ ppm):
8.12 (1H, d, J = 1.3 Hz), 7.79 (1H, d, J = 1.3 Hz), 6.08 (2H, s), 4.71 (4H, s), 4.59 (2H, s), 4.51 (2H, s), 4.48
(2H, q, J = 7.1 Hz), 4.30 (4H, q, J = 7.1 Hz), 4.29 (2H, q, J = 7.1 Hz), 1.43 (3H, t, J = 7.1 Hz), 1.31 (9 H, t,
J = 7.1 Hz).
13C-NMR (CDCl3, δ ppm): 167.92, 167.79, 164.52, 156.29, 133.27, 125.98, 125.08, 96.09, 93.92,
93.76, 88.21, 66.22, 65.59, 65.43, 62.04, 61.62, 61.51, 32.96, 14.22, 14.07, 14.03. MS(ESI-TOF) calculated for
C29H32BrNO11[M + H]+ 650.13 and 652.13; found 651.08 and, 653.02.
Compound 45. A mixture of compound 37 (0.41 g, 0.90 mmol), diBoc-TACN (0.14 g, 0.82 mmol),
dry K2CO3 (0.23 g, 1.62 mmol) and dry MeCN (8 mL) was stirred for 24 h at RT. After filtration and
washing the solid material with CH2Cl2, the filtrate was evaporated to dryness. The product was
purified by silica gel column chromatography using EtOH/CH2Cl2 (from 1:99 to 3:97) as an eluent.
1
Yield: 0.31 g (53%). H-NMR (DMSO-d6,
δ ppm): 11.48 (1H, s), 7.97 (1 H, s), 7.78–7.85 (3H, m), 7.66
(2H, d, J = 8.3 Hz), 4.38 (2H, q, J = 7.1 Hz); 3.80–3.85 (2H, m), 3.10–3.45 (8H, m), 2.65–2.75 (2H, m),
2.65–2.55 (2H, m), 1.43 (3H, s), 1.42 (3H, s), 1.40 (6H, s), 1.39 (6H, s), 1.34 (3 H, t, J = 7.1 Hz). 13C-NMR
(DMSO-d6,
δ ppm): 164.09, 155.78, 154.96, 154.80, 154.70, 154.56, 154.37, 154.08, 147.22, 137.51, 132.74,
132.54, 129.33, 127.03, 124.69, 120.85, 118.97, 116.69, 114.39, 112.62, 93.89, 86.35, 78.71, 61.44, 61.29,

Molecules 2017, 22, 1807
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51.42, 50.18, 49,69, 28.03, 14.02. Both spectra indicate the existence of rigid compound having different
structural isomers. MS(ESI-TOF) calculated for C35H44F3N5O7[M + H]+ 704.33; found 705.09.
Compound 46. A mixture of compound 45 (0.29 g, 0.41 mmol) and TFA (2 mL) was stirred for 2 h at RT,
evaporated to dryness and triturated with Et2O (40 mL). The product (0.34 g, 89%) was centrifuged,
1
washed with Et2O (2
×
15 mL) and dried. H-NMR (DMSO-d6,
δ
ppm): 11.54 (1H, s), 7.82 (2 H, d,
J = 8.4 Hz), 7.81 (1 H, s), 7.80 (1H, s), 7.71 (2 H, d, J = 8.4 Hz), 4.44 (2H, q, J = 7.0 Hz), 4.17 (2H, s),
3.69 (4H, bs), 3,26 (4H, bs), 2.97 (4H, bs), 1.39 (3H, t, J = 7.0 Hz). 13C-NMR (DMSO-d6, δ ppm): 154.41,
160.60, 155.48, 155.18, 154.89, 154.59, 147.17, 138.30, 133.24, 133.06, 129.87, 128.40, 125.22, 121.41, 118,57,
116.19, 114.84, 112.54, 95.54, 86.36, 62.47, 57.68, 50.42, 45.90, 45.36, 14,45. MS(ESI-TOF): calculated
C25H28F3N5O3 [M + 2H]+ 505.54; found 505.31.
General Procedure for the Synthesis of Ligand Esters 47 and 48. A mixture of compound 46 (0.12 g, 0.15 mmol),
43 or 44 (0.32 mmol), di-isopropylethylamine (0.40 mL) and dry MeCN (3.0 mL) was stirred for 5.5 h at
RT and evaporated to dryness. The product was purified by silica gel column chromatography.
Ligand Ester 47. The product was purified by silica gel column chromatography using EtOH/CH2Cl2/
TEA (from 10:90:0 to 10:89:1) as an eluent. Yield: 84%. As the product contains 2–3 rigid isomers, the
NMR spectra were too complicated to assign the isomers. MS(ESI-TOF) calculated for C75H78F3N7O19
[M + H]+ 1438.54; found 1439.41.
Ligand Ester 48. The product was purified by silica gel column chromatography using EtOH/CH2Cl2/
TEA (from 10:90:0 to 15:80:5) as an eluent. Yield: 79%. As the product contains 2–3 rigid isomers,
the NMR spectra were too complicated to assign the isomers. MS(ESI-TOF): calculated [M + H]+1642.60;
found 1643.57.
General Procedure for the Synthesis of Eu(III) Chelates 49 and 50. A mixture of the compound 47 or 48
(64 µmol) and 0.5 M KOH in EtOH (6.5 mL) was stirred for 1 h at RT and H2O (3 mL) was added.
After stirring for 4 h at RT, EtOH was evaporated, some H2O (2 mL) added and the residue was stirred
for 24 h at RT. After addition of citric acid (41 mg, 0.21 mmol) in H2O (0.25 mL), the pH was adjusted
to ca. 6.5 with 6 M HCl. Europium(III) chloride (26 mg, 71 µmol) in H2O (0.25 mL) was added within
10 min and the pH was adjusted to ca. 9.5 with 1 M NaOH. The mixture was stirred for 4–6 weeks
◦
at 95 C (after the analytical HPLC chromatogram showed completed complexation), the pH was
adjusted to ca. 7.0 with 1 M HCl, evaporated to dryness, dissolved in 20 mmol Triethylammonium
acetate (TEAA) buffer (1 mL) and purified with HPLC.
Eu(III) Chelate 49. Rf(HPLC) = 19.2 min. UV: 350 nm. MS(ESI-TOF) calculated for C59H48EuN7O18
[M + H]+ 1296.24; found 1295.85. Ligand isomers shown in the HPLC during the Eu(III) loading:
(1) Rf(HPLC) = 21.7 min, UV: 347 nm; (2) Rf(HPLC) = 22.3 min, UV: 339 nm; (3) Rf(HPLC) = 23.6 min,
UV: 343 nm. All of these peaks finally gave the product peak at Rf(HPLC) = 19.2 min. UV: 350 nm.
Eu(III) Chelate 50. Rf(HPLC) = 16.0 min. UV: 360 nm MS(ESI-TOF) calculated for C63H62EuN7O24
[M + H]+ 1444.24; found 1443.96. Ligand isomers shown in HPLC during the Eu(III) loading:
(1) Rf(HPLC) = 18.5 min, UV: 345 nm; (2) Rf(HPLC) = 20.4 min, UV: 347 nm; (3) Rf(HPLC) = 21.7 min,
UV: 347 nm. All of these peaks finally gave the product peak at Rf = 16.0 min, UV = 360 nm.
The Eu complex formation caused the observed bathochromic shift of 13–15 nm at UV. This was
separately secured with the complex 50 by an additional HPLC purification of the ligand isomers
and loadings of Eu(III) ion to each isomers. All of these tests gave finally the same product at
Rf(HPLC) = 16.0 min.
General Procedure for the Synthesis of Eu(III) Chelates
49 or 50 (21 mol) was added within 5 min to a mixture of CSCl2 (22
(28 mg, 0.33 mmol) and CHCl3 (1 mL). After stirring for 40 min at RT, the two phases were separated
and the aqueous phase was washed with CHCl3 (3 1 mL). The product was precipitated with acetone
7
and
8
. An aqueous solution (1 mL) of the chelate
µ
µL, 0.29 mmol) and NaHCO3
×

Molecules 2017, 22, 1807
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(ca. 45 mL), isolated by centrifugation, washed with acetone (2
a vacuum desiccator. The products were used as such for next phase or for labeling the antibodies.
×
10 mL), and dried overnight in
Eu(III) Chelate 7. Rf(HPLC) = 19.9 min. UV: 350 nm.
Eu(III) Chelate 8. Rf(HPLC) = 17.6 min. UV: 356 nm.
General Procedure for the Synthesis of Eu(III) Chelates 7a and 8a. A mixture of chelate
7
or
8 (2 mg) and
taurine (2 mg) in 50 mM Na2CO3 buffer (300
purified by using HPLC.
µL, pH 9.8) was stirred overnight at RT. The product was
Eu(III) Chelate 7a. Rf(HPLC) = 17.2 min. UV = 348 nm.
Eu(III) Chelate 8a. Rf(HPLC) = 15.2 min. UV = 354 nm.
3.3. Coupling of the Chelates 2–8 to Protein
The activated chelates were coupled to a troponin I antibody with a similar method as described
in [15] by using 100–300 fold excess of the labelling reagents. The reagents were incubated with the
antibody (1 mg) in 100–350 mM carbonate buffer (500–1000 µL, pH 9.8) overnight at RT. Labeled
antibody was separated from the chelate excess on a Superdex 200 GL 10/30 gel filtration column
(GE Healthcare Life Sciences) by using TRIS buffer (TRIS 50 mM, NaCl 0.9%, pH 7.75) as an eluent.
The fractions containing the antibody were pooled and the europium concentration was measured
by UV and confirmed by ICP-MS to obtain the labeling degrees i.e., Eu content per antibody. Finally,
bovine serum albumin was added to a concentration of 1g/L to the solutions containing the europium
labeled antibodies. The solutions were stored at 4 ◦C.
3.4. Fluorescence Measurements of the Eu Chelates 2a–8a and the Antibody Conjugates (2b–8b)
The fluorescence parameters for the Eu Chelates 2a–8a as well as the labeled antibodies (2b–8b)
were analyzed in 50 mM TRIS buffer (pH 7.75) and the Eu concentrations were measured by using
an ICP-MS (inductively coupled plasma mass spectrometer) instrument, ELAN 6100 DRC Plus
(PerkinElmer, Waltham, MA, USA), in quantitative mode. The analyzing parameters were: the Peak
Hopping mode, 20 sweeps/reading, 7 replicates, the Dwell time and the integration time was 50 ms and
1000 ms, respectively. Rhodium was used as the internal standard and the europium was measured on
Mass 152.929. A commercial multi-standard IMS–101, ICP-MS calibration standard 1 (ULTRA Scientific,
North Kingstown, RI, USA) was used for the calibration. The sample preparation for the ICP-MS
was done by using a digestion procedure i.e., the Microwave Sample preparation System, Multiwave
3000 (Anton Paar GmbH, Graz, Austria). The Eu chelate in the 50 mM TRIS buffer was digested with
microwave in mixture of Suprapur acids, HNO3 (5 mL) and H2O2 (1 mL). Afterwards, the sample was
diluted with deionized water (100 mL).
The fluorescence measurements were standardized using the Chelate 1a and 1b as a reference.
The photochemical values are based on earlier measurements [15,20]. The emission intensities of the
solutions were measured using the most intense emission line at 613–618 nm. Fluorescence efficiencies
and decay times were determined with VICTORTM X4 multilabel reader (PerkinElmer) and Cary
Eclipse spectrofluorometer (Agilent Technologies, Santa Clara, CA, USA), respectively.
3.5. Immunoassay and Signal Measurement from the Dried Microwell Surface
The troponin I antibody labeled with the chelate 2b
cardiac troponin I. A troponin I antibody labelled with 1b was used as a reference compound.
General assay procedure: 10 L of diluted tracer antibody (5 ng/ L) and 20 L of troponin I
–8b was tested in sandwich immunoassay for
µ
µ
µ
standard solution were pipetted to a pre-coated assay well (single wells in 96 well plate format,
wells coated with streptavidin and a biotinylated capture antibody against troponin I, Radiometer
◦
Turku Oy). The reaction mixtures were incubated 20 min at 36 C with shaking. The wells were washed

Molecules 2017, 22, 1807
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6 times and dried prior to measurement with VICTOR Plate fluorometer. More exact details have been
described in literature [15,20].
4. Conclusions
Altogether, seven new Eu(III) labeling chelates having two to three 4-(phenylethynyl)pyridine
chromophors in three different basic structural designs were synthesized and their key photophysical
properties were studied in this work. The focus of the study was to improve the signal level of the label
currently used in immunoassays, and, thus, to clarify the possibilities to enhance the assay sensitivity.
The significance of ligand antennas ILCT state as an alternative sensitization process on the label’s
brightness has been demonstrated, and the enhanced luminescence intensities were unprecedentedly
large especially when the signal was measured on the dried surface. These new labeling chelates
provide a solution to improve the assay sensitivity up-to tenfold from the present concepts.
Acknowledgments: The authors would like to thank Sirpa Oksman for technical synthesis support. Moreover, the
authors appreciate the contribution of the troponin I research team including Jari-Matti Sirkka, Virpi Leppänen,
Susann Eriksson, Sanna Rönnmark, Timo Valta, Heidi Vehmaan-Kreula, Titta Seppä and all project technicians for
the technical immunoassay support.
Author Contributions: All compounds were designed and prepared by Henri Sund, Harri Takalo and Niko Meltola.
The photochemical measurements were performed by Henri Sund and Kaj Blomberg’s research team. All authors
are aware of this manuscript and have agreed on this publication.
Conflicts of Interest: The authors declare no conflict of interest. A part of this research project has resulted in
issued patents Meltola N.; Takalo H.; WO2013026790 and Meltola N.; Takalo H.; Sund H.; WO2013092992.
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31. Compound 16 was prepared using the method described by [14]. H-NMR (DMSO-d6,
1
δ ppm): 7.65 (2H, d,
J = 1.8 Hz), 7.45 (2H, d, J = 1.8 Hz), 6.79 (2H, d, J = 8.4 Hz), 8.35 (2H, J = 8.4 Hz), 4.80 (2H, s), 4.05 (8H, q,
J = 7.1 Hz), 3.91 (4H, s), 3.76 (4H, s), 3.55 (8H, s), 2.66–2.58 (4H, m), 1.16 (12H, t, J = 7.1Hz). 13C-NMR
(DMSO-d6, δ ppm): 171.18, 161.14, 160.90, 147.07, 133.44, 129.43, 127.37, 124.29, 124.07, 114.46, 60.43, 59.50,
59.43, 56.63, 55.09, 32.59, 14.54. MS(ESI-TOF) calculated for C38H50Br2N6O8 [M + 2H]+ 878.23, 880.23, 882.23;
found 879.56, 881.57, 883.57.
Sample Availability: Not available.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).