Synthesis, photophysical and electrochemical properties, and protein-binding studies of luminescent cyclometalated iridium(III) bipyridine estradiol conjugates

Article abstract of DOI:10.1002/chem.200700530

A new series of luminescent cyclometalated iridium(III) bipyridine estradiol conjugates [Ir(N-C)₂(N-N)]-(PF₆) (N-N = 5-(4-(17α-ethynylestradiolyl)phenyl)-2,2′-bipyridine, bpy-est, HN-C = 2-phenylpyridine, Hppy (1a), 1-phenylpyrazole,

Full text of DOI:10.1002/chem.200700530

DOI: 10.1002/chem.200700530
Synthesis, Photophysical and Electrochemical Properties, and Protein-
Binding Studies of Luminescent Cyclometalated Iridium
Estradiol Conjugates
A
Kenneth Kam-Wing Lo,* Kenneth Yin Zhang, Chi-Keung Chung, and
Karen Ying Kwok[a]
Abstract: A new series of luminescent
cyclometalated iridium(III) bipyridine
estradiol conjugates [Ir(N-C)2(N-N)]-
(PF6) (N-N = 5-(4-(17a-ethynylestra-
diolyl)phenyl)-2,2’-bipyridine,bpy-est,
HN-C = 2-phenylpyridine,Hppy ( 1a),
1-phenylpyrazole,Hppz ( 2a),78,-ben-
zoquinoline,Hbzq ( 3a),2-phenylquino-
line,Hpq ( 4a),2-((11, ’-biphenyl)-4-yl)-
benzothiazole,Hbsb ( 5a); N-N = 4-
(N-(6-(4-(17a-ethynylestradiolyl)ben-
zoylamino)hexyl)aminocarbonyl)-4’-
methyl-2,2’-bipyridine,bpy-C6-est,HN-
thesized,characterized,and their pho-
tophysical and electrochemical proper-
ties studied. Upon photoexcitation,all
the complexes displayed intense and
long-lived emission in fluid solutions at
298 K and in low-temperature glass.
The emission of complexes 1a–3a and
1b–3b was assigned to a triplet metal-
to-ligand charge-transfer (3MLCT)
(dp(Ir)!p*(bpy-est and N-CÀ)) state
mixed with some triplet intraligand
(3IL) (p!p*) (N-CÀ and N-N) charac-
ter. However,the emissive states of the
pqÀ and bsbÀ complexes 4a, 4b, 5a,
AHCTREUNG
G
ACHTREUNG
3
E
and 5b showed substantial IL (p!p*)
(pqÀ/bsbÀ) character. The lipophilicity
of all the complexes was determined by
reversed-phase HPLC. Upon binding
to estrogen receptor a,all of these
iridium(III) estradiol conjugates exhib-
G
ited emission enhancement and life-
time extension,rendering them a novel
series of luminescent probes for this re-
ceptor.
Keywords: estrogen
·
iridium
·
C
= Hppy (1b),Hppz ( 2b),Hbzq
luminescence · N ligands · probes
(3b),Hpq ( 4b),Hbsb ( 5b)) was syn-
Introduction
for nonisotopic reagents. Different estradiol derivatives
equipped with reporters such as organic fluorophores,[7–10]
biotin,[10–12] transition-metal complexes,[13,14] and organome-
tallic compounds[15] have been reported. In particular,a
wide range of estradiol derivatives containing chromium,
rhenium,technetium,cobalt,iron,and manganese carbonyl
complexes have been investigated.[5,15a,c,d,f–h] Recently,we
studied the interactions of luminescent rhenium(I) polypyri-
dine estradiol complexes with estrogen receptors; binding of
the complexes to the biomolecules leads to an enhancement
of their emission intensities of between five- and sixfold.[16]
Our current aim is to design new probes with more favora-
ble emission properties and higher sensitivity. In view of the
Estradiol is the most potent natural estrogen responsible for
the development and maintenance of the secondary sexual
characteristics and functions of the reproductive system in
females.[1] The physiological effects of estradiol are triggered
by its binding to estrogen receptors.[2] Owing to the impor-
tant roles of both estradiol and its receptor in female physi-
ology,the design of new biological probes for estradiol-bind-
ing proteins has attracted much attention.[3–16] Radioactive
[3,4]
hormone derivatives,such as those containing tritium
and
technetium,[5,6] are the most frequently used reagents. How-
ever,the potential health hazards of these reagents and the
long experimental time required have prompted the search
interesting emissive behavior of iridium(III) polypyridine
A
complexes,[17–33] in particular their high environment-sensi-
tivity,diverse emissive-state character due to a wide choice
of both cyclometalating and diimine ligands,and our interest
in using these complexes as biological labels and probes,[33]
[a] Dr. K. K.-W. Lo,K. Y. Zhang,Dr. C.-K. Chung,K. Y. Kwok
Department of Biology and Chemistry,City University of Hong Kong
Tat Chee Avenue,Kowloon,Hong Kong,Peopleꢀs Republic of China
Fax: (852)2788-7406
we believe that cyclometalated iridium(III) polypyridine es-
A
tradiol conjugates have a high potential to serve as new lu-
minescent probes for estrogen receptors.
E-mail: bhkenlo@cityu.edu.hk
7110
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
Chem. Eur. J. 2007, 13,7110 – 7120

FULL PAPER
Here we report the synthesis,characterization,and photo-
physical and electrochemical properties of a series of lumi-
mined by reversed-phase HPLC. Additionally,the binding
of the conjugates to estrogen receptor a (ERa) was investi-
gated by emission titration experiments. To increase the
ERa-induced emission enhancement,ferricyanide was used
as a quencher to suppress the emission intensities of the free
complexes,resulting in more sensitive responses to protein
binding.
nescent cyclometalated iridium
A
complexes [Ir(N-C)2(N-N)](PF6) (N-N = 5-(4-(17a-ethyny-
A
N
ACHTREUNG
lestradiolyl)phenyl)-2,2’-bipyridine,bpy-est,HN-C = 2-phe-
nylpyridine,Hppy ( 1a),1-phenylpyrazole,Hppz ( 2a),78,-
benzoquinoline,Hbzq ( 3a),2-phenylquinoline,Hpq ( 4a),2-
((1,1’-biphenyl)-4-yl)benzothiazole,Hbsb ( 5a); N-N = 4-
(N-(6-(4-(17a-ethynylestradiolyl)benzoylamino)hexyl)ami-
nocarbonyl)-4’-methyl-2,2’-bipyridine,bpy-C6-est,HN-C
Hppy (1b),Hppz ( 2b),Hbzq ( 3b),Hpq ( 4b),Hbsb ( 5b))
=
Results and Discussion
(Scheme 1). The lipophilicity of all the conjugates was deter-
Synthesis: The estradiol-containing bipyridine ligands bpy-
est and bpy-C6-est were synthesized from Sonogashira reac-
tion of 17a-ethynylestradiol with 5-(4-bromophenyl)-2,2’-bi-
pyridine and 4-(N-(6-(4-iodobenzoylamino)hexyl)aminocar-
bonyl)-4’-methyl-2,2’-bipyridine,respectively. All the
iridium
ACHTREUNG
action of [Ir
AHCTREUNG
ing methanol/dichloromethane,followed by anion exchange
À
to the PF6 salts and purification by column chromatogra-
phy and recrystallization. They were isolated as air-stable
yellow to orange-red crystals. The complexes were charac-
1
terized by H NMR spectroscopy,positive-ion ESI-MS,and
IR spectroscopy and gave satisfactory microanalysis.
Electronic absorption spectroscopy: The electronic absorp-
tion spectral data of the complexes are listed in Table 1. The
electronic absorption spectra of complexes 1a–5a and 1b–
5b in CH2Cl2 at 298 K are shown in Figures 1 and 2,respec-
tively. All the complexes showed intense absorption bands
and shoulders at about 251–340 nm (e in the order of
104 dm3 molÀ1 cmÀ1) and less intense bands and shoulders at
>345 nm,which were assigned to spin-allowed intraligand
(1IL) (p!p*) (N-N and N-CÀ) and metal-to-ligand charge
transfer (1MLCT) (dp(Ir)!p*(N-N and N-CÀ)) transitions,
respectively.[17b,c,18–20,21a,c,d,22a,c,23b,c,24a,25–27,28b,29–31,32b,33] However,
the mixing of ligand-to-ligand charge transfer (1LLCT) (p-
A
G
and sigma-bond-to-
(s (N-
(Ir-C)!p*
ACHTREUNG
N))[18,19d,20,25b,27b] character into the CT transitions cannot be
completely excluded. All the complexes also exhibited weak
absorption tailing at about 450–550 nm that was assigned to
spin-forbidden MLCT (dp(Ir)!p*(N-N and N-CÀ)) transi-
3
tions.[19a,b,20a,c,d,21d,23b,c,24a,25,26a,27a,29,30a,31a,b,33]
Interestingly,the bpy-est complexes
1a–5a displayed
more intense absorption at about 333–340 nm than the bpy-
C6-est complexes 1b–5b (Figures 1 and 2). These absorption
features are mainly associated with the bpy-est ligand be-
cause the uncoordinated ligand also showed an intense ab-
sorption band in a similar region.
Luminescence properties: Upon photoexcitation,all the
complexes exhibited intense and long-lived greenish-yellow
to orange emission in fluid solutions under ambient condi-
tions and in low-temperature glass. The photophysical data
of the complexes are summarized in Table 2. The emission
spectra of complexes 2a, 2b, 5a,and 5b in CH2Cl2 at 298 K
Scheme 1. Complexes 1a–5a and 1b–5b.
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7111

K. K.-W. Lo et al.
Table 1. Electronic absorption spectral data of the iridium
ACHTREUNG
Complex
Medium
1a
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
260 (52,995), 270 sh (50,925), 314 sh (31,390), 339 (34,840), 400 sh (5,485), 469 sh (785)
259 (58,235), 269 (53,705), 313 sh (37,855), 334 (40,555), 406 sh (4,340), 468 sh (850)
268 (76,485), 337 sh (10,800), 361 sh (8,865), 381 sh (8,115), 419 sh (3,455), 471 sh (1,040)
264 (78,570), 317 sh (19,315), 335 sh (10,560), 380 sh (7,465), 411 sh (3,705), 475 sh (875)
261 (37,230), 315 sh (23,730), 339 (30,185), 458 sh (665)
257 (37,190), 316 sh (27,605), 334 (32,320), 443 sh (650)
261 (54,460), 317 sh (15,630), 353 sh (7,170), 453 sh (910)
260 (56,270), 315 sh (16,455), 347 sh (3,470), 443 sh (935)
251 sh (45,930), 257 (48,480), 315 sh (27,270), 333 (32,135), 415 sh (4,440)
252 sh (48,975), 259 (50,370), 316 sh (32,530), 340 (36,380), 416 sh (4,115)
260 (55,145), 286 sh (30,680), 322 sh (15,655), 360 sh (9,035), 418 sh (3,930), 490 sh (575)
251 sh (49,270), 259 (51,260), 281 sh (30,585), 317 sh (15,190), 351 sh (9,060), 414 sh (3,330)
262 (37,660), 281 (38,045), 337 (31,830), 438 sh (3,765), 527 sh (565)
1b
2a
2b
3a
3b
4a
4b
5a
5b
261 (33,595), 278 (33,095), 333 (28,515), 435 (2,925), 522 sh (285)
264 (55,280), 282 sh (51,420), 320 sh (18,940), 337 (18,655), 438 (3,670), 522 sh (570)
263 (59,935), 281 sh (52,030), 315 sh (19,270), 337 (18,640), 435 (3,985), 516 (495)
271 (54,335), 338 (96,030), 385 sh (26,435), 426 (12,825), 442 sh (12,165)
263 (55,830), 334 (103,280), 389 sh (19,170), 426 sh (11,320), 447 (9,360)
267 (65,860), 322 sh (53,960), 337 (61,870), 389 sh (21,385), 423 (11,930), 441 sh (11,365)
263 (68,105), 321 sh (55,915), 334 (61,870), 389 sh (18,860), 422 (10,910), 440 sh (10,229)
[Ir
A
G
the emission
3
usually originates from a MLCT state that may be mixed
with some 3LLCT[20b–d,21c,d,23b,25b,28b] or 3SBLCT[18,19d,20,25b,27b]
character in some cases. If more-conjugated cyclometalating
ligands such as pqÀ[19a,33b,c] and bsbÀ[33d] are used,the emis-
sive state involves predominant IL (p!p*) (pqÀ or bsbÀ)
character. Thus,the complexes in this work can be tentative-
ly grouped into two classes: Class I includes complexes 1a–
3a and 1b–3b,whose emission energies,lifetimes,and band
shapes in fluid solutions at 298 K are very similar to those
of common cyclometalated iridium(III) polypyridine com-
G
plexes. The emission was assigned to a 3MLCT (dp(Ir)!
p*(N-N and N-CÀ)) emissive state. This assignment is sup-
ported by the reduced emission lifetimes and quantum
yields upon changing the solvent from CH2Cl2 to more polar
CH3CN and aqueous buffer. Interestingly,the bpy-est com-
plexes 1a–3a showed substantially longer emission lifetimes
Figure 1. Electronic absorption spectra of complexes 1a (c), 2a (a),
3a (g), 4a (d),and 5a (-··) in CH2Cl2 at 298 K.
than their bpy-C6-est counterparts,complexes
1b–3b
(Table 2),suggesting the presence of 3IL (p!p*) (bpy-est)
character in the emissive states of complexes 1a–3a due to
the electron-withdrawing phenylacetylene substituent on the
bipyridine ligand.[19c,20a,21a,c,d,22a,c,23b,c,25b,26,27a,b,29a,b,30a,b,33] Class
II includes the four pqÀ and bsbÀ complexes. Among them,
complexes 4a, 5a,and 5b exhibited structured emission
spectra and very long emission lifetimes (to ꢀ1.4–5.5 ms) in
fluid solutions at 298 K (Table 2),indicating that their emis-
sive states should possess substantial 3IL (p!p*) (pqÀ or
bsbÀ) character. Similar features have been observed in re-
lated iridium
(III)-pq[19a,33b,c] and -bsb systems.[33d] Unexpect-
G
edly,the emission properties of complex 4b are very differ-
Figure 2. Electronic absorption spectra of complexes 1b (c), 2b (a),
3b (g), 4b (d),and 5b (-··) in CH2Cl2 at 298 K.
ent from those of its bpy-est counterpart and other iridium-
(III)-pq complexes reported in the literature.[19a,33b,c] First,
the emission spectrum of this complex did not display rich
structural features. Second,the emission quantum yield
showed strong dependence on the polarity of solvents. Addi-
tionally,the emission lifetimes ( <1 ms) were noticeably
and in low-temperature alcohol glass are shown in Figure 3.
According to previous studies on the luminescence proper-
ties of cyclometalated iridium(III) polypyridine complexes
A
7112
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Chem. Eur. J. 2007, 13,7110 – 7120

Iridium(III) Bipyridine Estradiol Conjugates
A
FULL PAPER
Table 2. Photophysical data of the iridium
A
deaerated solutions at 298 K and alcohol glass at 77 K.
Complex Medium (T
lem [nm]
to [ms]
Fem
[K])
1a
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
glass[b] (77)
596
605
578
0.74
0.39
0.15
5.56
0.22
0.091
0.038
530 (max),565,617
sh
1b
2a
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
glass[b] (77)
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
glass[b] (77)
598
604
590
538,569 sh
566
577
0.40
0.24
0.12
5.00
1.38
0.68
0.18
10.25
0.14
0.058
0.0060
0.40
0.16
0.092
540 sh,571
521 (max),561,608
sh
2b
3a
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
glass[b] (77)
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
glass[b] (77)
577
588
574
520,556 sh
599
608
581
528 (max),562,617
sh
601
606
592
535,568 sh
564,602 sh
564,602 sh
570,606 sh
545 (max),587,639
sh
0.68
0.31
0.13
5.34
0.66
0.33
0.15
9.85 (25%),
3.57 (75%)
0.34
0.18
0.11
4.45
1.66
1.40
0.38
5.65
0.30
0.11
0.013
Figure 3. Emission spectra of complexes 2a, 2b, 5a,and 5b in CH2Cl2 at
298 K (c) and in EtOH/MeOH (4:1) at 77 K (a).
0.18
0.070
0.064
3
cited state of MLCT (dp(Ir)!p*(N-N and N-CÀ)) charac-
ter. Again,the rich vibronic structures of the emission bands
and long emission lifetimes of complexes 1a–3a relative to
those of the bpy-C6-est analogues,complexes
1b–3b
3b
4a
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
glass[b] (77)
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
glass[b] (77)
0.11
0.054
0.0061
(Table 2 and Figure 3),reveal that the excited states of these
complexes should bear a high parentage of 3IL (p!p*)
(bpy-est) character. Complex 3a showed bi-exponential
decay with lifetimes of about 10 and 4 ms that are possibly
0.30
0.29
0.23
associated with 3MLCT (dp(Ir)!p*
G
(dp(Ir)!p*
U
ence to related complexes in the literature.[17b,c,18a,19d,20,23
b,30a,33]
4b
5a
5b
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
glass[b] (77)
574
582
582
0.76
0.56
0.20
5.05
0.30
0.12
0.011
Class II complexes 4a, 4b, 5a,and 5b showed a
highly structured emission band and a much smaller blue
shift upon cooling to 77 K (Table 2 and Figure 3). It is con-
ceivable that the emission is derived from a 3IL (p!p*)
(pqÀ or bsbÀ) state that may be mixed with some CT charac-
ter.[19a,33b–d]
539 (max),582,635
sh
553,594 sh,646 sh
554,596 sh,644 sh
562 (max),601,648
sh
543 (max),588,643
554,595 sh,645 sh
554,595 sh,645 sh
563,601 sh,645 sh
541 (max),585,638
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
5.49
4.93
0.28
0.39
0.31
0.30
Electrochemical properties: The electrochemical properties
glass[b] (77)
9.72
4.56
4.02
0.20
9.28
of all the iridium(III) estradiol complexes were studied by
E
CH2Cl2 (298)
CH3CN (298)
buffer[a] (298)
glass[b] (77)
0.51
0.24
0.10
cyclic voltammetry. The electrochemical data are listed in
Table 3. Complexes 1a–4a and 1b–4b showed an irreversi-
ble wave at about +1.17 to +1.23 V versus a saturated calo-
mel electrode (SCE). This was assigned to the oxidation of
the estradiol moiety because a wave at a similar potential
was observed for free estradiol under the same experimental
conditions. Complexes 1a, 1b, 2a, 2b, 4a,and 4b exhibited
a quasireversible couple at about +1.25 to +1.33 V,attribut-
[a] Potassium phosphate (50 mm,pH 7.4) containing 10% MeOH,except
for the quantum yield measurements in which 70% MeOH was used
(MeOH was used for solubility reasons). [b] EtOH/MeOH (4:1).
shorter than those of other iridium
(III)-pq complexes.[19a,33b,c]
A
On the basis of these observations,we argue that the emis-
sive state of this complex possesses predominant 3MLCT
(dp(Ir)!p*(bpy-C6-est)) character. A shift of excited-state
able to a metal-centered iridium
N
AHCTREUNG
did not appear in the cyclic voltammograms of complexes
3a and 3b; probably it is embedded in the estradiol-based
irreversible oxidation wave. Interestingly,complexes 5a and
5b are different from the other complexes in that they dis-
played an irreversible oxidation wave at about +1.3 V and a
quasireversible couple at about +1.5 V versus SCE. Al-
though the first irreversible oxidation wave occurred at a
3
3
character from the expected IL to MLCT of this iridium-
(III)-pq bipyridine complex is probably a consequence of
the electron-withdrawing amide substituent that stabilizes
the p* orbitals of the diimine ligand.
Upon cooling to 77 K,Class I complexes 1a–3a and 1b–
3b showed significant blue shifts in their emission maxima
(Table 2 and Figure 3). The emission was assigned to an ex-
potential similar to that of the iridium(IV/III) oxidation,its
Chem. Eur. J. 2007, 13,7110 – 7120
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7113

K. K.-W. Lo et al.
Table 3. Electrochemical data of the iridium
(III) estradiol complexes.[a]
A
than its bpy-C6-est counterpart
complex 1b,even though the
latter includes a C6 spacer arm.
Complex
Oxidation, E1/2 or Ea [V]
Reduction, E1/2 or Ec [V]
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
+1.17,[b] +1.25[c]
+1.19,[b] +1.27[c]
+1.23,[b] +1.32[c]
+1.21,[b] +1.33[c]
+1.20[b]
À1.30, À1.77,[b] À1.90,[c] À2.26,[c] À2.52[c]
À1.26, À1.90,[c] À2.19,[c] À2.45,[c] À2.62[b] This could be attributed to the
À1.31, À1.74,[b] À1.88,[c] À2.36,[c] À2.52[b]
very nonpolar bpy-est ligand
À1.29, À2.00,[b] À2.17,[b] À2.60[b]
and the additional polar amide
À1.29, À1.65,[b] À2.22,[c] À2.36[b]
moieties of the bpy-C6-est
ligand. Nevertheless,the differ-
+1.17[b]
À1.25, À2.28,[c] À2.64[c]
+1.23,[b] +1.31[c]
+1.19,[b] +1.32[c]
+1.32,[b] +1.49[c]
+1.32,[b] +1.45[c]
À1.29, À1.91,[c] À2.35[c]
À1.26, À1.97,[c] À2.12,[c] À2.23,[b] À2.59[c]
À1.22, À1.72,[c] À1.95,[c] À2.15,[c] À2.29[c]
À1.20, À1.78,[c] À1.95,[c] À2.19,[c] À2.56[c]
ence is negligible for the bzqÀ
and bsbÀ complexes because
their lipophilicity is dominated
by the hydrophobic cyclometa-
lating ligands.
[a] In CH3CN (0.1 moldmÀ3 nBu4NPF6) at 298 K,glassy carbon electrode,sweep rate 100 mVs À1,all potentials
versus SCE. [b] Irreversible waves. [c] Quasireversible couples.
irreversible nature and the finding of a similar irreversible
Table 4. Lipophilicity (logPo/w) of the iridium(III) complexes and 17a-
A
wave for the free Hbsb ligand imply that it is associated
with the oxidation of the coordinated bsbÀ ligands.[33d] The
estradiol-based oxidation wave was not observable and is
probably unresolved with this bsbÀ-based signal. The second
oxidation couple at about +1.45 to +1.49 V was tentatively
ethynylestradiol.
Compound
logPo/w
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
5.56
5.06
1.12
4.79
4.61
1.00
6.10
6.09
2.39
6.75
6.37
2.46
10.15
10.11
5.48
3.20
assigned to iridium
more positive than that of common cyclometalated iridium-
(III) polypyridine complexes due to the strong p-accepting
properties of the conjugated bsbÀ ligand. The first reduction
couples of all the iridium(III) estradiol complexes appeared
N
ACHTREUNG
AHCTREUNG
at about À1.20 to À1.31 V and are reversible in nature. They
were tentatively assigned to the reduction of the coordinat-
ed diimine li
gands.[18a,19d,20,22a,c,23b,c,25b,30b,33] The reduction
U
waves at more negative potentials are highly irreversible,
and were ascribed to the reduction of the diimine and the
cyclometalating ligands.
17a-ethynylestradiol
Lipophilicity: The lipophilicity of an estradiol derivative is
an important property with regard to the ability of the com-
pound to permeate biological membranes.[34] It is commonly
estimated by the partition coefficient (Po/w) of the derivative
in n-octanol/water.[35] The lipophilicity (logPo/w values) of
Emission titrations: The ERa-binding properties of all the
complexes were investigated by emission titrations using the
receptor as the titrant. All the estradiol complexes displayed
emission enhancement in the presence of ERa. As an exam-
ple,the emission spectral traces of complex 4a upon addi-
tion of the receptor are shown in Figure 4. The emission in-
tensities of the estradiol complexes were increased about 1.3
the iridium
ACHTREUNG
bipyridine counterparts,[Ir
A
G
A
=
Hppy (1c),Hppz ( 2c),Hbzq ( 3c),Hpq ( 4c),Hbsb ( 5c),
bpy = 2,2’-bipyridine),and 17 a-ethynylestradiol was deter-
mined and the data are listed in Table 4. The lipophilicity of
all the estradiol complexes (from 4.61 to 10.15) is signifi-
cantly higher than that of 17a-ethynylestradiol (3.20),even
though they all carry a formal cationic charge. The lipophi-
licity of the estradiol-free complexes 1c–5c is lower than
those of their estradiol-containing counterparts by about
3.6–4.7 units,reflecting the hydrophobic nature of the estra-
diol moiety and the linkers. The logPo/w values of the
iridium(III) complexes depend strongly on the cyclometalat-
N
ing ligands,and the observed order (ppz À <ppyÀ <bzqÀ <
pqÀ !bsbÀ) is in accordance with the hydrophobic nature of
the cyclometalating ligands. It can be seen that the introduc-
tion of a fused-benzene or phenyl ring to the cyclometalat-
À
ing ligand (for example,from ppy to bzqÀ or pqÀ) has sub-
Figure 4. Emission spectral traces of complex 4a in potassium phosphate
buffer (50 mm,pH 7.4)/methanol (9:1) at 298 K in the presence of 0–
375 nm ERa.
stantially increased the lipophilicity of the iridium
plex. Interestingly,complex 1a shows higher lipophilicity
(III) com-
7114
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ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
Chem. Eur. J. 2007, 13,7110 – 7120

Iridium(III) Bipyridine Estradiol Conjugates
A
FULL PAPER
to 4.8 times (Table 5). Although the emission wavelengths
did not change substantially,the emission lifetimes were ex-
tended about 2.4 to 7.0 times (Table 5). We ascribed these
vironment on the basis of the photophysical data of the
complexes in solvents of different polarities (Table 2). Inter-
À
estingly,the control bzq complex 3c and bsbÀ complex 5c
also showed small emission enhancement in the presence of
ERa (I/Io =1.2 and 1.3,respectively; t/to =1.2 in both cases,
Table 2). To study further the protein-binding specificity of
all the complexes,bovine serum album (BSA) was used as a
model protein and the titrations were performed under the
same experimental conditions. The majority of the estradiol
complexes did not show noticeable changes in the presence
of BSA (data not shown),suggesting that the ER a-induced
emission changes are due to specific binding of the com-
plexes to the receptor. The only exception is the control
complex 5c that showed increased emission intensity and
lifetime upon addition of BSA (I/Io =1.60; t/to =1.24). From
these results,we tentatively assigned the emission enhance-
ment and lifetime elongation shown by complexes 3c and
5c in the presence of ERa to nonspecific binding of the
complexes to the protein. The binding is likely to be hydro-
phobic in nature given the high lipophilicity of the com-
Table 5. Emission titration results and parameters for the binding of the
iridium(III) complexes to ERa in potassium phosphate buffer (50 mm,
A
pH 7.4)/methanol (9:1) at 298 K.
[a]
Complex
I/Io
to[b] [ms]
t[b] [ms]
Ka [mÀ1
]
nH
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
1.8
1.3
1.0
2.3
1.7
1.0
3.6
2.4
1.2
4.8
2.0
1.0
2.2
1.9
1.3
<0.1
<0.1
<0.1
0.10
<0.1
<0.1
<0.1
<0.1
<0.1
0.25
0.50
0.28
<0.1
0.67
0.33
<0.1
0.41
0.35
0.12
1.45
0.77
0.59
0.28
0.24
1.24
1.2”107
1.5”107
1.6
1.9
1.3”107
2.0”107
1.7
2.2
1.0”107
1.7”107
1.1
1.6
1.0”107
1.6”107
1.6
1.2
0.11
0.57
0.10
0.10
1.7”107
2.1”107
1.2
2.1
À
1.00
plexes,in particular the bsb conjugates (Table 4). The bind-
[a] Io and I are the emission intensities of the complexes (5 mm) in the ab-
sence and presence (375 nm),respectively,of ER a. [b] to and t are the
emission lifetimes of the complexes (5 mm) in the absence and presence
(375 nm),respectively,of ER a.
ing constants (Ka) of all the iridium(III) estradiol complexes
C
changes in photophysical properties to the specific binding
of the estradiol moieties of the complexes to ERa because
most of the estradiol-free control complexes 1c–5c did not
give similar observations (Table 5; see below for excep-
tions). To illustrate this,the emission titration curves for
complexes 4a, 4b,and 4c are shown in Figure 5. The specif-
Figure 6. Hill plot of log(Y/
G
G
binding parameters of all the iridium(III) estradiol com-
G
plexes are listed in Table 5. Similar to other estradiol sys-
tems,[3,8,37] positive cooperativity was observed for the bind-
ing of the estradiol complexes to ERa,as evidenced by
convex Scatchard plots (data not shown) and that the nH
values are >1. The binding constants of these complexes
range from 1.0 to 2.1”107 mÀ1,which are smaller than that
of unmodified estradiol (Ka =5”109 mÀ1).[38] The reduced
binding affinity is probably a consequence of the bulky [Ir-
Figure 5. Luminescence titration curves for the titrations of complexes 4a
~
&
*
( ), 4b ( ),and 4c ( ) (5 mm). The emission intensities of complexes 4a,
4b,and 4c were monitored at 566,584,and 560 nm,respectively.
A
N
icity of the binding was supported by the fact that similar
changes were absent when excess estradiol was present in
the titration mixture from the outset (I/Io <1.1). We attribut-
ed the protein-induced emission enhancement and lifetime
elongation of the estradiol complexes 1a–5a and 1b–5b to
the increase in hydrophobicity and rigidity of their local en-
between the complexes and the receptor. The binding of the
bpy-est complexes to ERa is slightly weaker than that of
the bpy-C6-est complexes (Table 5) due to the lack of C6
spacer arms. Nevertheless,the binding constants of all the
complexes are comparable to or larger than those of other
related estradiol derivatives such as 17a-[(L)Re(CO)3]-estra-
Chem. Eur. J. 2007, 13,7110 – 7120
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
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7115

K. K.-W. Lo et al.
diol (L=4’,4’-bis(ethanethio)-4’-carboxybutyn-1’-yl,6 ’,6’-bi-
s(ethanethio)-6’-carboxyhexyn-1’-yl; Ka =1.3 and 1.1”
repeated the emission titration experiments using
[Fe(CN)6]3À (100 mm) as a quencher in the solution. Remark-
[5]
107 mÀ1,respectively),
17a-[(C CCH2N
(CH3)C2H4N-
ably,in the presence of the quencher,all the iridium (III) es-
N
ꢁ
(CH3)2)Pt(X)]-estradiol (X=diiodide,malonato; Ka =1.0”
tradiol complexes exhibited much more significant ERa-in-
duced emission-intensity enhancement (Table 6). As exam-
ples,the emission spectra of complexes 1a, 2a, 3a,and 4a
in the absence and presence of ERa are shown in Figure 7.
107 and 2.5”106 mÀ1,respectively), and [Re
(N-N)(CO)3(py-
A
[6]
C6-est)](CF3SO3) (py-C6-est=4-(N-(6-(4-(17a-ethynylestra-
T
diolyl)benzoylamino)hexanoyl)aminomethyl)pyridine) (1.5
to 2.0”107 mÀ1).[16]
The intrinsic increase in emission intensities of the com-
plexes upon binding to the receptor is moderate (about 1.3
to 4.8 times,Table 5). To develop homogeneous assays for
ERa,it is desirable to maximize the difference in emission
intensities between the free and protein-bound forms of the
iridium(III) estradiol complexes. We anticipate that the use
E
of a quencher will improve the luminescence response if it
preferentially quenches the free form of the complexes
rather than the ERa-bound form. We selected ferricyanide,
[Fe(CN)6]3À,as the quencher because it can effectively
quench the emission of common d6 metal polypyridine com-
plexes.[39] The emission intensity quenching of the iridium-
A
ated buffer solutions in the absence and presence (375 nm)
of ERa. The Stern–Volmer constants (KSV) are listed in
Table 6. In the absence of the protein,the emission of all
Figure 7. Emission spectra of complexes 1a, 2a, 3a,and 4a in the ab-
sence (a) and presence (c) (375 nm) of ERa in potassium phos-
phate buffer (50 mm,pH 7.4)/methanol (9:1) at 298 K containing 100 mm
Table 6. Stern–Volmer constants for the emission quenching of the
iridium
(III) estradiol complexes by [Fe(CN)6]3À in the absence and pres-
A
[Fe(CN)6]3À
.
ence of ERa,and emission-enhancement factors of iridium (III) estradiol
E
complexes upon binding to ERa in the presence of [Fe(CN)6]3À
.
[b]
[c]
Complex
KSV[a] [mÀ1
]
KSV [mÀ1
]
I/Io
1.4”105
7.4”105
1.1”105
5.4”105
1.2”105
6.1”105
1.6”105
5.3”105
9.1”104
2.7”105
1.1”103
2.4”103
8.3”102
1.7”103
1.3”103
2.3”103
4.3”102
2.0”103
7.7”102
1.1”103
17.2
7.7
16.0
8.5
12.7
10.4
48.7
16.4
14.4
10.2
The emission intensities of all the complexes were increased
by at least 7.7 times (Table 6),which is larger than those of
the rhenium(I) estradiol complexes that we reported recent-
ly.[16] In particular,due to the largest difference in KSV in the
absence and presence of ERa,the emission intensity of
complex 4a was substantially increased by about 49 times
upon binding to the biological receptor (Table 6 and
Figure 7),rendering it to serve as a “light switch” for this
protein. These interesting changes in the photophysical
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
[a] [ERa]=0 nm. [b] [ERa]=375 nm. [c] Io and I are the emission intensi-
ties of the complexes (5 mm) in the absence and presence (375 nm),re-
spectively,of ER a in potassium phosphate buffer (50 mm,pH 7.4)/metha-
properties of the luminescent iridium(III) estradiol com-
G
plexes upon binding to ERa,together with the reasonably
strong binding affinities,indicate that they act as effective
luminescent probes for this biological receptor and are
useful in the development of new homogeneous bioassays.
nol (9:1) at 298 K containing 100 mm [Fe(CN)6]3À
.
the iridium(III) estradiol complexes was significantly
U
quenched by [Fe(CN)6]3À,with KSV ranging from 9.1”104 to
7.4”105 mÀ1 (Table 6). Interestingly,in the presence of ER a,
the emission quenching became much less effective,and the
KSV values were reduced by two to three orders of magni-
Conclusion
In this work,a series of luminescent cyclometalated iridium-
(III) bipyridine estradiol conjugates was designed. Their
2
tude,ranging from 4.3”10 to 2.4”103 mÀ1 (Table 6). We at-
A
tribute the lower emission-quenching efficiency to the spe-
photophysical and electrochemical properties and lipophilic-
ity were studied. All the complexes exhibited rich photolu-
minescence behavior. The most important result is that all
cific binding of the iridium(III) estradiol complexes to ERa
A
because when excess unmodified estradiol was initially pres-
ent,the emission quenching was similar to that in the ab-
sence of the protein. It is likely that immobilization of the
complexes to the protein matrix renders the quenching by
[Fe(CN)6]3À more difficult. Based on these observations,we
these iridium(III) estradiol conjugates displayed emission
A
enhancement and lifetime extension upon binding to ERa.
The binding is specific as revealed by results from the con-
trol experiments. When ferricyanide was used as a quencher,
7116
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Chem. Eur. J. 2007, 13,7110 – 7120

Iridium(III) Bipyridine Estradiol Conjugates
A
FULL PAPER
4-(N-(6-(4-Iodobenzoylamino)hexyl)aminocarbonyl)-4’-methyl-2,2’-bipyr-
the increase of emission intensities of all the estradiol com-
plexes upon binding to ERa was much more significant.
idine:
A mixture of N-(4-iodobenzoyl)-1,6-diaminohexane (390 mg,
1.13 mmol),succinimidyl-4-carboxy-4 ’-methyl-2,2’-bipyridine (350 mg,
1.13 mmol),and triethylamine (5 mL) in DMF (40 mL) was stirred under
nitrogen at RT for 12 h. The clear yellow solution was evaporated to dry-
ness under vacuum to give a yellow solid. The product was recrystallized
from MeOH/diethyl ether to give white crystals. Yield: 260 mg (43%).
ESI-MS (positive mode): m/z: 543 [M++H].
From these results we can conclude that these iridium(III)
A
estradiol conjugates are promising candidates in the devel-
opment of novel biosensors for receptors,related estrogen-
binding proteins,and endocrine disruptors.
Bpy-C6-est: 17a-Ethynylestradiol (70 mg,0.24 mmol) was added as a
solid to a mixture of 4-(N-(6-(4-iodobenzoylamino)hexyl)aminocarbon-
yl)-4’-methyl-2,2’-bipyridine (130 mg,0.24 mmol),[Pd (PPh3)2Cl2] (3.4 mg,
A
4.8 nmol),and CuI (1.8 mg,9.6 nmol) suspended in diethylamine (5 mL)
and DMF (15 mL) under an inert atmosphere of nitrogen. A black solid
was formed immediately. The mixture was stirred at RT for 10 min and
then refluxed for 12 h. After cooling to RT,the mixture was filtered and
the filtrate was evaporated to dryness to give a brown solid that was
washed with CH2Cl2 and diethyl ether. Recrystallization of the crude
product from MeOH/diethyl ether afforded bpy-C6-est as pale-yellow
crystals. Yield: 100 mg (59%). 1H NMR (500 MHz,[D 6]acetone,243 K,
TMS): d=9.19 (s,1H; H3 pyridyl ring),9.05 (brs,1H; bpy-4-CON H),
8.93 (s,1H; H3 ’ pyridyl ring),8.81 (d, J=5.0 Hz,1H; H6 pyridyl ring),
8.64–9.62 (m,1H; H6 ’ pyridyl ring),8.59 (d, J=5.0 Hz,1H; H5 pyridyl
Experimental Section
Materials and synthesis: All solvents were of analytical reagent grade
and purified according to standard procedures. IrCl3·3H2O,Hppy,Hppz,
Hbzq,Hpq,17 a-ethynylestradiol,triphenylphosphine,1-octanol,4-me-
thoxyaniline,4-methoxyphenol,phenol,acetophenone,naphthalene,
tert-
butylbenzene,anthracene,and pyrene were purchased from Aldrich. N-
Boc-1,6-diaminohexane hydrochloride (Boc=tert-butyl oxycarbonyl),2-
aminothiophenol,and 4-biphenylcarboxaldehyde were purchased from
International Laboratory. Palladium(II) chloride,copper(I) iodide,4-io-
dobenzoic acid, N-hydroxysuccinimide, N,N’-dicyclohexylcarbodiimide,
ring),8.35 (s,1H; 3-OH estradiol),7.93–7.91 (m,3H; H5
’ pyridyl ring,
KPF6,and K [Fe(CN)6] were purchased from Acros. K3[Fe(CN)6] was re-
3
H2,H6 phenyl ring),7.54 (d, J=8.2 Hz,2H; H3,H5 phenyl ring),7.34
crystallized from hot water and dried in a vacuum desiccator thoroughly
before use. 5-(4-Bromophenyl)-2,2’-bipyridine was obtained from Wako.
Succinimidyl-4-carboxy-4’-methyl-2,2’-bipyridine,[40] 4-iodobenzoic acid
N-hydroxysuccinimidyl ester,[41] Hbsb,[42] and all the precursor complexes
(brs,1H; bpy-4-CONHC H12NH),7.08 (d, J=8.7 Hz,1H; H1 estradiol),
6
6.54 (d, J=8.7 Hz,1H; H2 estradiol),6.46 (s,1H; H4 estradiol),5.60 (s,
1H; 17-OH estradiol),3.37–3.31 (m,4H; NHC H2C4H8CH2NH),2.77–
2.67 (m,2H; H6 estradiol),2.46 (s,3H; CH
pyridyl ring),2.35–2.12 (m,
3
[Ir2
(N-C)4Cl2][17a] were prepared according to reported procedures. Lamb
A
4H; H9a,H11 a,H12 ,H16 estradiol),2.00–1.79 (m,5H; H7 b,H8 ,H11 ,
b
a
b
b
uteri cytosol was used as the source of ERa.[15] The organ tissues ob-
tained from the HKSAR slaughterhouse in Sheung Shui,New Territories,
Hong Kong,were frozen immediately after isolation and stored at À708C
prior to purification. ERa was purified and quantitated according to re-
ported procedures.[15,43]
H15a,H16
estradiol),1.59–1.29 (m,12H; NHCH 2C4H8CH2NH,H7 ,
a
b
H12a,H14 a,H15 estradiol),0.86 ppm (s,3H; CH estradiol); IR (KBr):
b
3
À1
n˜ =3400 (O-H),3290 (N-H),2919 (C-H),2852 (C-H),1653 cm
ESI-MS (positive mode): m/z: 711 [M++H].
(C=O);
[Ir
A
2(N-N)]
G
N
Synthesis of bpy-est: 17a-Ethynylestradiol (444 mg,1.50 mmol) was
est or bpy-C6-est (0.06 mmol) in methanol/dichloromethane (30 mL,1:1)
was refluxed under an inert atmosphere of nitrogen in the dark for 4 h.
The yellow solution was then cooled to RT and KPF6 (0.07 mmol) was
added. The mixture was then evaporated to dryness. The solid was dis-
solved in dichloromethane and purified by column chromatography on
silica gel. The desired product was eluted with dichloromethane/acetone
(1:1) and recrystallized from acetone/diethyl ether.
added as
a
solid to
(449 mg,1.50 mmol),[Pd
a
mixture of 5-(4-bromophenyl)-2,2’-bipyridine
(PPh3)2Cl2] (21 mg,30 nmol),and CuI (11 mg,
AHCTREUNG
60 nmol) suspended in diethylamine (25 mL) under an inert atmosphere
of nitrogen. A black solid formed immediately. The mixture was stirred
at RT for 10 min and then refluxed for 12 h. After cooling to RT,the
mixture was filtered and the filtrate was evaporated to dryness to give a
grey solid that was washed with a small amount of cold CH2Cl2. Recrys-
tallization of the crude product from MeOH/diethyl ether afforded bpy-
est as off-white crystals. Yield: 229 mg (29%). 1H NMR (500 MHz,
[D6]acetone,243 K,TMS): d=9.03 (s,1H; H6 pyridyl ring),8.71 (d, J=
4.3 Hz,1H; H6 ’ pyridyl ring),8.58 (d, J=7.9 Hz,1H; H3 pyridyl ring),
8.54–8.52 (m,2H; 3-OH estradiol,H3 ’ pyridyl ring),8.28 (d, J=8.5 Hz,
1H; H4 pyridyl ring),7.98 (t, J=7.7 Hz,1H; H4 ’ pyridyl ring),7.86 (d,
[Ir
A
G
N
in 72% yield. 1H NMR (300 MHz,[D 6]acetone,298 K,TMS): d=8.93–
8.88 (m,2H; H3,H3 ’ pyridyl rings bpy-est),8.58 (dd, J=8.5,2.3 Hz,1H;
H6’ pyridyl ring bpy-est),8.35–8.22 (m,4H; H3 pyridyl ring ppy À,H6
pyridyl ring bpy-est,3-OH estradiol),8.14 (dd, J=5.6,0.9 Hz,1H; H4
pyridyl ring bpy-est),8.04–7.88 (m,7H; H3 phenyl ring ppy À,H4,H6 pyr-
idyl ring ppyÀ,H4 ’ pyridyl ring bpy-est),7.74 (ddd, J=7.6,5.6,1.2 Hz,
1H; H5’ pyridyl ring bpy-est),7.52 (d, J=8.5 Hz,2H; H3,H5 phenyl
ring bpy-est),7.42 (d, J=8.5 Hz,2H; H2,H6 phenyl ring bpy-est),7.21–
7.03 (m,5H; H4 phenyl ring ppy À,H5 pyridyl ring ppy À,H1 estradiol),
6.99–6.92 (m,2H; H5 phenyl ring ppy À),6.61 (dd, J=8.5,2.6 Hz,1H; H2
estradiol),6.54 (d, J=2.6 Hz,1H; H4 estradiol),6.44–6.38 (m,2H; H6
phenyl ring ppyÀ),4.57 (s,1H; 17-OH estradiol),2.82–2.77 (m,2H; H6
J=7.6 Hz,2H; H3,H5 phenyl ring),7.62 (d,
J=6.9 Hz,2H; H2,H6
phenyl ring),7.48 (t, J=6.2 Hz,1H; H5 ’ pyridyl ring),7.14 (d, J=8.1 Hz,
1H; H1 estradiol),6.59 (d, J=8.4 Hz,1H; H2 estradiol),6.51 (s,1H; H4
estradiol),5.73 (s,1H; 17-OH estradiol),2.78–2.70 (m,2H; H6 estra-
diol),2.39–2.16 (m,4H; H9 a,H11 a,H12 b,H16 estradiol),1.86–1.80 (m,
a
5H; H7b,H8 ,H11 ,H15 a,H16 estradiol),1.48–1.28 (m,4H; H7 a,H12 ,
a
b
b
b
H14a,H15 estradiol),0.93 ppm (s,3H; CH
estradiol); IR (KBr): n˜ =
3
b
3421 (O-H),2919 (C-H),2863 cm À1 (C-H); ESI-MS (positive mode): m/z:
527 [M++H].
estradiol),2.40–2.09 (m,4H; H9 a,H11 a,H12 b,H16 estradiol),2.01–1.76
a
(m,5H; H7 b,H8 b,H11 b,H15 a,H16 estradiol),1.54–1.29 (m,4H; H7
,
a
b
H12a,H14 a,H15 estradiol),0.95 ppm (s,3H; CH estradiol); IR (KBr):
b
3
N-(4-Iodobenzoyl)-1,6-diaminohexane: A mixture of 4-iodobenzoic acid
N-hydroxysuccinimidyl ester (480 mg,1.39 mmol), N-boc-1,6-diaminohex-
ane hydrochloride (420 mg,1.66 mmol),and triethylamine (10 mL) in
DMF (80 mL) was stirred under an inert atmosphere of nitrogen at RT
for 12 h. The solution was evaporated to dryness to give a white solid
that was then washed with diethyl ether. The solid was dissolved in
CH2Cl2 (20 mL) and the mixture was filtered. The filtrate was evaporated
to dryness to give a white solid. The solid was dissolved in trifluoroacetic
acid (5 mL) and the solution was stirred at RT for 12 h. The solution was
evaporated to dryness to give a white solid. The product was recrystal-
lized from CH2Cl2/diethyl ether to give white crystals (390 mg,81%).
ESI-MS (positive mode): m/z: 347 [M++H].
(PF6À); ESI-MS (posi-
À1
n˜ =3423 (O-H),2925 (C-H),2868 (C-H),845 cm
tive mode): m/z: 1026 [M+] [Ir
N
N
(%) for IrC58H50N4O2PF6·2H2O: C 57.66,H 4.50,N 4.64; found: C 57.60,
H 4.43,N 4.66.
[Ir
A
G
1
crystals in 66% yield. H NMR (300 MHz,[D ]acetone,298 K,TMS): d=
6
9.14 (s,1H; H3 pyridyl ring bpy-C6-est),8.80 (s,1H; H3 ’ pyridyl ring
bpy-C6-est),8.40 (t, J=5.5 Hz,1H; bpy-4-CON H),8.24 (d, J=7.7 Hz,
2H; H3 pyridyl ring ppyÀ),8.18 (d, J=5.6 Hz,1H; H6 pyridyl ring bpy-
C6-est),8.11 (s,1H; 3-OH estradiol),8.04–8.02 (m,2H; H4 pyridyl ring
ppyÀ),7.99–7.82 (m,9H; H3 phenyl ring ppy À,H6 pyridyl ring ppy À,H5,
Chem. Eur. J. 2007, 13,7110 – 7120
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7117

K. K.-W. Lo et al.
H6’ pyridyl rings bpy-C6-est,H2,H6 phenyl ring bpy-C6-est,bpy-4-
CONHC6H12NH),7.56 (d, J=5.6 Hz,1H; H5 ’ pyridyl ring bpy-C6-est),
7.48 (d, J=8.1 Hz,2H; H3,H5 phenyl ring bpy-C6-est),7.16–7.10 (m,
3H; H5 pyridyl ring ppyÀ,H1 estradiol),7.07–7.02 (m,2H; H4 phenyl
ring ppyÀ),6.94–6.90 (m,2H; H5 phenyl ring ppy À),6.60 (dd, J=8.1,
2.3 Hz,1H; H2 estradiol),6.54 (d, J=2.3 Hz,1H; H4 estradiol),6.36–
6.31 (m,2H; H6 phenyl ring ppy À),4.63 (s,1H; 17-OH estradiol),3.46–
3.39 (m,4H; NHC H2C4H8CH2NH),2.82–2.76 (m,2H; H6 estradiol),2.58
bzqÀ,H5 ’ pyridyl ring bpy-est),7.42 (d, J=8.2 Hz,2H; H3,H5 phenyl
ring bpy-est),7.26 (d, J=8.5 Hz,2H; H2,H6 phenyl ring bpy-est),7.22–
7.18 (m,2H; H8 bzq À),7.11 (d, J=8.2 Hz,1H; H1 estradiol),6.60 (d, J=
8.2 Hz,1H; H2 estradiol),6.54 (s,1H; H4 estradiol),6.47–6.42 (m,2H;
H9 bzqÀ),4.53 (s,1H; 17-OH estradiol),2.89–2.77 (m,2H; H6 estradiol),
2.33–1.91 (m,4H; H9 a,H11 a,H12 b,H16 estradiol),1.84–1.78 (m,5H;
a
H7b,H8 b,H11 b,H15 a,H16 estradiol),1.49–1.37 (m,4H; H7 a,H12
,
a
b
H14a,H15 estradiol),0.94 ppm (s,3H; CH
estradiol); IR (KBr): n˜ =
b
3
À1
(s, 3H; CH3 pyridyl ring bpy-C6-est),2.34–2.15 (m,4H; H9 a,H11 a,H12 ,
3421 (O-H),2919 (C-H),2858 (C-H),835 cm
(PF6À); ESI-MS (positive
b
H16a estradiol),1.93–1.82 (m,5H; H7 b,H8 ,H11 ,H15 a,H16 estradiol),
mode): m/z: 1074 [M+] [Ir
N
N
b
b
b
1.62–1.29 (m,12H; NHCH 2C4H8CH2NH,H7 a,H12 a,H14 a,H15 estra-
for IrC62H50N4O2PF6·2H2O: C 59.27,H 4.33,N 4.46; found: C 59.14,H
b
diol),0.96 ppm (s,3H; CH
(N-H),2923 (C-H),2864 (C-H),1676 (C =O),843 cm
(positive mode): m/z: 1210 [M+] [Ir
ysis calcd (%) for IrC67H66N6O4PF6·H2O: C 58.55,H 4.99,N 6.11; found:
C 58.30,H 4.87,N 6.04.
estradiol); IR (KBr): n˜ =3420 (O-H),3288
4.43,N 4.29.
3
(PF6À); ESI-MS
À1
[Ir
A
U
(ppy)2(bpy-C6-est)]+; elemental anal-
1
crystals in 70% yield. H NMR (300 MHz,[D ]acetone,298 K,TMS): d=
9.18 (s,1H; H3 pyridyl ring bpy-C6-est),8.85 (s,1H; H3 ’ pyridyl ring
bpy-C6-est),8.56 (d, J=8.1 Hz,2H; H4 bzq À),8.40–8.32 (m,2H; bpy-4-
6
[Ir
A
2(bpy-est)]
A
CONH,H6 pyridyl ring bpy-C6-est),8.26 (dd, J=5.6,1.3 Hz,1H; H6
pyridyl ring bpy-C6-est),8.18 (d, J=5.6 Hz,1H; H5 pyridyl ring bpy-C6-
est),8.07 (s,1H; 3-OH estradiol),7.99 (d,
’
1
crystals in 83% yield. H NMR (300 MHz,[D ]acetone,298 K,TMS): d=
8.92–8.87 (m,2H; H3,H3 ’ pyridyl rings bpy-est),8.73 (d, J=2.6 Hz,1H;
H5 pyrazole ring ppzÀ),8.70 (d, J=3.0 Hz,1H; H5 pyrazole ring ppz À),
8.58 (dd, J=8.5,2.6 Hz,1H; H6 ’ pyridyl ring bpy-est),8.46 (d, J=1.7 Hz,
1H; H6 pyridyl ring bpy-est),8.36–8.28 (m 2H; H4,H4 ’ pyridyl rings
bpy-est),8.03 (s,1H; 3-OH estradiol),7.76–7.71 (m,1H; H5 ’ pyridyl ring
bpy-est),7.68–7.63 (m,2H; H3 phenyl ring ppz À),7.54–7.47 (m,4H; H2,
H3,H5,H6 phenyl ring bpy-C6-est),7.45 (d, J=2.6 Hz,1H; H3 pyrazole
ring ppzÀ),7.31 (d, J=2.1 Hz,1H; H3 pyrazole ring ppz À),7.13–7.05 (m,
3H; H4 phenyl ring ppzÀ,H1 estradiol),6.94–6.86 (m,2H; H5 phenyl
ring ppzÀ),6.71–6.69 (m,2H; H4 pyrazole ring ppz À),6.61 (dd, J=8.5,
2.6 Hz,1H; H2 estradiol),6.54 (d, J=2.6 Hz,1H; H4 estradiol),6.44
(dd, J=7.3,1.3 Hz,1H; H6 phenyl ring ppz À),6.38 (dd, J=7.3,1.3 Hz,
1H; H6 phenyl ring ppzÀ),4.58 (s,1H; 17-OH estradiol),2.82–2.76 (m,
J=7.7 Hz,2H; H2 bzq À),
7.95–7.87 (m,5H; H5,H6 bzq À,bpy-4-CONHC 6H12NH),7.84 (d, J=
8.5 Hz,2H; H2,H6 phenyl ring bpy-C6-est),7.62–7.55 (m,4H; H3,H7
bzqÀ),7.49–7.46 (m,3H; H3,H5 phenyl ring bpy-C6-est,H5 ’ pyridyl ring
bpy-C6-est),7.21–7.15 (m,2H; H8 bzq À),7.11 (d, J=8.1 Hz,1H; H1 es-
tradiol),6.60 (dd, J=8.5,2.3 Hz,1H; H2 estradiol),6.55 (d,
J=2.3 Hz,
1H; H4 estradiol),6.37–6.33 (m,2H; H9 bzq À),4.60 (s,1H; 17-OH estra-
diol),3.44–3.37 (m,4H; NHC H2C4H8CH2NH),2.79–2.75 (m,2H; H6 es-
tradiol),2.57 (s,3H; CH
pyridyl ring bpy-C6-est),2.38–2.15 (m,4H;
3
H9a,H11 a,H12 b,H16 estradiol),1.93–1.82 (m,5H; H7 b,H8 b,H11
,
b
a
H15a,H16
estradiol),1.63–1.29 (m,12H; NHCH 2C4H8CH2NH,H7 ,
a
b
H12a,H14 a,H15 estradiol),0.96 ppm (s,3H; CH estradiol); IR (KBr):
b
3
n˜ =3416 (O-H),3285 (N-H),2930 (C-H),2846 (C-H),1647 (C
=O),
2H; H6 estradiol),2.38–2.13 (m,4H; H9 a,H11 a,H12 b,H16 estradiol),
1.99–1.78 (m,5H; H7 b,H8 b,H11 b,H15 a,H16 estradiol),1.50–1.29 (m,
849 cmÀ1 (PF6À); ESI-MS (positive mode): m/z: 1258 [M+] [Ir
ACHTREUNG
C6-est)]+; elemental analysis calcd (%) for IrC66H54N4O2PF6·CH3OH: C
4H; H7a,H12 a,H14 a,H15 estradiol),0.95 ppm (s,3H; CH
estradiol);
60.20,H 4.91,N 5.85; found: C 60.26,H 4.85,N 5.63.
b
3
(PF6À);
À1
IR (KBr): n˜ =3425 (O-H),2918 (C-H),2854 (C-H),850 cm
[Ir(pq)
N
N
ESI-MS (positive mode): m/z: 1004 [M+] [Ir
N
N
in 83% yield. 1H NMR (300 MHz,[D 6]acetone,298 K,TMS): d=8.66–
8.63 (m,2H; H3,H3 ’ pyridyl rings bpy-est),8.59–8.45 (m,4H; H3 quino-
line pqÀ,H6,H6 ’ pyridyl rings bpy-est),8.40–8.36 (m,2H; H3 phenyl
ring pqÀ),8.29 (d, J=7.0 Hz,2H; H4 quinoline pq À),8.17–8.11 (m,1H;
analysis calcd (%) for IrC54H48N6O2PF6·2H2O: C 54.68,H 4.42,N 7.08;
found: C 54.58,H 4.44,N 7.10.
[Ir
N
N
yellow crystals in 69% yield. 1H NMR (300 MHz,[D 6]acetone,298 K,
TMS): d=9.13 (s,1H; H3 pyridyl ring bpy-C6-est),8.79 (s,1H; H3 ’ pyr-
idyl ring bpy-C6-est),8.72 (d, J=2.1 Hz,2H; H5 pyrazole ring ppz À),
8.38–8.35 (m,2H; bpy-4-CON H,H6 pyridyl ring bpy-C6-est),8.10–8.12
(m,3H; H5,H6 ’ pyridyl rings bpy-C6-est,3-OH estradiol),7.94 (t, J=
5.3 Hz,1H; bpy-4-CONHC 6H12NH),7.86 (d, J=8.5 Hz,2H; H2,H6
phenyl ring bpy-C6-est),7.64 (d, J=7.3 Hz,2H; H3 phenyl ring ppz À),
7.57 (d, J=5.6 Hz,1H; H5 ’ pyridyl ring bpy-C6-est),7.49 (d, J=8.5 Hz,
2H; H3,H5 phenyl ring bpy-C6-est),7.36 (d, J=1.8 Hz,1H; H3 pyrazole
ring ppzÀ),7.25 (d, J=1.8 Hz,1H; H3 pyrazole ring ppz À),7.13–7.04 (m,
3H; H4 phenyl ring ppzÀ,H1 estradiol),6.90–6.85 (m,2H; H5 phenyl
ring ppzÀ),6.71–6.68 (m,2H; H4 pyrazole ring ppz À),6.60 (dd, J=8.5,
2.8 Hz,1H; H2 estradiol),6.55 (d, J=2.8 Hz,1H; H4 estradiol),6.36–
6.31 (m,2H; H6 phenyl ring ppz À),4.58 (s,1H; 17-OH estradiol),3.50–
3.40 (m,4H; NHC H2C4H8CH2NH),2.83–2.76 (m,2H; H6 estradiol),2.61
H4 pyridyl ring bpy-est),8.07 (s,1H; 3-OH estradiol),7.92 (dt,
J=8.4,
1.5 Hz,2H; H8 quinoline pq À),7.71 (t, J=7.6 Hz,1H; H4 ’ pyridyl ring
bpy-est),7.61 (d, J=8.2 Hz,2H; H3,H5 phenyl ring bpy-est),7.56–7.41
(m,7H; H5,H7 quinoline pq À,H2,H6 phenyl ring bpy-est,H5 ’ pyridyl
ring bpy-est),7.24–7.12 (m,5H; H4 phenyl ring pq À,H6 quinoline pq
,
À
H1 estradiol),6.89–6.82 (m,2H; H5 phenyl ring pq À),6.66 (d, J=7.9 Hz,
1H; H2 estradiol),6.62 (s,1H; H4 estradiol),6.59–6.55 (m,2H; H6
phenyl ring pqÀ),4.62 (s,1H; 17-OH estradiol),2.84–2.79 (m,2H; H6 es-
tradiol),2.39–2.16 (m,4H; H9 a,H11 a,H12 b,H16 estradiol),2.02–1.83
a
(m,5H; H7 b,H8 b,H11 b,H15 a,H16 estradiol),1.53–1.29 (m,4H; H7
,
a
b
H12a,H14 a,H15 estradiol),0.98 ppm (s,3H; CH estradiol); IR (KBr):
b
3
À1
n˜ =3420 (O-H),2926 (C-H),2853 (C-H),848 cm
(PF6À); ESI-MS (posi-
tive mode): m/z: 1126 [M+] [Ir(pq)2(bpy-est)]+; elemental analysis calcd
A
(%) for IrC66H54N4O2PF6·2H2O: C 60.59,H 4.47,N 4.28; found: C 60.72,
H 4.52,N 4.31.
(s, 3H; CH3 pyridyl ring bpy-C6-est),2.39–2.14 (m,4H; H9 a,H11 a,H12 ,
b
[Ir(pq)2(bpy-C6-est)](PF6) (4b): Complex 4b was isolated as orange crys-
G
tals in 74% yield. 1H NMR (300 MHz,[D 6]acetone,298 K,TMS): d=
8.75 (s,1H; H3 pyridyl ring bpy-C6-est),8.56–8.49 (m,4H; H3 quinoline
pqÀ,H3 ’ pyridyl ring bpy-C6-est bpy-4-CONH),8.45 (d, J=6.0 Hz,1H;
H6 pyridyl ring bpy-C6-est),8.40 (s,1H; 3-OH estradiol),8.26–8.23 (m,
3H; H3 phenyl ring pqÀ,H6 ’ pyridyl ring bpy-C6-est),8.18 (d, J=6.0 Hz,
1H; H5 pyridyl ring bpy-C6-est),8.05–8.02 (m,2H; H4 quinoline pq À),
H16a estradiol),1.93–1.79 (m,5H; H7 b,H8 ,H11 ,H15 a,H16 estradiol),
b
b
b
1.64–1.29 (m,12H; NHCH 2C4H8CH2NH,H7 a,H12 a,H14 a,H15 estra-
b
diol),0.96 ppm (s,3H; CH
(N-H),2927 (C-H),2853 (C-H),1636 (C =O),847 cm
(positive mode): m/z: 1188 [M+] [Ir
estradiol); IR (KBr): n˜ =3426 (O-H),3279
3
(PF6À); ESI-MS
À1
(ppz)2(bpy-C6-est)]+; elemental anal-
ysis calcd (%) for IrC63H64N8O4PF6·1.5H2O: C 55.58,H 4.96,N 8.23;
found: C 55.60,H 4.89,N 8.32.
7.94–7.88 (m,3H; H8 quinoline pq À,bpy-4-CONHC H12NH),7.82 (d, J=
6
[Ir
A
2(bpy-est)]
T
8.5 Hz,2H; H2,H6 phenyl ring bpy-C6-est),7.55 (d, J=5.6 Hz,1H; H5 ’
pyridyl ring bpy-C6-est),7.46 (d, J=5.6 Hz,2H; H3,H5 phenyl ring bpy-
C6-est),7.44–7.38 (m,4H; H5,H7 quinoline pq À),7.20–7.09 (m,5H; H4
phenyl ring pqÀ,H6 quinoline pq À,H1 estradiol),6.85–6.81 (m,2H; H5
phenyl ring pqÀ),6.63–6.53 (m,4H; H6 phenyl ring pq À,H2,H4 estra-
diol),4.59 (s,1H; 17-OH estradiol),3.44–3.33 (m,4H;
in 72% yield. 1H NMR (300 MHz,[D 6]acetone,298 K,TMS): d=8.95–
8.90 (m,2H; H3,H3 ’ pyridyl rings of bpy-est),8.58–8.46 (m,3H; H4
bzqÀ,H6,H6 ’ pyridyl rings bpy-est),8.31–8.28 (m,3H; H2 bzq À,3-OH
estradiol),8.11 (d, J=5.6 Hz,1H; H4 pyridyl ring bpy-est),8.02–7.84 (m,
5H; H5,H6 bzq À,H4 ’ pyridyl ring bpy-est),7.64–7.56 (m,5H; H3,H7
7118
www.chemeurj.org
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
Chem. Eur. J. 2007, 13,7110 – 7120

Iridium(III) Bipyridine Estradiol Conjugates
A
FULL PAPER
NHCH2C4H8CH2NH),2.84–2.72 (m,2H; H6 estradiol),2.44 (s,3H; CH
pyridyl ring bpy-C6-est),2.39–2.15 (m,4H; H9 a,H11 a,H12 ,H16 estra-
cients (nH) of the iridium(III) estradiol complexes were determined from
A
3
the Hill equation:[36]
b
a
diol),1.92–1.82 (m,5H; H7 b,H8 b,H11 b,H15 a,H16 estradiol),1.61–1.29
b
(m,12H; NHCH 2C4H8CH2NH,H7 a,H12 a,H14 a,H15
estradiol),
b
log ððY=ð1ÀYÞÞ ¼ nH log ½ERaŠ þ nH log Ka
0.96 ppm (s,3H; CH estradiol); IR (KBr): n˜ =3429 (O-H),3273 (N-H),
3
2921 (C-H),2863 (C-H),1659 (C =O),853 cm (PF6À); ESI-MS (positive
À1
Y=(IobsÀImin)/(ImaxÀImin),in which Iobs, Imin,and Imax are the emission in-
mode): m/z: 1310 [M+] [Ir(pq)2(bpy-C6-est)]+; elemental analysis calcd
(%) for IrC75H70N6O4PF6·2H2O: C 60.35,H 5.00,N 5.63; found: C 60.23,
H 4.89,N 5.68.
tensities of the apparent,free,and bound forms of the iridium
plex,respectively.
A
[Ir
A
G
A
1
crystals in 81% yield. H NMR (300 MHz,[D ]acetone,298 K,TMS): d=
6
8.94–8.89 (m,2H; H3,H3 ’ pyridyl rings bpy-est),8.62 (dd, J=8.3,2.3 Hz,
1H; H6’ pyridyl ring bpy-est),8.55 (d, J=2.1 Hz,1H; H6 pyridyl ring
bpy-est),8.48 (dd, J=4.7,2.1 Hz,1H; H4 pyridyl ring bpy-est),8.37 (dt,
J=7.7,1.7 Hz,1H; H4 ’ pyridyl ring bpy-est),8.23–8.19 (m,2H; H4 ben-
zothiazole ring bsbÀ),8.13 (d, J=7.7 Hz,1H; H5 biphenyl ring bsb À),
8.10 (d, J=7.7 Hz,1H; H5 biphenyl ring bsb À),8.03 (s,1H; 3-OH estra-
diol),7.84 (dt, J=7.7,1.3 Hz,1H; H5 ’ pyridyl ring bpy-est),7.53–7.42
(m,8H; H2 ’,H6 ’ biphenyl ring bsbÀ,H2,H3,H5,H6 phenyl ring bpy-
est),7.27–7.18 (m,12H; H5,H6 benzothiazole ring bsb À,H3 ’,H4 ’,H5 ’,
H6 biphenyl rings bsbÀ),7.11 (d, J=8.1 Hz,1H; H1 estradiol),6.77–6.74
(m,2H; H2 biphenyl ring bsb À),6.61 (dd, J=8.5,3.0 Hz,1H; H2 estra-
diol),6.58 (d, J=8.1 Hz,1H; H7 benzothiazole ring bsb À),6.54 (d, J=
2.6 Hz,1H; H4 estradiol),6.48 (d, J=8.1 Hz,1H; H7 benzothiazole ring
bsbÀ),4.56 (s,1H; 17-OH estradiol),2.83–2.76 (m,2H; H6 estradiol),
Acknowledgements
We thank the Hong Kong Research Grants Council (Project No. CityU
101605) for financial support. K.Y.Z. and C.K.C. acknowledge the receipt
of a postgraduate studentship administered by the City University of
Hong Kong.
[1] See,for example: a) C. Behl, Estrogen—Mystery Drug for the
Brain? The Neuroprotective Activities of the Female Sex Hormone,
Springer-Verlag,New York, 2001; b) P. M. Parker,J. N. Parker, Es-
tradiol—A Medical Dictionary, Bibliography, and Annotated Re-
search Guide to Internet References,ICON Health Publications,San
Diego,CA, 2004.
2.38–2.13 (m,4H; H9 a,H11 a,H12 b,H16 estradiol),1.96–1.78 (m,5H;
[2] See,for example: a) E. von Angerer, The Estrogen Receptor as a
Target for Rational Drug Design,Springer,Heidelberg,Germany,
1995; b) F. F. Parl, Estrogens, Estrogen Receptor, and Breast Cancer,
a
H7b,H8 b,H11 b,H15 a,H16 estradiol),1.52–1.26 (m,4H; H7 a,H12
,
a
b
H14a,H15 estradiol),0.94 ppm (s,3H; CH
estradiol); IR (KBr): n˜ =
b
3
À1
3419 (O-H),2926 (C-H),2846 (C-H),855 cm
(PF6À); ESI-MS (positive
IOS Press,Amsterdam,Oxford,Tokyo,
2000; c) S. Nilsson,J.-Š
mode): m/z: 1290 [M+] [Ir
N
N
Gustafsson, Crit. Rev. Biochem. Mol. Biol. 2002, 37,1–28.
[3] J. A. Schwartz,D. F. Skafar, Biochemistry 1994, 33,13267—13273.
[4] P. Liang,B. Adhyaru,W. L. Pearson,K. R. Williams, J. Chem. Educ.
2006, 83,294–295.
[5] L. G. Luyt,H. M. Bigott,M. J. Welch,J. A. Katzenellenbogen,
Bioorg. Med. Chem. 2003, 11,4977–4989.
for IrC74H58N4O2S2PF6·1.5H2O: C 60.73,H 4.20,N 3.83; found: C 60.55,
H 4.28,N 4.00.
[Ir
A
G
1
crystals in 46% yield. H NMR (300 MHz,[D ]acetone,298 K,TMS): d=
6
9.17 (s,1H; H3 pyridyl ring bpy-C6-est),8.82 (s,1H; H3 ’ pyridyl ring
bpy-C6-est),8.53 (d, J=5.6 Hz,1H; H6 pyridyl ring bpy-C6-est),8.38 (t,
J=5.4 Hz,1H; bpy-4-CON H),8.26–8.19 (m,3H; H4 benzothiazole ring
bsbÀ,H6 ’ pyridyl ring bpy-C6-est),8.13 (d, J=4.7 Hz,1H; H5 pyridyl
ring bpy-C6-est),8.07 (dd, J=6.7,1.2 Hz,2H; H5 biphenyl ring bsb À),
[6] C. Cassino,E. Gabano,M. Ravera,G. Cravotto,G. Palmisano,A.
Vessi›res,G. Jaouen,S. Mundwiler,R. Alberto,D. Osella,
Inorg.
Chim. Acta 2004, 357,2157–2166.
[7] Y. J. Lee,A. C. Notides,Y.-G. Tsay,A. S. Kende, Biochemistry 1977,
16,2896–2901.
8.02 (s,1H; 3-OH estradiol),7.93 (t,
J=5.5 Hz,1H; bpy-4-
[8] K. Ohno,T. Fukushima,T. Santa,N. Waizumi,H. Tokuyama,M.
Maeda,K. Imai, Anal. Chem. 2002, 74,4391–4396.
[9] K. Kuningas,T. Ukonaho,H. Pakkila,T. Rantanen,J. Rosenberg,T.
Lovgren,T. Soukka, Anal. Chem. 2006, 78,4690–4696.
CONHC6H12NH),7.83 (d, J=8.2 Hz,2H; H2,H6 phenyl ring bpy-C6-
est),7.67 (d, J=5.4 Hz,1H; H5 ’ pyridyl ring bpy-C6-est),7.49–7.42 (m,
6H; H2’,H6 ’ biphenyl ring bsbÀ,H3,H5 phenyl ring bpy-C6-est),7.27–
7.18 (m,12H; H5,H6 benzothiazole ring bsb À,H3 ’,H4 ’,H5 ’,H6 biphen-
yl rings bsbÀ),7.12 (d, J=8.2 Hz,1H; H1 estradiol),6.67 (dd, J=8.5,
1.8 Hz,2H; H2 biphenyl ring bsb À),6.60 (dd, J=8.2,2.3 Hz,1H; H2 es-
tradiol),6.55 (d, J=2.3 Hz,1H; H4 estradiol),6.49 (d, J=8.2 Hz,1H;
H7 benzothiazole ring bsbÀ),6.43 (d, J=8.5 Hz,1H; H7 benzothiazole
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7120
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