Minocycline-based europium(III) chelate complexes: Synthesis, luminescent properties, and labeling to streptavidin

Article abstract of DOI:10.1002/hlca.200900175

Two chelate ligands for europium(III) having minocycline (=(4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,10, 12,12a-tetrahydroxy-1,11-dioxonaphthacene-2-carboxamide; 5) as a VIS-light-absorbing group were synthesized as possible VIS-light-excitable stable Eu³⁺ complexes for protein labeling. The 9-amino derivative 7 of minocycline was treated with H₆TTHA (= triethylenetetraminehexaacetic acid=3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12- tetraazatetradecanedioic acid) or H₅DTPA (= diethylenetriaminepentaacetic acid=N,N-bis{2-[bis(carboxymethyl)amino]ethyl} glycine) to link the polycarboxylic acids to minocycline. One of the Eu ³⁺ chelates, [Eu³⁺(minocycline-TTHA)] (13), is moderately luminescent in H₂O by excitation at 395 nm, whereas [Eu ³⁺(minocycline-DTPA)] (9) was not luminescent by excitation at the same wavelength. The luminescence and the excitation spectra of [Eu ³⁺(minocycline-TTHA)] (13) showed that, different from other luminescent EuIII chelate complexes, the emission at 615 nm is caused via direct excitation of the Eu³⁺ ion, and the chelate ligand is not involved in the excitation of Eu³⁺. However, the ligand seems to act for the prevention of quenching of the Eu³⁺ emission by H₂O. The fact that the excitation spectrum of [Eu³⁺(minocycline-TTHA)] is almost identical with the absorption spectrum of Eu³⁺ aqua ion supports such an excitation mechanism. The high stability of the complexes of [Eu³⁺(minocycline-DTPA)] (9) and [Eu³⁺(minocycline-TTHA)] (13) was confirmed by UV-absorption semi-quantitative titrations of H ₄(minocycline-DTPA) (8) and H5(minocycline-TTHA) (12) with Eu ³⁺. The titrations suggested also that an 1:1 ligand Eu³⁺ complex is formed from 12, whereas an 1:2 complex was formed from 8 minocycline-DTPA. The H₅(minocycline-TTHA) (12) was successfully conjugated to streptavidin (SA) (Scheme 5), and thus the applicability of the corresponding Eu³⁺ complex to label a protein was established.

Full text of DOI:10.1002/hlca.200900175

Helvetica Chimica Acta – Vol. 92 (2009)
2357
Minocycline-Based Europium(III) Chelate Complexes: Synthesis,
Luminescent Properties, and Labeling to Streptavidin
a
a
a
b
by Takuya Nishioka ), Yuji Yamamoto ), Kimikazu Hashino ), and Kazuko Matsumoto* )
a) Department of Chemistry, Waseda University, Okubo, Shinjuku-ku, Tokyo 169-8555, Japan
b) Tokyo Chemical Industry Co., Ltd., 4-10-2, Nihonbashi-honcho, Chuo-ku, Tokyo 103-0023, Japan
(
phone: þ 81356408859; fax þ 81356408868; e-mail: kmatsu@yf 6.so-net.ne.jp)
Dedicated to Professor Jean-Claude Bꢀnzli on the occasion of his 65th birthday
Two chelate ligands for europium(III) having minocycline (¼(4S,4aS,5aR,12aS)-4,7-bis(dimethyl-
amino)-1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tetrahydroxy-1,11-dioxonaphthacene-2-carboxamide;
3
þ
5
) as a VIS-light-absorbing group were synthesized as possible VIS-light-excitable stable Eu complexes
for protein labeling. The 9-amino derivative of minocycline was treated with H TTHA
7
6
(
¼triethylenetetraminehexaacetic acid ¼ 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecane-
dioic acid) or H DTPA (¼diethylenetriaminepentaacetic acid ¼ N,N-bis{2-[bis(carboxymethyl)amino]e-
5
3
þ
3þ
thyl}glycine) to link the polycarboxylic acids to minocycline. One of the Eu chelates, [Eu (minocy-
3
þ
cline-TTHA)] (13), is moderately luminescent in H O by excitation at 395 nm, whereas [Eu (minocy-
2
cline-DTPA)] (9) was not luminescent by excitation at the same wavelength. The luminescence and the
3
þ
excitation spectra of [Eu (minocycline-TTHA)] (13) showed that, different from other luminescent
III
3þ
Eu chelate complexes, the emission at 615 nm is caused via direct excitation of the Eu ion, and the
3
þ
chelate ligand is not involved in the excitation of Eu . However, the ligand seems to act for the
3
þ
prevention of quenching of the Eu emission by H O. The fact that the excitation spectrum of
2
3
þ
3þ
[
Eu (minocycline-TTHA)] is almost identical with the absorption spectrum of Eu aqua ion supports
3
þ
such an excitation mechanism. The high stability of the complexes of [Eu (minocycline-DTPA)] (9) and
3
þ
[
Eu (minocycline-TTHA)] (13) was confirmed by UV-absorption semi-quantitative titrations of
3
þ
H (minocycline-DTPA) (8) and H (minocycline-TTHA) (12) with Eu . The titrations suggested also
that an 1:1 ligand Eu complex is formed from 12, whereas an 1:2 complex was formed from 8
4
5
3
þ
minocycline-DTPA. The H (minocycline-TTHA) (12) was successfully conjugated to streptavidin (SA)
5
3
þ
(
Scheme 5), and thus the applicability of the corresponding Eu complex to label a protein was
established.
Introduction. – Time-resolved luminescence measurement of lanthanide chelate
labels is recognized as a sensitive detection principle suitable for the determination of
biological components in fluids and tissues, where the complex matrices of biological
samples cause substantial background fluorescence and interfere with the analysis with
normal non-time-resolved fluorescence detection [1 – 5]. Although a number of
3
þ
3þ
3þ
luminescent lanthanide chelate complexes (especially those of Eu , Tb , Sm , and
3þ
Dy ) have been synthesized [6 – 18], only a few complexes are satisfactory for the
practical use in biological analysis, such as in immunoassay, DNA-hybridization assay,
ligand – receptor-binding assay, cell and tissue imaging, microarray, and many other
biological analyses. Efforts to improve the luminescence properties still continue such
as to develop stronger luminescence, higher labeling efficiency while maintaining the
ꢀ
2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Helvetica Chimica Acta – Vol. 92 (2009)
original biological functions of the labeled proteins or nucleic acids, and higher water
solubility and stability of the chelate complex in biological matrices. A recent trend in
the research is also directed to multicolor labeling and lanthanide complexes emitting
3þ
3þ
3þ
in the near-infrared region (complexes of Nd , Er , and Yb ) [19] for biological
analysis as well as for light-source materials.
Lanthanide chelates are usually excited with UV light of the wavelength specific to
the ligand aromatic groups, and emit VIS light at the wavelength specific to the central
3þ
3þ
lanthanide ion (Eu 615 nm and Tb 545 nm). The excitation light (usually in the
range 300 – 340 nm) is absorbed by the aromatic moiety of the ligand, and the energy is
transferred from the ligand to the lanthanide ion, and the lanthanide ion finally emits
VIS light at the wavelength specific to the lanthanide energy level. Owing to such an
emission mechanism, the excitation and emission wavelengths are separated very much
from each other, leading to a lower fluorescence background level at the emission
wavelength, and thus a high detectability is obtained. In addition, the long lifetimes of
lanthanide complexes (often more than 1 ms) afford time-resolved measurements very
effective avoiding undesired short-lived background fluorescence caused by the matrix
materials and cuvette materials. However, in some applications, the UV-light excitation
damages biological samples, and excitation with VIS light is desired. Lanthanide
chelate complexes excitable with VIS light are strongly sought in the current research;
however, so far, only a few complexes have been reported, and even though these are
luminescent with ca. 400 nm excitation, the luminescence is significantly strong only in
organic solvents. For biological applications, lanthanide luminescence in H O is
2
necessary, but such complexes seem so far not easy to realize, due to the strong
quenching effect of H O. VIS-Light excitation is also desired because various laser and
2
diode light sources are available for the VIS region, and it is expected that more
compact instruments will be possible with such light sources, compared to the Xe lamp
currently used as a UV-light source.
We noted a recent report that addition of tetracycline (1) to Eu3þ gives moderately
3þ
strong luminescence in H O with excitation at 405 nm, and the [Eu (tetracycline)]
2
complex has been used for several analyses [20 – 25]. As far as we know, the
3
þ
3þ
[
Eu (tetracycline)] complex and analogous [Eu (doxycycline)] complexes [26][27]
3
þ
are the only Eu complexes which give moderate luminescence in H O by excitation at
2
405 nm. However, considering the corresponding ligand structure, it seems that the
stability of these metal-chelate structures in water and the luminescence intensities can
be improved to allow a wider range of bio-applications. In addition, these ligands have
not been used to label proteins and nucleic acids, since no group is attached to them
allowing such a labeling. We expected that the luminescence in H O would be enhanced
2
by attaching a more strongly and stable chelating group to the tetracycline moiety to
3þ
satisfy the usual coordination number of 9 or 10 of Eu . In the present study, an
attempt was made to link tetracycline to H EDTA (¼ethylenediaminetetraacetic
4
acid ¼ N,N’-ethane-1,2-diylbis[N-(carboxyethyl)glycine]) or H TTHA (triethylenete-
6
traminehexaacetic acid ¼ 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradeca-
nedioic acid). However, the amide N-atom of tetracycline was not reactive enough for
such a linking, and the target ligands were not obtained in practical yields. Therefore,
minocycline (¼4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahy-
dro-3,10,12,12a-tetrahydroxy-1,11-dioxonaphthacene-2-carboxamide; 5) was used in-

Helvetica Chimica Acta – Vol. 92 (2009)
2359
stead of tetracycline, since amination of the 9-position of minocycline was known in the
literature. The multidentate chelate ligands H DTPA and H TTHA were then attached
5
6
successfully to the amino group of 9-aminominocycline (7). Such multidentate chelate
structures were expected to enhance the complex stability of the metal chelate in

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Helvetica Chimica Acta – Vol. 92 (2009)
various buffers including phosphate and carbonate. In these buffers, the stability of
lanthanide chelate complexes decreases due to the high coordinating affinity of the
buffer materials to lanthanide ions [28][29]. High complex stability would exclude H O
2
molecules from the coordination sphere of the lanthanide ion, and thus enhance the
lanthanide emission.
Results and Discussion. – Attempt to Synthesize the Ligands H (tetracycline-EDTA)
4
(
2) and H (tetracycline-TTHA) (4). Tetracycline hydrochloride (1 · HCl; 6.25 mg,
5
1
3 mmol) was treated with H (aminobenzyl-EDTA) (5.17 mg, 13 mmol) and form-
4
aldehyde (11 ml, 136 mmol) in t-BuOH (8 ml) at room temperature for 30 min with
stirring, and after a further reaction for 15 min under reflux, the hot solution was
filtered and the filtrate cooled to give a yellow precipitate (Scheme 1). The proceeding
of the reaction was monitored by reversed-phase HPLC and ESI-MS, and two new
peaks were found in the MS. However, these peaks did not correspond to 2, and even on
varying several factors of the reaction conditions including the relative amount of the
reactants and different solvents and reaction times, the target compound 2 was not
obtained.
Scheme 1
It seemed that the direct introduction of the H (aminobenzyl-EDTA) to the amide
4
group at the 2-position of tetracycline via the reaction of Scheme 1 is difficult. In the
literature, no such reaction of an arenamine with the tetracycline amide N-atom is

Helvetica Chimica Acta – Vol. 92 (2009)
2361
reported. However, since the morpholine N-atom is reported to react with the
minocycline amide N-atom in the presence of formaldehyde [30], the cyclic piperidin-
4
-amine was selected as a linker to attach H TTHA to tetracycline to give
6
H (tetracycline-TTHA) (4), as shown in Scheme 2. Reversed-phase HPLC monitoring
5
of the reaction solution for the first step in Scheme 2 showed, however, only the peak of
tetracycline, and although the ESI-MS of the separated product showed a small peak of
the target compound 3 in addition to the main peak of tetracycline, the reaction was not
pursued any further because the yield of 3 seemed negligibly low.
Scheme 2
Ligands H (minocycline-DTPA) (8) and H (minocycline-TTHA) (12). From all of
4
5
the above unsuccessful attempts to link H EDTA or H TTHA to the amide N-atom of
4
6

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Helvetica Chimica Acta – Vol. 92 (2009)
tetracycline, we concluded that the amide group at the 2-position of tetracycline is not
suitable to link amino groups of other molecules, and we moved to minocycline (5)
instead of tetracycline (1) since amination at the 9-position of 5 had been reported [31],
and H DTPA (¼diethylenetriaminepentaacetic acid ¼ N,N-bis{2-[bis(carboxymethy-
5
l)amino]ethyl}glycine) and H TTHA were used as the chelating groups. The overall
6
reaction scheme to H (minocycline-DTPA) (8) is shown in Scheme 3.
4
Scheme 3
The first and second steps of the synthesis of 8 (5 ! 6 ! 7) were carried out
according to [31], and the product of each step was confirmed by ESI-MS. The yields
were 76.6 and 32.7%, respectively. The reaction of 9-aminominocycline (7) with
H DTPA dianhydride proceeded easily and gave the final product H (minocycline-
5
4
DTPA) (8) in 64.3% yield. The product was confirmed by ESI-MS after reversed-phase
HPLC purification. H (minocycline-DTPA) was then treated with EuCl · 6 H O in
4
3
2
3þ
Tris · HCl buffer (pH 6.5) to obtain the metal complex [Eu (minocycline-DTPA)] (9).
The ligand-to-metal 1:1 complex was confirmed by ESI-MS. The complex formation
reaction was also carried out in Et N(OAc) buffer (pH 5.0 and 6.0), NaHCO buffer
4
3
(
pH 6.5), and 0.1m NaOH; however, only Tris · HCl buffer gave the peaks correspond-

Helvetica Chimica Acta – Vol. 92 (2009)
2363
ing to the metal complex in the ESI-MS. In the reaction of 9-aminominocycline (7)
with the DTPA carboxyl group (Scheme 3), several solvents (0.1m Tris · HCl (pH 8.0),
Et N(OAc) buffer (pH 5.0 and 6.0), NaHCO buffer (pH 6.5), and 0.1m NaOH were
4
3
compared, and the final products were analyzed with ESI-MS; however, only 0.1m Tris ·
þ
HCl (pH 8.0) gave a product which showed the ESI-MS peak of [8 þ H] .
Compound 7 was similarly treated with H TTHA dianhydride 11 (from H TTHA
6
6
(
10) with DCC in dry pyridine) to obtain H (minocycline-TTHA) (12) (Scheme 4).
5
However, different from our expectation, the reversed-phase HPLC of the reaction
solution of 11 with 7 showed only one peak, which however, was identified as the
starting compound 9-aminominocycline (7). The reaction conditions were as follows:
To a solution of 11 (5.90 mmol) in dry DMF (2 ml), dry Et N (3 ml) was added. A dry
3
DMF solution (2 ml) containing 7 (2.0 mg) was added to the DMF solution of 11, and
the solution was stirred under Ar at room temperature for 2 h. Then Et N(OAc) buffer
4
(0.1m, pH 6.5; 2 ml) was added, and the solution was further stirred at room
temperature for 30 min. After evaporation of the solvent, a white powder was
obtained. However, reversed-phase HPLC analysis of the white compound showed
only the peak of 7.
Scheme 4
Since H TTHA dianhydride 11 did not react with 7, unmodified H TTHA (10) itself
6
6
was treated with 7 in the next attempt. The reactions described in the Exper. Part gave
the final product 12 after reversed-phase HPLC purification in 86.1% yield. The

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Helvetica Chimica Acta – Vol. 92 (2009)
product was confirmed by ESI-MS. The ligand 12 was treated with EuCl · 6 H O, and
3
2
3
þ
the [Eu (minocycline-TTHA)] complex 13 was obtained, which was confirmed by
ESI-MS.
These syntheses showed that the 2-amide group of tetracycline (1) is much less
reactive compared to the 9-amino group of 9-aminominocycline (7).
3þ
Photophysical Properties of the Minocycline-Based Eu Complexes 9 and 13. The
UVabsorption spectra of 9 and 13 are shown in Fig. 1. Both spectra are very similar to
each other, and suggest that the spectra are dominated by the minocycline moiety and
are not appreciably affected by the aminopolycarboxy moiety. The absorption maxima
and the molar extinction coefficients are listed in the Table, together with those of free
minocycline, free H (minocycline-DTPA) (8), and free H (minocycline-TTHA) (12).
4
5
The absorption bands of 8 or 12 at ca. 355 nm (Fig. 2) are significantly shifted to longer
wavelengths (ca. 395 nm) on metal-complex formation for both 8 and 12 (Fig. 1),
Fig. 1. UV Absorption spectra of [Eu3 (minocycline-DTPA)] (9; dark blue) and [Eu (minocycline-
þ
3þ
TTHA)] (13; purple). Both solns. were 100 mm in 0.1m Tris · HCl (pH 8.0).
Table. Properties of the UV-Absorption for the Free Ligands 8 and 12 and Eu3 Complexes
þ
ꢀ1
ꢀ1
lmax [nm] (e [cm
m
])
Buffer
4
4
Minocycline
375 (1.40 · 10 ), 273 (1.56 · 10 ), 20 mm · Tris · HCl, pH 8.5
2
4
44 (1.95 · 10 )
[
Eu3þ(minocycline)] (1 : 1)a)
H (minocycline-DTPA) (8)
Eu (minocycline-DTPA)] (9; 1 : 2) )
387 (1.17 · 10 ), 241 (1.52 · 10 )
4
4
20 mm Tris · HCl, pH 8.5
0.1m Tris · HCl, pH 7.0
0.1m Tris · HCl, pH 7.0
0.1m Tris · HCl, pH 7.0
0.1m Tris · HCl, pH 7.0
4
4
351 (1.10 · 10 ), 240 (1.51 · 10 )
4
3
þ
a
4
4
[
397 (1.39 · 10 ), 240 (1.50 · 10 )
3
4
H (minocycline-TTHA) (12)
351 (9.80 · 10 ), 240 (1.50 · 10 )
5
3
þ
a
4
4
[
Eu (minocycline-TTHA)] (13; 1 : 1) ) 395 (1.20 · 10 ), 240 (1.50 · 10 )
a) Ratio Eu /ligand.
3þ

Helvetica Chimica Acta – Vol. 92 (2009)
2365
Fig. 2. UV Absorption Spectra of SA (purple) and H (minocycline-TTHA) (12; dark blue) in 50 mm
5
AcONa (pH 3.5) adjusted to pH 7.0 with 1m Tris · HCl (pH 9.0)
whereas the absorption maximum of the free ligands at around 240 nm is not
significantly shifted on metal-complex formation.
The excitation and emission spectra of 9 and 13 are shown in Fig. 3. Although
complex 9 is not luminescent, 13 gives moderate luminescence with excitation at
3þ
Fig. 3. Excitation and emission spectra of [Eu (minocycline-DTPA)] (9; red and dark blue lines) and of
3þ
[
Eu (minocycline-TTHA)] (13; green and purple lines). Both solns. were 100 mm in 0.1m Tris · HCl
pH 8.0), and were excited at 395 nm and observed at 616 nm.
(

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Helvetica Chimica Acta – Vol. 92 (2009)
395 nm. The excitation spectrum of 13 is very unusual, since its spectral pattern in the
range 350 to 450 nm consists of several sharp peaks somewhat similar to the absorption
peaks of lanthanide metal ion. We recognized that the excitation spectrum is very
3
þ
similar to the reported absorption spectrum of Eu aqua ion [10][32]. The excitation
spectra of 9 and 13 in Fig. 3 also show that in the wavelength range below 350 nm, both
complexes are excited strongly by the ligand absorption via the ligand-to-metal
3þ
intramolecular energy transfer, similarly to many other luminescent Eu complexes.
Apparently the broad and strong absorption band around 400 nm of 9 and 13 in Fig. 1
are not effective for luminescence excitation.
At first we thought that the metal ion of 13 was dissociated from the ligand, and
3þ
thus the excitation and emission spectra of Eu aqua ion was observed in the spectra of
Fig. 1. To check this point, a 100 mm EuCl solution in Tris · HCl buffer at pH 8.0 was
3
prepared, and the emission spectrum was measured. However, the solution was not
luminescent. Hence, the metal ion of 13 was not dissociated in the solution of Fig. 3.
The absorption spectrum of 13 in Fig. 1 is definitely different from that of the free
3þ
ligand, and the spectrum is not an overlap of the spectra of free ligand and Eu ion,
which supports that the structure of the metal-coordinated complex is retained in the
solution under the spectral-measurement conditions. As for the excitation and emission
mechanism of 13, the minocycline moiety of 13 is not effective for energy transfer to
3þ
Eu , and the emission occurs via direct absorption by the metal ion at its strongest
absorption line at 395 nm [10][33]. For comparison, emission was examined for Tris ·
HCl buffer (pH 8.0) solutions containing 100 mm minocycline and EuCl · 6 H O at a
3
2
molar ratio of 1:1 and 1:0.5; however, the solutions were not luminescent. Minocycline
seems different from tetracycline and doxycycline in the energy-level diagram. The
latter ligands absorb excitation light at ca. 400 nm and transfer the energy from the
5
3þ
3þ
ligand triplet state to the D level of Eu . The central Eu ion finally emits
n
5
7
luminescence via the D ! F transition [20 – 25]. Free minocycline cannot transfer
0
2
3þ
the absorbed energy at ca. 395 nm to Eu , presumably because of the unsuitable
triplet-state-energy level. The excitation line at 395 nm in Fig. 3 is the transition of
7
5
5
7
F ! L [10][33], and the energy is emitted as the 616 nm line of the D ! F
0
6
0
2
3þ
transition of Eu .
To examine the complex stability of 9 and 13 in solution, and the stoichiometry of
3þ
the metal-complex formation, both ligands 8 and 12 were titrated with Eu by
monitoring the absorption spectra. To a 0.1 mm solution of 8 in 0.1m Tris · HCl (pH 7.0),
a solution of EuCl · 6 H O at suitable concentration was added, so that the molar ratio
of the ligand and Eu was varied stepwise from 1:0 to 1:20, and the absorption spectra
were measured. As the spectra in Fig. 4 and the titration curve (expressed as the
absorbance change at 397 nm) in Fig. 5 show, the complex formation was completed
3
2
3
þ
3þ
after 2 equiv. of Eu were added. Similar measurements were carried out for 12, and
the spectra and the titration curve are shown in Figs. 6 and 7.
For ligand H (minocycline-TTHA) (12), the complex formation was completed at a
5
1
:1 ratio, suggesting a 1:1 complex 13 is formed. However, for ligand H (minocycline-
4
3
þ
DPTA) (8), the Eu complex 9 seems to be a 1:2 complex, and such a difference of the
metal-to-ligand ratio between 13 and 9 suggests that complex 13 would be more stable
than 9. This difference may also explain the difference of the luminescence properties
of the two metal complexes. It is also highly probable that the metal complexes stably

Helvetica Chimica Acta – Vol. 92 (2009)
2367
Fig. 4. Spectral change in the titration of H (minocycline-DTPA) (8) with EuCl3
4
Fig. 5. Titration curve of H (minocycline-DTPA) (8) with EuCl , observed at the absorption at 397 nm
4
3
exist and are not dissociated in the solutions of Figs. 1 and 3, since the final spectra of
the titration solutions (Figs. 4 and 6) are identical to the spectra in Fig. 1. The excitation
spectrum such as that of 13 having several line-like peaks is very rare, compared to
3þ
those of other luminescent Eu complexes. The latter exhibit excitation spectra having
broad absorption bands almost identical to the absorption spectra of the aromatic
3þ
ligands. Measurements of absorption or emission spectra of a simple aqueous Eu

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Helvetica Chimica Acta – Vol. 92 (2009)
Fig. 6. Spectral change in the titration of H (minocycline-TTHA) (12) with EuCl
5
3
Fig. 7. Titration curve of H (minocycline-TTHA) (12) with EuCl , observed at the absorption at 391 nm
5
3
solution need higher concentrations than those used in the present experiments, and the
solutions must be acidic to avoid precipitation of lanthanide hydroxide. Thus, obviously
the ligand in 13 acts as inhibitor preventing precipitation and as protection or against
quenching of the emission of the metal complex by H O.
Conjugation of 13 to Streptavidin. To test the suitability of Eu complex 13 for the
labeling of proteins, streptavidin (SA) was conjugated with ligand 12 according to
2
3þ

Helvetica Chimica Acta – Vol. 92 (2009)
2369
Scheme 5. Streptavidin was selected as a typical protein to see whether the new labeling
reagent can be labeled to proteins without any inconvenience, since our past experience
3þ
showed that for some labeling reagents for Eu , the conjugation reaction causes
precipitation of the protein. If SA can be conjugated successfully to H (minocycline-
5
TTHA) (12), it means that the reagent can be used in many bio-analytical applications
taking advantage of the selective and strong binding of the conjugated SA to biotin.
Thus, one of the carboxy groups of compound 12 was succinimidylated (ester formation
with N-hydroxysuccinimide (NHS)) to give 14, which was treated with SA in 0.1m phos-
phate buffer (pH 8.0) at room temperature for 6 h. The details of the purification of the
conjugated streptavidin, H (SA-minocycline-TTHA), is described in the Exper. Part.
4
Scheme 5

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370
Helvetica Chimica Acta – Vol. 92 (2009)
Luminescence Properties of the Labeled SA. The absorption spectrum of the H4
SA-minocycline-TTHA) (Fig. 8) consists of the absorption bands almost identical to
(
those of SA and free H (minocycline-TTHA) (12; Fig. 2). The time-resolved
5
3þ
luminescence spectrum of [Eu (SA-minocycline-TTHA)] was measured after the
addition of 2 equiv. of EuCl · 6 H O and 1 h reaction at room temperature. The
3
2
concentration of H (SA-minocycline-TTHA) was determined with the UV spectrum,
4
assuming that the molar extinction coefficient of the conjugated H (SA-minocycline-
4
TTHA) is not changed from that of free H (minocycline-TTHA). The luminescence
5
3þ
spectrum of the labeled SA, i.e., of complex [Eu (SA-minocycline-TTHA)] is shown
3þ
in Fig. 9, together with that of [Eu (minocycline-TTHA)] at the same concentration
of the minocycline-TTHA moiety. Both spectra were measured with the excitation
wavelength 395 nm and the luminescence-peak wavelength 615 nm. It should be noted
3þ
that the luminescence of [Eu (minocycline-TTHA)] is 10-fold increased on con-
jugation to SA, in spite of the loss of one of the carboxy groups needed for the
conjugation.
Fig. 8. UV Spectrum of H (SA-minocycline-TTHA) in 50 mm AcONa (pH 3.5) adjusted to pH 7.0 with
4
1m Tris · HCl (pH 9.0)
Conclusions. – As a candidate for a VIS-light-excitable lanthanide complex,
3þ
3þ
[
Eu (minocycline-DTPA)] (9) and [Eu (minocycline-TTHA)] (13) were synthe-
sized, and the luminescence properties and suitability to label SAwere studied. While 9
was not luminescent in H O, 13 could be excited at 395 nm in H O to emit luminescence
2
2
at 615 nm. The luminescence intensity of 13 is moderate, and the excitation spectrum of
the complex suggests that the luminescence occurs not via the conventional energy
3þ
transfer from the ligand to the central Eu ion but by direct absorption of the
3
þ
7
5
5
7
excitation light by the Eu ion ( F ! L ) and emission at 615 nm as D ! F . The
0
6
0
2
effect of the ligand of 13 seems to consist of the protection of the metal ion from

Helvetica Chimica Acta – Vol. 92 (2009)
2371
Fig. 9. Time-resolved luminescence spectra of [Eu3 (SA-minocycline-TTHA)] (green) and [Eu (mi-
þ
3þ
nocycline-TTHA)] (red). Both solns. were 19.4 mm for the minocycline-TTHA moiety in 50 mm AcONa
(
pH 3.5) adjusted to pH 7.0 with 1m Tris · HCl (pH 9.0).
quenching by H O, since none of the simple aqueous solutions of EuCl , a 1:1 mixture
2
3
3þ
of EuCl and H TTHA, or a 1:1 mixture of EuCl and minocycline at the same Eu
3
6
3
concentration level did show any luminescence. The minocycline-DTPA ligand of 9
does not exert such a preventing function on H O quenching. It seems that the triplet-
2
3þ
state energy of minocycline is not suitable for energy transfer to Eu . Since the
3þ
molecular absorption coefficient of Eu is not large, the luminescence of 13 is not
strong; however, observation of the luminescence at such a low concentration was not
known previously, and complex 13 is noteworthy in this regard. The conjugation of 13
to SA showed that a protein can be labeled with 13 without troubles under usual
reaction conditions.
Experimental Part
General. All reactions with air- or humidity-sensitive materials were carried out under Ar by using
standard Schlenk technique or a glove box. All the reagents were used as received without purification.
HPLC: Waters-XTerra-MS-C18 column (f 2.5 mm 4.6 ꢁ 50 mm); mobile phase: MeCN containing 0.01%
of CF COOH (A) and H O containing 0.01% of CF COOH (B); flow rate 1.0 ml/min; gradient 0 min A/
3
2
3
B 5 :95, 5 min A/B 5 :95, and 50 min A/B 50 :50. UV/VIS Spectra: Jasco-Ubest-V-570 UV/VIS
spectrophotometer. ESI-MS: Finnigan-Thermo-Quest-LCQ-Deca spectrometer. Luminescence excita-
tion and emission spectra: Perkin-Elmer-LS-50B luminescence spectrometer.
(
4S,4aS,5aR,12S)-4,7-Bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tetrahydroxy-
9
-nitro-1,11-dioxonaphthacene-2-carboxamide (6). Minocycline hydrochloride (5 · HCl; 405 mg,
0
.821 mmol) was dissolved in conc. H SO (100 ml), and while the soln. was cooled with ice-water,
2
4
KNO (98.4 mg, 0.974 mmol) was added. After the soln. was stirred for 1.5 h at 08 (reversed-phase HPLC
3
(
see General) and ESI-MS monitoring), Et O (800 ml) cooled with ice-water was added dropwise. The
2
formed precipitate was filtered, washed with Et O, and vacuum dried at 408: 6 (76.6%). ESI-MS: 503.1
2
þ
(
[M þ H] ).

2
372
Helvetica Chimica Acta – Vol. 92 (2009)
(
4S,4aS,5aR,12aS)-9-Amino-4,7-bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tet-
rahydroxy-1,11-dioxonaphthacene-2-carboxamide (7). To the soln. of 6 (250 mg, 0.358 mmol) in 2m
H SO (10 ml) and 2-methoxyethanol (15 ml), 10% Pd/C (50 mg) was added, and the mixture was
2
4
stirred for 1.5 h under 2.72 atm of H . The soln. was filtered and the filtrate concentrated. A small amount
2
of MeOH was added to the residue, and the MeOH soln. of the product was dropwise added to a mixture
of i-PrOH (70 ml) and Et O (50 ml). The precipitate formed was collected by centrifugation, washed
2
with Et O, and vacuum dried at 408. The product was purified by reversed-phase HPLC and then
2
þ
vacuum-dried: 7 (32.7%). ESI-MS: 473.1 ([M þ H] ).
H (minocycline-DTPA) (8). The dianhydride of H DTPA (27 mg, 74.3 mmol) was dissolved in dry
4
5
DMF (2 ml), to which molecular-sieves-dried Et N (36 ml, 247.5 mmol) was added. Then 7 (25.2 mg,
3
4
9.5 mmol) in dry DMF (3 ml) was added, and the soln. was stirred for 2 h at r.t. under Ar. To this soln.
0
.1m Et N(OAc) (buffer pH 6.5; 2 ml) was added, and the soln. was stirred for 30 min at r.t. After the
4
solvent was evaporated, the product was purified by reversed-phase HPLC and dried under reduced
þ
pressure: 8 (64.3%). ESI-MS: 848.3 ([M þ H] ).
3
þ
[
Eu (minocycline-DTPA)] (9). To a soln. of 8 (3 mg, 3.54 mmol) in 0.1m Tris · HCl (pH 8.0; 2 ml), a
soln. (1 ml) of 0.1m Tris · HCl (pH 8.0) containing EuCl · 6 H O (1.95 mg, 5.31 mmol) was added, and the
3
2
soln. was stirred for 30 min at r.t. After adjusting the pH to 9.0 with 0.1m NaOH, the precipitate was
removed by filtration, and acetone (50 ml) was added to the filtrate to precipitate the metal complex. The
precipitate was collected by centrifugation, washed with acetone, and vacuum-dried: 9. ESI-MS: 998.2
þ
(
[M þ H] ).
H (TTHA Dianhydride) (11). H TTHA (665 mg, 1.35 mmol) and N,N’-dicyclohexylcarbodiimide
2
6
(
DCC; 555 mg, 2.7 mmol) were stirred in dry pyridine (20 ml) for 48 h at 408. The precipitate was
collected by filtration, washed with Et O, and vacuum-dried: 11 (almost 100%).
2
H (minocycline-TTHA) (12). To a soln. of H TTHA (265.2 mg, 537 mmol) in dry DMF (5 ml),
5
6
molecular-sieves-dried Et N (1.5 ml) was added. The soln. was stirred overnight at r.t. under Ar. While
3
the soln. was cooled with ice-water, a mixture of dry DMF (300 ml) and isobutyl chloroformate (¼2-
methylpropyl carbonochloridate) (76.7 ml, 537 mmol) was gradually added. After the soln. was stirred for
1
5 min at 08 under Ar, dry DMF (1 ml) containing 7 (27.3 mg, 53.7 mmol) was added, and the soln. was
stirred for 5 h at r.t. At this stage, reversed-phase HPLC and ESI-MS monitoring showed that 12 was
produced as the major product. The solvent was evaporated and the product purified by reversed-phase
þ
HPLC and vacuum-dried: 12 (86.1%). ESI-MS: 949.3 ([M þ H] ).
3
þ
[
Eu (minocycline-TTHA)] (13). As described for 9, with 0.1m Tris · HCl (pH 8.0; 2 ml) containing
1
5
2 (2.5 mg, 2.63 mmol) and a soln. (1 ml) of 0.1m Tris · HCl (pH 8.0) containing EuCl · 6 H O (1.93 mg,
3
2
þ
.26 mmol): 13 (1.7 mg). ESI-MS: 1099.3 ([M þ H] ).
Succinimidyl Derivative 14 of H (minocycline-TTHA). H (minocycline-TTHA) (12), N-hydroxy-
5
5
succinimide (NHS), and DCC were vacuum-dried for 24 h before use. To a soln. of dried 12 (4.6 mg,
.85 mmol) in dry DMF (1 ml), a soln. of dry DMF (1 ml) containing NHS (0.56 mg, 4.85 mmol) and DCC
1 mg, 4.85 mmol) was added. The soln. was stirred for 24 h at r.t. under Ar. After an undissolved
precipitate was removed by centrifugation, the filtrate was concentrated: H (Su-minocycline-TTHA)
4
(
4
(14). Since 11 is easily hydrolyzed, it was immediately used for the following conjugation to streptavidin
(
SA).
Conjugation of H (minocycline-TTHA) (14) to SA. Compound 14 was dissolved in 0.1m phosphate
4
buffer (pH 8.0; 700 ml), and to this soln., 0.1m phosphate buffer (pH 8.0; 200 ml) containing SA at the
concentration of 5 mg/ml was added. The soln. was stirred for 6 h to give a soln. of crude H (SA-
4
minocycline-TTHA). Since the product soln. contained unreacted 14, the target product was purified by
column chromatographies (Sephadex G25 and iminobiotin immobilized column) as follows. Sephadex
G25 was equilibrated with 0.1m Tris · HCl/0.1m NaCl buffer (pH 8.0). The crude H (SA-minocycline-
4
TTHA) soln. was eluted with 0.1m Tris · HCl/0.1m NaCl buffer (pH 8.0) (500 ml fractions, UV
monitoring). The product fractions were further purified with a iminobiotin immobilized column. The
column was equilibrated with 0.1m Tris · HCl/0.1m NaCl buffer (pH 8.0), and the Sephadex-G25-purified
product was eluted with the same buffer as the one used for equilibration (500 ml fractions). After the
elution of unreacted 14 UV monitoring, the column was washed, and the H (SA-minocycline-TTHA) on
4
the column was eluted with AcONa buffer (pH 3.5; 50 ml). Each 250 ml fraction was neutralized by the

Helvetica Chimica Acta – Vol. 92 (2009)
2373
addition of 1m Tris · HCl (pH 9.0; 20 ml). The purified H (SA-minocycline-TTHA) was confirmed by its
4
UV spectra having absorptions of both SA and H (minocycline-TTHA) (Figs. 2 and 8).
5
3
þ
Titrations of H (minocycline-DTPA) (8) and H (minocycline-TTHA) (12) with Eu . The titrations
4
5
were carried out by using 0.1 mm ligand solns. in 0.1m Tris · HCl (pH 7.0). Solns. of EuCl · 6 H O with
3
2
appropriate concentrations in 0.1m Tris · HCl (pH 7.0) were added to the ligand solns., and the UV spectra
were measured after stirring for 20 min at r.t. For construction of the titration curves, absorbance at 397
and 391 nm were used for 8 and 12, resp.
3
þ
3þ
Measurement of the Time-Resolved Spectra of [Eu (SA-minocycline-TTHA)] and [Eu (minocy-
3
þ
cline-TTHA)] (13). The luminescence spectrum of [Eu (SA-minocycline-TTHA)] was measured with
a time-resolved mode, to remove the background fluorescence of the SA moiety. The concentration of
the purified H (SA-minocycline-TTHA) was determined by UVabsorption, assuming that the molecular
4
absorption coefficient is not changed by conjugation to SA. For the luminescence-spectrum measure-
ment, 2 equiv. of EuCl · 6 H O were added to the H (SA-minocycline-TTHA) soln., and after the soln.
3
2
4
was stirred for 1 h at r.t., the spectrum was measured under the following conditions: window time 0.5 ms,
delay time after flash 0.1 ms, number of flash 200, and concentration of the ligand 19.4 mm in 50 mm
AcONa/1m Tris · HCl buffer at pH 7.0.
REFERENCES
[
[
[
[
[
[
[
1] I. Hemmilꢂ, Clin. Chem. 1985, 31, 359.
2] E. P. Diamandis, Clin. Biochem. 1988, 21, 139.
3] E. P. Diamandis, T. K. Chiristopoulos, Anal. Chem. 1990, 62, 1149A.
4] I. Hemmilꢂ, Appl. Fluoresc. Technol. 1989, 1, 1.
5] I. Hemmilꢂ, Scand. J. Clin. Lab. Invest. 1988, 48, 389.
6] P. R. Selvin, Ann. Rev. Biophys. Biomol. Struct. 2002, 31, 275.
7] G. F. de S a´ , O. L. Malta, D. de Mello Doneg a´ , A. M. Simas, R. L. Longo, P. A. Santa-Cruz, E. F. da
Silva Jr., Coord. Chem. Rev. 2000, 196, 165.
[
[
8] K. Senanayake, A. L. Thompson, J. A. K. Howard, M. Botta, D. Parker, Dalton Trans. 2006, 5423.
9] R. A. Poole, G. Bobba, M. J. Cann, J.-C. Frias, D. Parker, R. D. Peacock, Org. Biomed. Chem. 2005,
3, 1013.
[
[
[
[
[
10] N. Sabbatini, M. Guardigli, J.-M. Lehn, Coord. Chem. Rev. 1993, 123, 201.
11] M. Li, P. R. Selvin, J. Am. Chem. Soc. 1995, 117, 8132.
12] P. Ge, P. R. Selvin, Bioconjugate Chem. 2004, 15, 1088.
13] J. C. Rodrꢃguez-Ubis, M. T. Alonso, O. Juanes, R. Sedano, E. Brunet, J. Lumin. 1998, 79, 121.
14] C. Yang, L.-M. Fu, Y. Wang, J.-P. Zhang, W.-T. Wong, X.-C. Ai, Y.-F. Qiao, B.-S. Zou, L.-L. Gui,
Angew. Chem., Int. Ed. 2004, 43, 5010.
[15] D. Parker, R. S. Dickins, H. Puschmann, C. Crossland, J. A. K. Howard, Chem. Rev. 2002, 102, 1977.
[16] J.-C. G. Bꢁnzli, B. Klein, G. Chapuis, K. J. Schenk, Inorg. Chem. 1982, 21, 808.
[17] F. Renaud, C. Piguet, G. Bernardinelli, J.-C. G. Bꢁnzli, G. Hopfgartner, J. Am. Chem. Soc. 1999, 121,
9326.
[
18] S. I. Klink, G. A. Hebbink, L. Grave, P. G. B. Oude Alink, F. C. J. M. van Veggel, M. V. Werts, J.
Phys. Chem. A 2002, 106, 3681.
[
19] S. Comby, J.-C. G. Bꢁnzli, ꢄ Handbook of the Physics and Chemistry of Rare Earthsꢅ, Eds. K. A.
Gschneidner Jr., J.-C. G. Bꢁnzli, and V. K. Pecharsky, North-Holland, Amsterdam, 2007, Vol. 37,
Chapter 235, p. 217.
[
[
[
[
[
20] Y.-X. Ci, Y.-Z. Li, X.-J. Liu, Anal. Chem. 1995, 67, 1785.
21] M. Wu, Z. Lin, O. S. Wolfbeis, Anal. Biochem. 2003, 320, 129.
22] O. S. Wolfbeis, A. Dꢁrkop, M. Wu, Z. Lin, Angew. Chem., Int. Ed. 2002, 41, 4495.
23] Z. Lin, M. Wu, M. Schꢂferling, O. S. Wolfbeis, Angew. Chem., Int. Ed. 2004, 43, 1735.
24] C. Cano-Raya, M. D. Fern a´ ndez Ramos, L. F. Capit a´ n Vallvey, O. S. Wolfbeis, M. Schꢂferling, Appl.
Spectrosc. 2005, 59, 1209.
[
25] Z. Ling, M. Wu, O. S. Wolfbeis, M. Schꢂferling, Chem. – Eur. J. 2006, 12, 2730.

2
374
Helvetica Chimica Acta – Vol. 92 (2009)
[
[
[
26] C. Jiang, L. Luo, Anal. Chim. Acta 2004, 506, 171.
27] X.-J. Liu, Y.-Z. Li, Y.-X. Ci, Anal. Chim. Acta 1997, 345, 213.
28] T. Nishioka, J. Yuan, Y. Yamamoto, K. Sumitomo, Z. Wang, K. Hashino, C. Hosoya, K. Ikawa, G.
Wang, K. Matsumoto, Inorg. Chem. 2006, 45, 8460.
[
[
[
29] T. Nishioka, J. Yuan, K. Matsumoto, in ꢄBioMEMS and Biomedical Nanotechnologyꢅ, Eds. M.
Ozkan, M. J. Heller, and M. Ferrari, Springer, New York, 2006, Vol. 2, p. 437.
30] M. L. Nelson, B. H. Park, J. S. Andrews, V. A. Georgian, R. C. Thomas, S. B. Levy, J. Med. Chem.
1993, 36, 370; W. J. Gottstein, W. F. Minor, L. C. Cheney, J. Am. Chem. Soc. 1959, 81, 1198.
31] M. L. Nelson, M. Y. Ismail, L. McIntyre, B. Bhatia, P. Viski, P. Hawkins, G. Rennie, D. Andorsky, D.
Messersmith, K. Stapleton, J. Dumornay, P. Sheahan, A. K. Verma, T. Warchol, S. B. Levy, J. Org.
Chem. 2003, 68, 5838.; P. E. Sum, V. J. Lee, R. T. Testa, J. J. Hlavka, G. A. Ellestad, J. D. Bloom, Y.
Gluzman, F. P. Tally, J. Med. Chem. 1994, 37, 184.
[
[
32] B. Alpha, V. Balzani, J.-M. Lehn, S. Perathoner, N. Sabbatini, Angew. Chem., Int. Ed. 1987, 26, 1266.
33] W. T. Carnall, ꢄ Handbook of the Physics and Chemistry of Rare Earthsꢅ, Eds. K. A. Gschneidner Jr.,
and L. Eyring, North-Holland, Amsterdam, 1979, Vol. 3, Chapt. 24, p. 171.
Received May 8, 2009