Binuclear terbium(III) complex as a probe for tyrosine phosphorylation

Article abstract of DOI:10.1002/chem.200903379

By using the luminescence from binuclear complexes of Tb^III (Tb₂L¹ and Tb₂-L²), phosphorylated Tyr residue in peptides was selectively detected in neutral aqueous solutions. Neither the non-phosphorylated Tyr, pSer, pThr, nor the other phosphate-containing biomolecules tested affected the luminescence intensity to any notable extent. Upon the binding of the pTyr to these Tb^III complexes, the luminescence from the metal ion was notably promoted, as the light energy absorbed by the benzene ring of pTyr is efficiently transferred to the Tb^IIIcenter. The binding activity of the binuclear Tb ^III complexes towards pTyr is two orders of magnitude larger than that of the corresponding mononuclear complex. These binuclear complexes were successfully used for real-time monitoring of enzymatic phosphorylation of a peptide by a tyrosine kinase.

Full text of DOI:10.1002/chem.200903379

DOI: 10.1002/chem.200903379
Binuclear TerbiumACHTNUTGRNEUNG(III) Complex as a Probe for Tyrosine Phosphorylation
Hiroki Akiba, Jun Sumaoka,* and Makoto Komiyama*[a]
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Chem. Eur. J. 2010, 16, 5018 – 5025

FULL PAPER
Abstract: By using the luminescence
from binuclear complexes of TbIII (Tb2-
L1 and Tb2-L2), phosphorylated Tyr res-
idue in peptides was selectively detect-
ed in neutral aqueous solutions. Nei-
ther the non-phosphorylated Tyr, pSer,
pThr, nor the other phosphate-contain-
ing biomolecules tested affected the lu-
minescence intensity to any notable
extent. Upon the binding of the pTyr
to these TbIII complexes, the lumines-
cence from the metal ion was notably
promoted, as the light energy absorbed
by the benzene ring of pTyr is efficient-
ly transferred to the TbIII center. The
binding activity of the binuclear TbIII
complexes towards pTyr is two orders
of magnitude larger than that of the
corresponding mononuclear complex.
These binuclear complexes were suc-
cessfully used for real-time monitoring
of enzymatic phosphorylation of a pep-
tide by a tyrosine kinase.
Keywords: kinases · lanthanides ·
luminescence · phosphorylation ·
tyrosine
Introduction
very weak (the f–f transitions of the ions are Laporte-forbid-
den). By combining lanthanide complexes and antenna mol-
ecules, elegant systems to detect various anion guests have
already been prepared.[7–12] A sophisticated example in-
cludes the analysis of benzoic acid derivatives using the an-
tenna effect.[8] Phosphate-containing molecules were detect-
ed by attaching an antenna to lanthanide complexes. The
binding of the phosphate group(s) of the guest molecule in-
Phosphorylation of proteins controls many cellular events.
In the course of signal transduction, Ser, Thr, and Tyr resi-
dues in proteins are reversibly phosphorylated and dephos-
phorylated.[1] Although Tyr phosphorylation accounts for
only 0.05% of the total phosphorylation in cells (the majori-
ty occurs on Ser or Thr), it takes a crucial role in biological
functions.[2] For example, the autophosphorylation of a Tyr
in epidermal growth factor receptor (EGFR) triggers the
signal-cascade inside the cell.[3] In the downstream of this
kinase, there are Src family kinases, which are also con-
trolled by Tyr phosphorylation and in turn phosphorylate
Tyr residues in other proteins.[4] Excessive or insufficient Tyr
phosphorylation causes serious problems.[1,2] Thus, selective
detection of phosphorylated Tyr (pTyr) in proteins, with
minimized signals from phosphoserine (pSer) or phospho-
threonine (pThr), which exist more abundantly in biological
systems, is quite important. Several metal complexes have
been already reported to detect phosphorylation of pro-
teins.[5,6] However, although these complexes recognize the
phosphate groups of specimens,
duces environmental changes around the lanthanideACHTUNGTRENNUNG(III) ion
and alters the luminescence from the ion.[10–13] However, se-
lective detection of pTyr is difficult with this strategy, as co-
existing molecules that also have phosphate groups (e.g.,
pSer, pThr, ATP, and DNA) could show similar effects as
pTyr. These factors are more critical if a TbIII ion (without
any ligand) is used; nucleotides and nucleic acids show enor-
mous antenna effects.[14,15] To obtain the signal from pTyr
precisely, these background signals must be reduced.
Herein, we report binuclear TbIII complexes (Tb2-L1 and
Tb2-L2), which selectively detect pTyr with respect to i) non-
phosphorylated Tyr, ii) pSer and pThr, and iii) other coexist-
ing phosphate-containing biomolecules. We have previously
they barely discriminate pTyr
from any other phosphate-bear-
ing species, such as pSer and
pThr.
Lanthanide ions and their
complexes emit strong lumines-
cence, when
a chromophore
(“antenna”) is placed near
them and transfers its excitation
energy to the metal center.[7] In
the absence of this antenna,
however, the luminescence is
demonstrated by using Tb-DOTAM that the benzene ring
of pTyr in the target peptides can be used as an antenna to
enhance the emission from the TbIII center (see Figure 1).[16]
Of Tyr, Ser, Thr, and their phosphorylated products, only
pTyr possesses both the notable antenna effect (benzene
ring) and sufficient binding activity towards the TbIII com-
plex (phosphate group). Thus, the selectivities (i) and (ii) are
fulfilled. Furthermore, the selectivity (iii) to other coexisting
[a] H. Akiba, Dr. J. Sumaoka, Prof. Dr. M. Komiyama
Research Center for Advanced Science and Technology
The University of Tokyo
4-6-1 Komaba, Meguro-Ku, Tokyo, 153-8904 (Japan)
Fax : (+81)3-5452-5209
E-mail: sumaoka@mkomi.rcast.u-tokyo.ac.jp
komiyama@mkomi.rcast.u-tokyo.ac.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200903379.
molecules is accomplished by using
a
bulky ligand
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M. Komiyama, J. Sumaoka, and H. Akiba
Under the situation described above, pTyr satisfactorily en-
hances the luminescence from the binuclear complexes Tb2-
L1 and Tb2-L2 with a reasonable magnitude, because 1) the
electrostatic interactions are primarily responsible for the
binding of pTyr to the TbIII, 2) the doubled positive charges
of the binuclear TbIII complex induce tighter interaction,
and 3) the TbIII center and the benzene ring of pTyr are in
sufficient proximity for energy transfer. By using these binu-
clear TbIII complexes, Tyr-phosphorylated peptides are clear-
ly distinguished from non-phosphorylated peptides in neu-
tral aqueous solutions. Furthermore, the enzymatic phos-
phorylation of Tyr in the peptides can be successfully moni-
tored in real-time.
Results and Discussion
Figure 1. A phosphotyrosine-specific sensor based on the “antenna
effect”. a) The emission for the TbIII complex is promoted by the transfer
of excited energy of the benzene ring of pTyr to the TbIII ion. b) Non-
phosphorylated Tyr does not bind to the TbIII complex so that no energy
transfer occurs. c) pSer/pThr has no benzene ring to absorb the energy.
d) The guanine group of GMP is located far away from the TbIII complex
even when its phosphate interacts with the metal center. Ex: excitation;
ET: energy transfer; Em: emission.
Design and synthesis of TbIII complexes: The binuclear com-
plexes (Tb2-L1 and Tb2-L2) were synthesized according to
Scheme 1, and characterized as described in the Supporting
Information. These complexes, as well as their monomeric
analog Tb-DOTAM, have no aromatic moiety, which could
possibly work as an antenna, so that their intrinsic lumines-
cence is minimized. In addition, both of these binuclear
complexes carry a +6 net positive charge, which is able to
electrostatically attract the phosphate residue of pTyr. As
evidenced below, the Tb2-L1 and Tb2-L2 have one coordina-
tion water molecule on each of the TbIII ions. This argument
is also supported by the crystal structure analysis of Tb-
DOTAM complex.[18,19]
(DOTAM), which extensively surrounds the metal ion and
suppresses the coordination of various phosphate-bearing
molecules. To further improve the detection ability, binu-
clear complexes (Tb2-L1 and Tb2-L2) were developed.[17]
Scheme 1. The synthetic procedures for Tb2-L1 (n=1) and Tb2-L2 (n=2). Reagents and conditions: i) chloroacetyl chloride, CHCl3, reflux, 6 h;
ii) (Boc)2O, NEt3, CHCl3, 12 h; iii) NaHCO3, KI, MeCN, reflux, 96 h; iv) 4m HCl, 1,4-dioxane, 2 h; v) 2-bromoacetamide, NaHCO3, KI, MeCN, reflux,
72 h; vi) TbACHTUNGTRENNUNG(OTf)3, MeCN, reflux, 24 h.
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TerbiumACHTUNGTRENNUNG(III) as a Probe for Tyrosine Phosphorylation
FULL PAPER
Phosphotyrosine-selective promotion of luminescence from
Tb2-L1, Tb2-L2, and Tb-DOTAM: Shown in Figure 2a are
the emission spectra of Tb2-L1 in the presence or the ab-
sence of phenyl phosphate (PhOP), which is a model com-
pound for the side chain of pTyr in proteins. The excitation
wavelength was l=262.5 nm, which the benzene ring of
PhOP absorbs. When PhOP was added to the solution, the
mononuclear Tb-DOTAM and another binuclear complex
Tb2-L2 (Figure 2b). It is noteworthy that these dimeric TbIII
complexes enhanced the luminescence much more remarka-
bly than the monomeric complex Tb-DOTAM. There was
not much difference in luminescence enhancement between
Tb2-L1 and Tb2-L2. This is primarily because the interactions
between TbIII complexes and PhOP are electrostatic, as de-
scribed below.
Similar enhancement of the luminescence was observed
with the use of pTyr in place of PhOP (Figure 2c). In con-
trast, non-phosphorylated Tyr showed virtually no improve-
ment, since not much of its excitation energy transfers to
the TbIII complexes. The promotion of luminescence was se-
lective to pTyr, and all other amino acid derivatives investi-
gated (pSer, pThr, Ser, Thr, Phe, and Trp) were inactive (see
Figure 2c). Apparently, both the benzene ring (an antenna)
of the pTyr residue and its phosphate (metal-binder) are es-
sential to promote the luminescence, exactly as proposed in
Figure 1. Although the TbIII complexes also bind pSer and
pThr, they do not affect the emission, owing to the absence
of an antenna function.
5
luminescence assignments for the D4–7FJ (J=6, 5, 4) transi-
tions of the TbIII. Similar enhancement was observed for
These results strongly suggest that the present system is
applicable to the detection of pTyr even if various phos-
phate-bearing species coexist in the same solution. Consis-
tently, GMP (guanosine-5’-monophosphate), UMP, AMP,
CMP, ADP, ATP, and a-d-glucose-1-phosphate hardly pro-
moted the luminescence from Tb2-L1 (Table 1). These spe-
Table 1. The luminescence intensity at 545 nm in the presence of various
phosphate-bearing species.[a]
Tb2-L1
Tb-DOTAM
TbCl3
[b]
–
3.03
187
1.60
14.7
0.40
1.00
0.85
0.69
0.64
0.44
1.54
1.03
313
9.36
16.3
900
30.4
10.1
0.61
1.27
PhOP
AMP
UMP
GMP
CMP
0.82
3.60
1.02
1.56
1.13
1.18
3.67
ADP
ATP
Glucose-P[c]
[a] Conditions: [TbIII complex]=[additive]=100 mm, HEPES buffer
(10 mm, pH 7.0), lex =262.5 nm. [b] The value without any additive.
[c] Glucose-P=a-d-glucose-1-phosphate.
cies and their analogues, such as DNA and RNA, are abun-
dant in cells. In marked contrast, the luminescence from the
TbIII ion (without ligands) is notably enhanced by these spe-
cies. The effect of GMP is especially remarkable. It has
been proposed that GMP binds to a TbIII ion through simul-
taneous coordination of the phosphate residue and the gua-
nine ring (N7 and O6), using its guanine group as an anten-
na to promote the emission.[13] A similar coordination
should occur for DNA and RNA. In the present TbIII com-
plexes, the coordination of these molecules to the TbIII ion
is prevented by the bulky ligands (Figure 1d). This deprives
these species of proximity to the TbIII center. On the other
hand, pTyr has a benzene ring adjacent to the phosphate
Figure 2. a) The luminescence spectra of Tb2-L1 in the presence and the
absence of PhOP. b) The luminescence intensity at 545 nm of the solu-
tions of Tb2-L1, Tb2-L2, and Tb-DOTAM. The luminescence was largely
enhanced by PhOP. c) The luminescence intensity at 545 nm in the pres-
ence of PhOP or amino acids. The addition of PhOP or pTyr explicitly in-
creased the emission. Conditions: [TbIII complex]=[additive]=100 mm,
HEPES buffer (10 mm, pH 7.0), lex =262.5 nm.[20]
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M. Komiyama, J. Sumaoka, and H. Akiba
group and sufficient proximity is maintained even if it is
bound by the metal ions through electrostatic interactions
(see below). The vital necessity of the ligands for selective
detection of pTyr is evident.
ment of the H2O molecules with D2O. Therefore the q
value, which represents the number of coordinated water
molecules, can be estimated by using the following Equa-
tion (1).[23] If the phosphate group of PhOP is directly coor-
dinated with the TbIII ion, the q value would be reduced,
owing to its replacement with the corresponding coordinat-
ed water molecule.
Promoted phosphate-binding by binuclear complex Tb2-L1:
The Jobꢁs plot between Tb2-L1 and PhOP indicates the exis-
tence of an equimolar complex (see Figure S1 in the Sup-
porting Information).[21] To compare the strength of interac-
tions, an apparent dissociation constant (KD) was estimated
in terms of 1:1 complex formation. The change in lumines-
cence intensity with increasing Tb2-L1 concentration satisfac-
torily fits a curve based on this model (Figure 3). The KD of
q ¼ 5:0ðkH O ꢀ kD O ꢀ 0:06Þ
ð1Þ
2
2
Here, kH O and kD O represent the rate constants of lumines-
2
2
cence decay, measured in H2O and D2O, respectively (the
decay curves are presented in Figures S2 and S3 in the Sup-
porting Information). For both Tb2-L1 and Tb-DOTAM, the
q value was about 1, either before or after the addition of
PhOP (Table 2).[24] Apparently, no ligand exchange with co-
Table 2. Half-lives of luminescence from the TbIII complexes in H2O
(tH O) and in D2O (tD O), and the q values calculated.[a]
2
2
tH O [ms]
tD O [ms]
q
2
2
Tb-DOTAM
+PhOP
Tb2-L1
1.65
1.65
1.52
1.53
3.17
3.20
2.62
2.60
1.15
1.17
1.08
1.04
+PhOP
[a] See the Experimental Section for the experimental conditions.
ordination water occurs in the binding of PhOP to the TbIII
complexes. Therefore, it is concluded that the interaction
between the TbIII complexes and PhOP is by means of ion-
pairing, rather than a direct coordination.[25,26] Consistently,
the luminescence from Tb2-L1 in the presence of PhOP
gradually decreased with increasing concentration of NaCl
or KCl (see Figure S4 in the Supporting Information). Tb2-
L1 binds PhOP much more strongly (two orders of magni-
tude) than Tb-DOTAM, probably through simultaneous in-
teractions of the phosphate with its two TbIII ions. The elec-
trostatic interactions of the Tb2-L1 are also stronger, because
the net positive charges of this binuclear complex (+6) are
two times as large as that for Tb-DOTAM (+3).
Figure 3. The titration of a) Tb-DOTAM and b) Tb2-L1 to PhOP (100 mm)
in HEPES buffer (10 mm, pH 7.0). lex =262.5 nm.
Detection of phosphorylated Tyr in peptides by Tb2-L1: If
the Tyr-phosphorylated P1 (Glu-Glu-Glu-Ile-Tyr-Glu-Glu-
Phe-Asp) was added to a solution of Tb2-L1, the lumines-
cence was considerably enhanced (Figure 4). The lumines-
cence intensity was tenfold larger than that obtained if non-
phosphorylated P1 was added. This peptide comes from a
sequence that is heavily phosphorylated by the cancer-relat-
ed protein v-Src.[27] The phosphorylated Tyr is surrounded
by negatively charged amino acid residues, as is often ob-
served in peptide sequences that can be tyrosine-phosphory-
lated by tyrosine kinases.[28] Assumedly, these negative
charges promote the binding of the pTyr residue to Tb2-L1,
resulting in the efficient detection of the Tyr phosphoryla-
tion of this peptide. Consistently, Tyr-phosphorylated P2
(Ser-Ala-Ala-Pro-Tyr-Leu-Lys-Thr-Lys), which is from an-
other cancer-related protein STAT3 and has positive charges
the Tb2-L1/PhOP complex was estimated to be 29 mm. This
value is 110 times lower than corresponding value (3.3 mm)
for Tb-DOTAM. Clearly, Tb2-L1 binds pTyr more strongly
than Tb-DOTAM, so that the pTyr-induced emission en-
hancement is larger for Tb2-L1 (Figure 2b).[22]
To shed light on the manner of the interactions between
the TbIII complexes and PhOP, the number of the water
molecules coordinated to the TbIII ion was estimated. As the
coordination of water molecules to TbIII ion reduces the lu-
minescence lifetime, owing to the energy transfer from the
excited state of TbIII to the higher O H vibration overtones
ꢀ
of the water, the lifetime would be increased by the replace-
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TerbiumACHTUNGTRENNUNG(III) as a Probe for Tyrosine Phosphorylation
FULL PAPER
Figure 5. Change in luminescence intensity at 545 nm from Tb2-L1 after
the addition of Src kinase to the solution of P1 peptide. For red and
green circles, the concentrations of P1 were 5 and 2.5 mm, respectively,
and Src kinase (1.2 mgmLꢀ1) was added at time=0. Note that the back-
ground signal satisfactorily remained constant before the addition of the
enzyme (time <0). Conditions: [Tb2-L1]=100 mm, [MnCl2]=1 mm,
[ATP]=10 mm, HEPES buffer (5 mm, pH 7.4), lex =262.5 nm, lem
=
545 nm. The blue squares show the result without substrate peptide.
addition of kinase (data not shown). Apparently, the Tyr-
phosphorylation of the substrate peptide was successfully
monitored by Tb2-L1 in the presence of ATP which competi-
tively bound to the complex. The light absorption by the
protein and ATP imposed no critical effects on this analysis.
The present method is advantageous in that neither radio-
labeling of the peptide nor its chemical labeling is re-
quired.[13,31,32]
Figure 4. The luminescence intensity at 545 nm from Tb2-L1 in the pres-
ence of phosphorylated (orange) and nonphosphorylated (light blue)
peptides. The peptides used are shown in (a) with the phosphorylated
residue underlined. Conditions: [Tb2-L1]=[peptide]=100 mm, HEPES
buffer (10 mm, pH 7.0), lex =262.5 nm.
near the Tyr,[29] exhibits only a 2 fold increase in the lumi-
nescence intensity of Tb2-L1. The luminescence enhance-
ment by Ser-phosphorylated P3 (Phe-Pro-Gln-Phe-Ser-Tyr-
Ser-Ala-Ser) from c-Akt (PKB), which bears Tyr next to
phosphorylated Ser,[30] was also small, as expected.
Conclusion
With the use of pTyr-induced enhancement of luminescence
from binuclear Tb2-L1 and Tb2-L2 complexes, pTyr in pep-
tides has been selectively detected even in the presence of
pSer, pThr, and other potential coexisting molecules (e.g.,
other amino acids, GTP, DNA, and RNA). The benzene
ring of pTyr works as an antenna and enhances the lumines-
cence from the TbIII complex, which is otherwise very weak.
The pTyr-induced enhancement of the luminescence from
Tb2-L1 was far larger than that observed for monomeric Tb-
DOTAM complex. The process of enzymatic phosphoryla-
tion of Tyr in peptide was successfully monitored in real-
time by Tb2-L1. These results have opened a way to molecu-
lar design of pTyr-specific sensors.
The high pTyr selectivity of the present method, with re-
spect to pSer/pThr and other phosphate-containing mole-
cules, is certainly an advantage for various applications. The
pTyr-detection limit is further improved by designing TbIII
complexes that can bind pTyr still more strongly, even under
high salt conditions. Direct coordination of phosphate to the
TbIII center, in place of electrostatic interaction, could be ef-
fective there. Although the rather short wavelength of exci-
tation light used (l=262.5 nm) provides some limitations to
the potential applications, use of multi-photon excitation
systems could reduce some of these limitations. In vitro
analyses, demonstrated here with a kinase, would be very ef-
fective in the biochemical analysis of Tyr-phosphorylation,
Real-time monitoring of enzymatic phosphorylation of Tyr
in the P1 peptide: The process of phosphorylation of P1
peptide by Src kinase in aqueous solutions was directly
monitored by fluorimetric analysis of Tb2-L1 in real-time
(Figure 5). To the solutions containing Tb2-L1 and P1, as
well as ATP and MnCl2 (essential factors in this enzymatic
reaction), the kinase was added and the luminescence at
545 nm was measured. The luminescence intensity increased
time-dependently (red and green circles in Figure 5). The
magnitude of increase in luminescence intensity exactly re-
flects the difference in the concentration of P1. To confirm
that the increase of luminescence is owed to the increase in
the amount of Tyr-phosphorylated P1, this enzymatic phos-
phorylation was independently analyzed under the same
conditions by polyacrylamide gel-electrophoresis by using
TAMRA-labeled P1. As presented in Figures S5–S7 in the
Supporting information, the results of the two methods
almost agree with each other, providing evidence for the ar-
gument. The Tb2-L1 induced no notable change in the rate
of Tyr-phosphorylation (compare the red squares with the
blue ones in Figure S7 in the Supporting Information). In no
experiment was the luminescence enhanced without the sub-
strate peptide (blue squares in Figure 5). If Tyr-phosphory-
lated P1 was used as the substrate, the luminescence was
strong from the beginning and not enhanced even after the
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M. Komiyama, J. Sumaoka, and H. Akiba
3073; b) S. J. Parsons, J. T. Parsons, Oncogene 2004, 23, 7906–7909;
c) W. Luo, R. J. Slebos, S. Hill, M. Li, J. Brꢃbek, R. Amanchy, R.
Chaerkady, A. Pandey, A.-J. L. Ham, S. K. Hanks, J. Proteome Res.
2008, 7, 3447–3460.
including the activation or inactivation of kinases and phos-
phatases.
[5] a) B. Schulenberg, R. Aggeler, J. M. Beechem, R. A. Capaldi, W. F.
Patton, J. Biol. Chem. 2003, 278, 27251–27255; b) B. Schulenberg,
T. N. Goodman, R. Aggeler, R. A. Capaldi, W. F. Patton, Electro-
phoresis 2004, 25, 2526–2532.
Experimental Section
General: The reagents and compounds were purchased from Tokyo
Chemical Industries, Wako Pure Chemicals, Sigma–Aldrich, or Nacalai
Tesque at the highest grades available. Luminescence measurements
were conducted in HEPES-NaOH buffer (pH 7.0, 10 mm) unless other-
wise stated. The spectra were measured by using a FP-6500 fluorimeter
(JASCO) with excitation at l=262.5 nm, soon after the additives were
added. The procedures for the synthesis of the TbIII complexes are in-
cluded in the Supporting Information.
[6] a) T. Sakamoto, A. Ojida, I. Hamachi, Chem. Commun. 2009, 141–
152; b) A. Ojida, M. Inoue, Y. Mito-oka, I. Hamachi, J. Am. Chem.
Soc. 2003, 125, 10184–10185; c) A. Ojida, Y. Mito-oka, K. Sada, I.
Hamachi, J. Am. Chem. Soc. 2004, 126, 2454–2463; d) E. Kinoshita,
M. Takahashi, H. Takeda, M. Shiro, T. Koike, Dalton Trans. 2004,
1189–1193; e) E. Kinoshita, E. Kinoshita-Kikuta, K. Takiyama, T.
Koike, Mol. Cell. Proteomics 2006, 5, 749–757; f) T. Zhang, N. Y.
Edwards, M. Bonizzoni, E. V. Anslyn, J. Am. Chem. Soc. 2009, 131,
11976–11984; g) A. Riechers, F. Schmidt, S. Stadlbauar, B. Kçnig,
Bioconjugate Chem. 2009, 20, 804–807.
[7] a) F. S. Richardson, Chem. Rev. 1982, 82, 541–552; b) I. Hemmilꢃ,
V.-M. Mukkala, Crit. Rev. Clin. Lab. Sci. 2001, 38, 441–519; c) J.-
C. G. Bꢄnzli, C. Piguet, Chem. Soc. Rev. 2005, 34, 1048–1077;
d) C. M. G. dos Santos, A. J. Harte, S. J. Quinn, T. Gunnlaugsson,
Coord. Chem. Rev. 2008, 252, 2512–2527.
Lifetime measurements: Luminescence lifetime measurements of the
TbIII complexes were conducted in H2O or D2O solutions (10 mm
HEPES buffer, pH 7 for H2O solution). The D2O solution was prepared
by completely drying up this solution followed by the addition of the
same volume of D2O to the residue. The concentrations of TbIII com-
plexes were 100 and 500 mm, respectively, in the presence and the absence
of PhOP (1 mm). Excitation was made at l=355 nm by pulse laser by
means of a Nd:YAG laser (Spectra-Physics KK. INDI-40–10-HG-TRI-
T). Luminescence at l=545 nm was measured by using a monochroma-
tor (JASCO CT-25TP) and a photomultiplier (Hamamatsu Photonics
R446).
[8] T. Gunnlaugsson, A. J. Harte, J. P. Leonard, M. Nieuwenhuyzen, Su-
pramol. Chem. 2003, 15, 505–519.
[9] a) S. J. A. Pope, B. P. Burton-Pye, R. Berridge, T. Khan, P. J. Ska-
bara, S. Faulkner, Dalton Trans. 2006, 2907–2912; b) M. L. Cable,
J. P. Kirby, D. J. Levine, M. J. Manary, H. B. Gray, A. Ponce, J. Am.
Chem. Soc. 2009, 131, 9562–9570; c) R. S. Dickins, T. Gunnlaugsson,
D. Parker, R. D. Peacock, Chem. Commun. 1998, 1643–1644.
[10] a) M. D. Best, E. V. Anslyn, Chem. Eur. J. 2003, 9, 51–57; b) M.
Schꢅferling, O. S. Wolfbeis, Chem. Eur. J. 2007, 13, 4342–4349.
[11] A TbIII complex responding to nucleoside triphosphates was used
for kinetic analysis of enzymes: a) C. M. Spangler, C. Spangler, M.
Gçttle, Y. Shen, W.-J. Tang, R. Seifert, M. Schꢅferling, Anal. Bio-
chem. 2008, 381, 86–93; b) C. Spangler, C. M. Spangler, M. Spoern-
er, M. Schꢅferling, Anal. Bioanal. Chem. 2009, 394, 989–996.
[12] a) P. Atkinson, Y. Bretonniere, D. Parker, Chem. Commun. 2004,
438–439; b) P. Atkinson, Y. Bretonniere, D. Parker, G. Muller, Helv.
Chim. Acta 2005, 88, 391–405.
The preparation of the peptides: P1-TMR was TAMRA-labeled on its
N-terminal and amidated on C-terminal (see the Supporting Informa-
tion). All other peptides were acetylated on their N-terminal and amidat-
ed on C-terminal. The Tyr-phosphorylated P1, Ser-phosphorylated P3,
and P1-TMR were synthesized by standard Fmoc-coupling chemistry fol-
lowed by purification by using RP-HPLC; solid phase: ODS column;
mobile phase: H2O/MeCN (0.1% TFA). The other peptides were ob-
tained from Life Technologies Japan Ltd. and used without further purifi-
cation.
The conditions of the kinase reaction: The Src kinase, expressed as full-
length human Src with a N-terminal GST-fusion protein using the baculo-
virus expression system, was obtained from Carna Biosciences, Inc, and
stored in Tris-HCl (50 mm, pH 7.5) containing NaCl (150 mm), Brij 35
(0.05%), DTT (1 mm), and glycerolACTHNUTRGENUGN(10%). The reaction was assayed in
HEPES-NaOH (5 mm, pH 7.4) containing Tb2-L
G
[13] Lanthanide complexes were directly conjugated to kinase substrate
peptides and used to assess kinase activity: a) M. S. Tremblay, Q.
Zhu, A. A. Marti, J. Dyer, M. Halim, S. Jockusch, N. J. Turro, D.
Sames, Org. Lett. 2006, 8, 2723–2726; b) M. S. Tremblay, M. Lee, D.
Sames, Org. Lett. 2008, 10, 5–8.
ATP (10 mm), P1 (2.5 or 5 mm), and Src kinase (1.2 mgmLꢀ1) as the final
concentrations.
[14] D. P. Ringer, J. L. Etheredge, B. L. Dalrymple, J. S. Niedbalski, Bio-
chem. Biophys. Res. Commun. 1990, 168, 267–273.
Acknowledgements
[15] a) P. K.-L. Fu, C. Turro, J. Am. Chem. Soc. 1999, 121, 1–7; b) D. P.
Ringer, S. Burchett, D. E. Kizer, Biochemistry 1978, 17, 4818–4825;
c) M. D. Topal, J. R. Fresco, Biochemistry 1980, 19, 5531–5537.
[16] H. Akiba, J. Sumaoka, M. Komiyama, ChemBioChem 2009, 10,
1773–1776.
[17] Cationic binuclear lanthanide complexes were described for anion
recognition: a) K. Nwe, C. M. Andolina, J. R. Morrow, J. Am. Chem.
Soc. 2008, 130, 14861–14871; b) A. J. Harte, P. Jensen, S. E. Plush,
P. E. Kruger, T. Gunnlaugsson, Inorg. Chem. 2006, 45, 9465–9474.
[18] CCDC 756622 contains the supplementary crystallographic data of
Tb-DOTAM complex for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
We thank Prof. Kazuyuki Ishii for luminescence lifetime measurements;
Dr. Hidetake Seino and Prof. Yasushi Mizobe for crystal structure analy-
sis. This work was partially supported by a Grant-in-Aid for Scientific
Research and Global COE Program (Chemistry Innovation through Co-
operation of Science and Engineering) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
[1] a) T. Hunter, Cell 2000, 100, 113–127; b) P. Cohen, Nat. Cell Biol.
2002, 4, E127-E130.
[2] a) T. Hunter, Philos. Trans. R. Soc. London Ser. B 1998, 353, 583–
605; b) T. Hunter, Curr. Opin. Cell Biol. 2009, 21, 140–146.
[3] a) S. Morandell, T. Stasyk, S. Skvortsov, S. Ascher, L. A. Huber,
Proteomics 2008, 8, 4383–4401; b) A. Guo, J. Villꢂn, J. Kornhauser,
K. L. Lee, M. P. Stokes, K. Rikova, A. Possemato, J. Nardone, G. In-
nocenti, R. Wetzel, Y. Wang, J. MacNeill, J. Mitchell, S. P. Gygi, J.
Rush, R. D. Polakiewicz, M. J. Comb, Proc. Natl. Acad. Sci. USA
2008, 105, 692–697.
[19] The DOTAM complexes of GdIII, EuIII, and YbIII have similar struc-
tures: a) J. Vipond, M. Woods, P. Zhao, G. Tircsꢆ, J. Ren, S. G. Bott,
D. Ogrin, G. E. Kiefer, Z. Kovacs, A. D. Sherry, Inorg. Chem. 2007,
46, 2584–2595; b) G. Bombieri, N. Marchini, S. Clattini, A. Mortil-
laro, S. Aime, Inorg. Chim. Acta 2006, 359, 3405–3411; c) S. Amin,
D. A. Voss, Jr., W. D. Horrocks, Jr., C. H. Lake, M. R. Churchill,
J. R. Morrow, Inorg. Chem. 1995, 34, 3294–3300.
[4] a) J. R. Engen, T. E. Wales, J. M. Hochrein, M. A. Meyn, S. B.
Ozkan, I. Bahar, T. E. Smithgall, Cell. Mol. Life Sci. 2008, 65, 3058–
[20] The notable background fluorescence of Trp was subtracted.
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Chem. Eur. J. 2010, 16, 5018 – 5025

TerbiumACHTUNGTRENNUNG(III) as a Probe for Tyrosine Phosphorylation
FULL PAPER
[21] As shown in Figure S1 in the Supporting Information, the Job plot
was not sharp enough to conclude that only 1:1 complex is formed.
This is probably owed to the fact that the interactions between the
TbIII complex and PhOP are not direct coordination, but electrostat-
ic interaction as described below. It is therefore statistically possible
to form other modes as well. However, a 1:1 complex formation
model sufficiently explains the difference in luminescence change
between Tb-DOTAM and Tb2-L1, as described in the text.
[22] Attempts to determine the KD value for Tb2-L2/PhOP system were
unsuccessful. The titration data showed notable deviation at larger
concentrations of Tb2-L2, probably owing to precipitation of the
complex there.
[23] A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker, L.
Royle, A. S. de Sousa, J. A. G. Williams, M. Woods, J. Chem. Soc.
Perkin Trans. 2 1999, 493–503.
[24] As described in text, the solid Tb-DOTAM complex (without pTyr
or PhOP) has one coordination water molecule, according to the X-
ray crystallography.
Cantley, Nature 1995, 373, 536–539; b) Z. Songyang, L. C. Cantley,
Trends Biochem. Sci. 1995, 20, 470–475.
[28] A. Kreegipuu, N. Blom, S. Brunak, J. Jꢅrv, FEBS Lett. 1998, 430,
45–50.
[29] The phosphorylation of this Tyr is crucial for the activation of tran-
scription, and also overactivated in cancer cells; see a) D. E. Levy,
J. E. Darnell, Jr., Nat. Rev. Mol. Cell Biol. 2002, 3, 651–662; b) H.
Yu, R. Jove, Nat. Rev. Cancer 2004, 4, 97–105.
[30] The phosphorylation of this Ser, combined with T308 phosphoryla-
tion, activates signalling pathways including MAPK pathway to
induce many cellular actions, such as proliferation and cell growth:
a) D. P. Brazil, B. A. Hemmings, Trends Biochem. Sci. 2001, 26, 657–
664; b) B. D. Manning, L. C. Cantley, Cell 2007, 129, 1261–1274.
[31] a) J. Inglese, R. L. Johnson, A. Simeonov, M. Xia, W. Zheng, C. P.
Austin, D. S. Auld, Nat. Chem. Biol. 2007, 3, 466–479; b) R. M.
Eglen, T. Reisine, Assay Drug Dev. Technol. 2009, 7, 22–43; c) J.
Comley, Drug Discovery World 2006, 7, 27–50.
[32] a) D. S. Lawrence, Q. Wang, ChemBioChem 2007, 8, 373–378; b) Y.
Li, W. Xie, G. Fang, Anal. Bioanal. Chem. 2008, 390, 2049–2057;
c) M. D. Shults, B. Imperiali, J. Am. Chem. Soc. 2003, 125, 14248–
14249; d) Q. Wang, Z. Dai, S. M. Cahill, M. Blumenstein, D. S. Law-
rence, J. Am. Chem. Soc. 2006, 128, 14016–14017; e) T. Anai, E.
Nakata, Y. Koshi, A. Ojida, I. Hamachi, J. Am. Chem. Soc. 2007,
129, 6232–6239; f) H. Sahoo, A. Hennig, M. Florea, D. Roth, T. En-
derle, W. M. Nau, J. Am. Chem. Soc. 2007, 129, 15927–15934.
[25] This result further confirms that the luminescence enhancement by
PhOP is the result of energy transfer from the benzene ring of pTyr.
The possibility that the removal of water molecules, which quench
the luminescence, is responsible for the enhancement is unlikely.
[26] Similar outer-sphere interaction of phosphate diester with EuIII com-
plex was reported: C.-H. Huang, J. R. Morrow, J. Am. Chem. Soc.
2009, 131, 4206–4207.
[27] a) Z. Songyang, K. L. Carraway III, M. J. Eck, S. C. Harrison, R. A.
Feldman, M. Mohammadi, J. Schlessinger, S. R. Hubbard, D. P.
Smith, C. Eng, M. J. Lorenzo, B. A. J. Ponder, B. J. Mayer, L. C.
Received: December 9, 2009
Revised: February 18, 2010
Published online: April 13, 2010
Chem. Eur. J. 2010, 16, 5018 – 5025
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www.chemeurj.org
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