Inorganica Chimica Acta 360 (2007) 293302

www.elsevier.com/locate/ica

Luminescent ruthenium(II) amidodipyridoquinoxaline biotin

complexes that display higher avidin-induced emission enhancement
Kenneth Kam-Wing Lo *, Terence Kwok-Ming Lee
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, PR China

Received 20 June 2006; accepted 13 July 2006

Available online 27 July 2006

Inorganic Chemistry The Next Generation.

Abstract

A series of luminescent ruthenium(II) amidodipyridoquinoxaline biotin (dpq-B) complexes [Ru(NN)2(NN 0 )](PF6)2 (NN = 2,2 0 -
bipyridine (bpy), 1,10-phenanthroline (phen), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen); NN 0 = 2-((2-biotinamido)ethyl)amidodi-
pyrido[3,2-f:2 0 ,3 0 -h]quinoxaline (dpq-C2-B), 2-((6-biotinamido)hexyl)amidodipyrido[3,2-f:2 0 ,3 0 -h]quinoxaline (dpq-C6-B)) has been
designed as new luminescent probes for avidin. The electrochemical and photophysical properties of these complexes have been inves-
tigated. Upon irradiation, all the complexes exhibited metal-to-ligand charge-transfer (3MLCT) (dp(Ru) ! p*(diimine)) emission in uid
solutions at 298 K and in low-temperature glass. In aqueous buer, the emission was extremely weak, probably a consequence of hydro-
gen-bonding interactions between the amide moiety of the dpq-B ligands and the water molecules. The avidin-binding properties of all
the complexes have been studied by 4 0 -hydroxyazobenzene-2-carboxylic acid (HABA) assays, luminescence titrations, kinetics experi-
ments and confocal microscopy using avidin-conjugated microspheres.
 2006 Elsevier B.V. All rights reserved.

Keywords: Avidin; Biotin; Luminescence; Probes; Ruthenium complexes

1. Introduction or biotin for various detection purposes [4]. We have pre-

The avidinbiotin system is one of the most widely
exploited tools for biological analysis, purication and
recognition [1]. The remarkable advantages are the
extraordinarily high binding anity (Kd = ca. 1015 M)
of biotin to avidin and the four biotin-binding sites of
the biological host that can increase detection sensitivity
[1a,2]. The avidinbiotin adduct is stable under strong
chemically denaturing conditions over a wide range of
temperature and pH [3]. Also, modication of biomole-
cules with biotin seldom aects the biological activities
of both the biotin and the molecules. For these reasons,
a number of reporters have been conjugated to avidin
*
Corresponding author. Tel.: +852 2788 7231; fax: +852 2788 7406.
E-mail address: bhkenlo@cityu.edu.hk (Kenneth K.-W. Lo).
viously reported that luminescent transition metal biotin
conjugates can function as eective probes because of
their characteristic photophysical properties, in particular,
large Stokes shifts and environment-dependent emission
behaviour [5]. Emission enhancement and lifetime elonga-
tion have been observed when these complexes bind to
avidin. Our current target is to search for more ecient
and sensitive probes that display higher avidin-induced
emission enhancement.
Luminescent ruthenium(II) polypyridine complexes
have been extensively utilised as biomolecular sensors
[6] because they are easily prepared, possess rich photo-
redox properties and emit in the visible region with a rela-
tively long lifetime under ambient conditions [7]. However,
the emission properties of these complexes are less sensi-
tive to the change in the hydrophobicity of their local

0020-1693/$ - see front matter  2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2006.07.040
294 Kenneth K.-W. Lo, Terence K.-M. Lee / Inorganica Chimica Acta 360 (2007) 293302

surroundings compared to other d6 metal complexes [8]; for 2. Results and discussion
example, we have previously synthesised two ruthenium(II)
polypyridine biotin complexes that exhibit emission 2.1. Synthesis
enhancement factors of only 1.2 and 1.4 upon binding to
avidin [5d]. To increase the environment-sensitive lumines- The ligand dpq-C2-B was synthesised according to a
cence properties, specially designed polypyridine ligands reported procedure [5h]. The ligand dpq-C6-B with a
are required. Kelly and co-workers have recently reported longer spacer-arm between the dpq and biotin units was
a ruthenium(II) amidodipyrido[3,2-f:2 0 ,3 0 -h]quinoxaline prepared from the reaction of 2-((6-amino)hexyl)amidodi-
(dpqa) complex that is non-emissive in aqueous solution pyrido[3,2-f:2 0 ,3 0 -h]quinoxaline (dpq-C6-NH2) with bioti-
but emits strongly in non-aqueous solutions or after bind- nyl-N-hydroxysuccinimidyl ester. All the ruthenium(II)
ing to double-stranded DNA molecules [9]. On the basis of dpq-B complexes were obtained from the reactions of cis-
this study, we have recently demonstrated that rhenium(I) [Ru(NN)2Cl2] 2H2O [11] with the corresponding dpq-B
[5g] and iridium(III) [5h] amidodipyrido[3,2-f:2 0 ,3 0 -h]qui- ligands in reuxing aqueous ethanol, followed by metathe-
noxaline biotin (dpq-B) complexes show much larger emis- sis with KPF6 and recrystallisation from acetone/diethyl
sion enhancement and lifetime extension after binding to ether. The complexes were characterised by 1H NMR spec-
avidin. It is anticipated that related ruthenium(II) dpq-B troscopy, positive-ion ESI-MS, IR spectroscopy, and gave
complexes will behave in a similar manner and can serve satisfactory microanalysis.
as sensitive luminescent probes for avidin. Additionally,
it is interesting to investigate the eects of ancillary diimine 2.2. Electrochemical properties
ligands on the photophysical properties of the complexes in
dierent environments because electron localisation in the The electrochemical properties of all the ruthenium(II)
excited state of mixed-ligand ruthenium(II) polypyridine complexes have been studied by cyclic voltammetry. The
complexes has been observed [10]. electrochemical data are listed in Table 1. The complexes
We report herein the synthesis, characterisation, electro- displayed a reversible/quasi-reversible ruthenium(III/II)
chemical and photophysical properties of a series of oxidation couple at ca. +1.26 to +1.32 V versus SCE.
luminescent ruthenium(II) dpq-B complexes [Ru(NN)2- The rst reversible reduction couples at ca. 1.1 V of all
(NN 0 )](PF6)2 (NN = 2,2 0 -bipyridine (bpy), NN 0 = 2- the complexes are assigned to the reduction of the dpq-B
((2-biotinamido)ethyl)amidodipyrido[3,2-f:2 0 ,3 0 -h]quinox- ligands because similar couples have been observed in
aline (dpq-C2-B) (1a), 2-((6-biotinamido)hexyl)amidodi- related dpqa and dpq systems [5g,5h,12]. It is possible that
pyrido[3,2-f:2 0 ,3 0 -h]quinoxaline (dpq-C6-B) (1b); NN = the quinoxaline moiety of the ligands plays a key role in
1,10-phenanthroline (phen), NN 0 = dpq-C2-B (2a), this reduction owing to the resemblance of the LUMOs
dpq-C6-B (2b); NN = 4,7-diphenyl-1,10-phenanthroline of quinoxaline and dpq (and its derivatives) [12]. The sec-
(Ph2-phen), NN 0 = dpq-C2-B (3a), dpq-C6-B (3b)) (Chart ond and third reduction couples and waves are ascribed
1). The avidin-binding properties of these complexes have to the reduction of the ancillary diimine ligands.
been studied by 4 0 -hydroxyazobenzene-2-carboxylic acid
(HABA) assays, luminescence titrations, kinetics experi- 2.3. Electronic absorption spectroscopy
ments and confocal microscopy using avidin-conjugated
microspheres. The electronic absorption spectral data of all the com-
plexes in CH3CN are listed in Table 2. The electronic
absorption spectra of complexes 1a, 2a and 3a are shown
O in Fig. 1. All the spectra featured intense absorption bands
b HN NH at ca. 258286 nm (e on the order of 104 dm3 mol1 cm1)
N a c O H H which are assigned to spin-allowed intraligand (1IL)
N N N NH
NH S
Ru n
d O
N N N
N g e Table 1
f
(PF 6)2 Electrochemical data of the ruthenium(II) complexesa
Complex Oxidation, E1/2 (V) Reduction, E1/2 or Ec (V)
1a +1.32 1.09, 1.39, 1.65,b 1.87b
1b +1.31 1.12, 1.38, 1.67,b 1.93b
N N N N 2a +1.31 1.11, 1.37,b 1.67,b 1.93b
= 2b +1.32 1.11, 1.36,b 1.67,b 1.94b
N N N N 3a +1.26 1.11, 1.33,b 1.58,c 1.70,c 1.90c
3b +1.28 1.12, 1.29,b 1.56,c 1.73,c 1.91c
a
In CH3CN (0.1 M nBu4NPF6) at 298 K, glassy carbon electrode, sweep
n = 1 (1a), 3 (1b) n = 1 (2a), 3 (2b) n = 1 (3a), 3 (3b) rate = 0.1 V s1, all potentials vs. SCE.
b
Quasi-reversible couple.
c
Chart 1. Structures of complexes. Irreversible wave.
 Kenneth K.-W. Lo, Terence K.-M. Lee / Inorganica Chimica Acta 360 (2007) 293302 295

Table 2
Electronic absorption spectral data of the ruthenium(II) complexes in CH3CN at 298 K
Complex kabs/nm (e/dm3 mol1 cm1)
1a 258 (53 380), 286 (63 865), 328 sh (12 610), 421 sh (12 370), 449 (14 160)
1b 258 (59 985), 286 (67 650), 328 sh (13 980), 420 sh (13 085), 448 (15 040)
2a 222 (69 320), 262 (109 755), 301 sh (29 310), 328 sh (8955), 418 sh (15 780), 444 (16 710)
2b 223 (72 515), 262 (115 750), 300 sh (31 910), 327 sh (10 520), 419 sh (16 650), 444 (17 600)
3a 222 sh (70 250), 263 sh (81 270), 276 (95 760), 303 sh (43 505), 432 (22 005), 458 (22 300)
3b 223 sh (79 860), 261 sh (90 920), 276 (108 280), 305 sh (48 280), 434 (24 600), 458 (24 980)

12 sion maxima of all the complexes occurred at ca. 611

616 nm in CH3CN and at ca. 617620 nm in buer solu-
10 tions at 298 K, and at ca. 570590 nm in alcohol glass at
1

77 K. The emission is attributed to a 3MLCT

e x 10- / dm mol- cm-

8 (dp(Ru) ! p*(diimine)) excited state. While the emission

1

energies of all the complexes are very similar, the Ph2-phen

complexes 3a and 3b displayed more intense and longer-
3

6
lived emission than the other complexes (Table 3). In addi-
4

4 tion, the photophysical properties of all the complexes are

comparable to those of their corresponding homoleptic
2 counterparts [Ru(bpy)3]2+ [13,17], [Ru(phen)3]2+ [18] and
[Ru(Ph2-phen)3]2+ [15] in CH3CN. All these results suggest
0 the possible involvement of the ancillary diimine ligands in
200 250 300 350 400 450 500 550 600 the 3MLCT emissive states of the current complexes.
Wavelength / nm It is important to note that the emission quantum yields
Fig. 1. Electronic absorption spectra of complexes 1a (), 2a (- - -) and of the ruthenium(II) complexes decreased substantially
3a (  ) in CH3CN at 298 K. from CH3CN to aqueous buer (Table 3). These quantum
yield values are much lower than that of the related com-
plex [Ru(bpy)2(dpq)]2+ in buer, arguing that the reduced
(p ! p*)(diimine) transitions. In particular, the intense brightness of the emission is a result of the interactions
absorption peaks at ca. 286 nm of complexes 1a and 1b between the amide moiety of the dpq-B ligands and the
are assigned to 1IL (p ! p*)(bpy) transitions, which are
commonly observed in [Ru(bpy)3]2+ and its derivatives
[13]. The absorption bands in the visible region (ca. 420 Table 3
Photophysical data of the ruthenium(II) complexes
458 nm) are associated with spin-allowed metal-to-ligand
charge-transfer (1MLCT) transitions (dp(Ru) ! p*(dii- Complex Medium (T/K) kem (nm)a so (ls)b Uema
mine)). The 1MLCT band for complexes 3a and 3b 1a CH3CN (298) 616 1.05 0.058
occurred at slightly longer wavelengths than complexes Buerc (298) 620 0.79 0.0009
Glassd (77) 582 (max), 629, 683 sh 5.83
1a, 1b, 2b and 2b owing to the lower-lying p* orbitals of
the Ph2-phen ligand. Interestingly, all these complexes did 1b CH3CN (298) 616 1.12 0.057
not show a remarkably low-energy 1MLCT absorption Buerc (298) 619 0.73 0.0010
Glassd (77) 583 (max), 626, 686 sh 5.98
band associated with the dpq-B ligands despite their
extended p-conjugation, suggestive of the bichromophoric 2a CH3CN (298) 612 0.85 0.067
Buerc (298) 617 0.83 0.0006
(i.e. bpy and quinoxaline-amide) character of these
Glassd (77) 574 (max), 618, 677 sh 7.65
ligands [12]. In fact, the 1IL and 1MLCT absorption bands
of all the complexes are similar to those of the correspond- 2b CH3CN (298) 611 1.09 0.073
Buerc (298) 615 0.89 0.0010
ing homoleptic complexes [Ru(bpy)3]2+ [13], [Ru(phen)3]2+ Glassd (77) 570 (max), 615, 677 sh 7.73
[14] and [Ru(Ph2-phen)3]2+ [15]. Similar observations have
3a CH3CN (298) 613 2.56 0.12
been reported for related ruthenium(II) dipyrido[3,2-
Buerc (298) 617 1.55 0.0009
a:2 0 ,3 0 -c]phenazine (dppz) systems [16]. Glassd (77) 589 (max), 634, 690 sh 7.98
3b CH3CN (298) 613 2.57 0.11
2.4. Luminescence properties Buerc (298) 617 1.62 0.0013
Glassd (77) 590 (max), 641, 692 sh 8.38
All the ruthenium(II) dpq-B complexes exhibited a
Excitation wavelength = 455 nm.
orange-red luminescence upon visible-light irradiation in b
Excitation wavelength = 355 nm.
uid solutions at 298 K and in alcohol glass at 77 K. The c
50 mM potassium phosphate buer pH 7.4/DMSO (9:1, v/v).
d
photophysical data are summarised in Table 3. The emis- EtOH/MeOH (4:1, v/v).
296 Kenneth K.-W. Lo, Terence K.-M. Lee / Inorganica Chimica Acta 360 (2007) 293302

water molecules [9]. Interestingly, Meyer and co-workers

reported that the solvent-induced light-switch behaviour
of [Ru(bpy)2(dppz)]2+ originates from a dynamic equilib-

Emission Intensity (A.U.)

rium between a dark and a bright MLCT state [19].
The dark MLCT state localises on the phenazine frag-
ment of dppz ligand, which is lower in energy than the
bpy-based bright MLCT state. A similar model could
account for the dierent emission properties of the current
ruthenium(II) dpq-B complexes in CH3CN and buer.
Although it is possible that the LUMOs of the complexes
involve the quinoxaline moiety of the dpq-B ligands as con-
cluded in related systems [12], the bpy-fragment of these
dpq-B ligands or the ancillary diimine ligands (especially 550 600 650 700 750 800
the Ph2-phen ligands in the cases of complexes 3a and 3b Wavelength / nm
due to their long lifetimes) may contribute to the bright Fig. 2. Emission spectra of complex 3b (2.8 lM) in the absence (- - -) and
MLCT emission in CH3CN. In aqueous solution, it is presence of 0.70 lM () of avidin in potassium phosphate buer at
probable that the dark state associated with the quinox- 298 K.
aline-fragment of the dpq-B ligands is stabilised by hydro-
gen-bonding interactions between their amide moieties and avidin and biotin-blocked avidin are shown in Figs. 35.
water molecules, resulting in ecient non-radiative decay At [avidin]:[Ru] = 0.25, the emission intensities of the
processes and thus very low emission quantum yields of complexes were increased by ca. 3.8- to 8.2-fold (Figs.
the complexes [9].

2.5. HABA assays

a
The assay is based on the competition between biotin 5
and HABA on binding to avidin [1a]. The binding of
HABA to avidin is associated with an absorption feature 4
at ca. 500 nm. Since the binding of HABA to avidin
(Kd = 6 106 M) is much weaker than that of biotin
I / Io

3
(Kd = ca. 1015 M), addition of biotin will replace the
bound HABA molecules from the protein, leading to a
decrease of the absorbance at 500 nm [1a]. Addition of 2
the current ruthenium(II) dpq-B complexes to a mixture
of avidin and HABA resulted in the decrease of absor-
1
bance at 500 nm. This indicates that the complexes bind
specically to the substrate-binding sites of avidin. The 0.0 0.1 0.2 0.3 0.4 0.5
plots of DA500 nm versus [Ru]:[avidin] for complexes [Avidin] : [1a]
1a, 1b, 2a and 2b showed that the equivalence points
occurred at [Ru]:[avidin] = ca. 4.24.7. Assuming the
b
binding stoichiometry is 4:1, the occurrence of an equiv-
4
alence point >4.0 reveals that the binding of the com-
plexes to avidin is not signicantly stronger than
HABA. Unfortunately, precipitation occurred before the
equivalence points were reached for complexes 3a and 3
I / Io

3b due to the highly hydrophobic nature of the Ph2-phen

ligand.
2
2.6. Luminescence titrations

Similar to other luminescent transition metal polypyri- 1

dine biotin complexes [5], addition of avidin to the current
ruthenium(II) dpq-B complexes in aqueous buer resulted 0.0 0.1 0.2 0.3 0.4 0.5
in emission enhancement. As an example, the emission [Avidin] : [1b]
spectra of complex 3b in the absence and presence of avi- Fig. 3. Luminescence titration curves for the titrations of (a) complex 1a
din in aqueous buer are shown in Fig. 2. Luminescence (2.8 lM) and (b) complex 1b (2.8 lM), respectively, with avidin (d) and
titration curves for the titrations of the complexes with biotin-blocked avidin (m).
 Kenneth K.-W. Lo, Terence K.-M. Lee / Inorganica Chimica Acta 360 (2007) 293302 297

a a 7
6
6
5
5
4
4
I / Io

I / Io
3
3
2
2

1 1

0 0
0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5
[Avidin] : [2a] [Avidin] : [3a]

b b
6 10

5 8

4
I / Io

6
I / Io
3
4
2
2
1
0
0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5
[Avidin] : [2b] [Avidin] : [3b]
Fig. 4. Luminescence titration curves for the titrations of (a) complex 2a Fig. 5. Luminescence titration curves for the titrations of (a) complex 3a
(2.8 lM) and (b) complex 2b (2.8 lM), respectively, with avidin (d) and (2.8 lM) and (b) complex 3b (2.8 lM), respectively, with avidin (d) and
biotin-blocked avidin (m). biotin-blocked avidin (m).

35 and Table 4). The enhancement is a result of the spe- Table 4

cic binding of the complexes to avidin because similar Relative emission intensities and excited-state lifetimes of the ruthe-
enhancement was not observed when biotin-blocked avi- nium(II) complexes in the absence and presence of avidin (and excess
biotin)a
din was used (Figs. 35 and Table 4). The excited-state
lifetimes of the complexes were also elongated upon the Complex I (s/ns)b I (s/ns)c I (s/ns)d
avidin-binding event (Table 4). The degree of emission 1a 1.00 (263) 4.57 (561) 1.14 (270)
intensity enhancement depends strongly on the diimine 1b 1.00 (298) 3.83 (848) 1.11 (324)
2a 1.00 (290) 5.26 (1017) 1.09 (286)
ligands and follows the order: Ph2-phen > phen > bpy 2b 1.00 (355) 5.89 (999) 1.05 (342)
(Table 4), which is in line with our previous ndings that 3a 1.00 (606) 5.94 (1149) 1.13 (607)
more hydrophobic transition metal biotin complexes exhi- 3b 1.00 (464) 8.24 (1264) 1.51 (484)
bit more signicant avidin-induced emission amplication a
In aerated 50 mM potassium phosphate buer pH 7.4/DMSO (9:1, v/v),
[5]. Most importantly, the avidin-induced emission [Ru] = 2.8 lM.
b
enhancement factors (ca. 3.88.2) of the current ruthe- [avidin] = 0 M, [biotin] = 0 M.
c
nium(II) dpq-B complexes are much larger than those of [avidin] = 0.70 lM, [biotin] = 0 M.
d
[avidin] = 0.70 lM, [biotin] = 70 lM.
two related ruthenium(II) bipyridinebiotin complexes
(1.2- and 1.4-fold) that we reported recently [5d]. The rea-
son is that in the absence of avidin, the current ruthe- 2.7. Avidin-binding anities
nium(II) dpq-B complexes showed extremely weak
emission in aqueous buer. Similar observations have The rst dissociation constants Kd of the ruthenium(II)
been reported in rhenium(I) [5g] and iridium(III) dpq-B avidin adducts have been estimated from the on-rates (kon)
systems [5h]. and o-rates (ko) of the ruthenium(II)avidin adducts
298 Kenneth K.-W. Lo, Terence K.-M. Lee / Inorganica Chimica Acta 360 (2007) 293302

Table 5 which is similar in energy to the 488-nm excitation of an

On- and o-rate constants for the ruthenium(II) complexes and rst argon laser, rendering them promising candidates as
dissociation constants for the ruthenium(II)avidin adductsa
probes for immunoassays and in vitro analysis using
Complex kon (M1 min1) ko (min1) Kd (M) laser-scanning confocal microscopy. To investigate these
1a 2.6 106 2.0 101 7.7 108 possibilities, we have preliminarily examined the binding
1b 6.5 106 4.0 102 6.2 109 of complex 1a to avidin molecules immobilised on micro-
2a 2.9 106 2.3 101 7.9 108
sphere particles. Specically, a solution of complex 1a
2b 6.0 106 4.2 102 6.4 109
3a 2.1 106 2.0 101 9.5 108 was added to avidin-coated microspheres in the absence
3b 5.3 106 5.0 102 9.4 109 and presence of excess biotin, respectively, and the samples
a
50 mM potassium phosphate buer pH 7.4/DMSO (9:1, v/v). were visualised with a confocal microscope. Bright eld,
uorescent and overlaid images of these two sample solu-
tions are shown in Fig. 6ac and df, respectively. Without
from kinetics experiments [4c,5eh]. The Kd values deter- excess biotin, the avidin-modied microspheres revealed
mined ranged from ca. 6.2 109 to 9.5 108 M (Table intense luminescence upon excitation owing to avidin-
5), which are about six to seven orders of magnitude larger bound complex 1a on the surface of the particles (Fig. 6b
than that of the native biotinavidin system (Kd = ca. and c). However, when excess biotin was initially present,
1015 M) [1a] and one to three orders of magnitude larger addition of complex 1a to the microspheres only resulted
than those of our previous ruthenium(II) bipyridinebiotin in a very small number of weakly luminescent particles
complexes [5d]. It is likely that the binding of the current (Fig. 6e and f), indicating the lack of substantial binding
complexes to avidin is hindered by the extended planar of the complexes to the immobilised protein. These results
moieties of the dpq-B ligands. On the contrary, the ancil- demonstrate that the current ruthenium(II) dpq-B com-
lary diimine ligands did not have signicant eects on the plexes can act as luminescent probes for immobilised avidin
protein-binding anities. The Kd values of the dpq-C6-B and can be employed in the development of new heteroge-
complexes are one order of magnitude smaller than those neous bioassays.
of the dpq-C2-B complexes as a consequence of their smal-
ler ko values (Table 5). This clearly illustrates the impor- 3. Conclusions
tance of the spacer-arms on the stability of the
rutheniumavidin adducts. We have designed a series of luminescent ruthenium(II)
amidodipyridoquinoxaline biotin complexes that contain
2.8. Confocal microscopy studies dierent spacer-arms and ancillary ligands. The electro-
chemical and photophysical properties of these complexes
The current ruthenium(II) dpq-B complexes showed sig- have been investigated. The avidin-binding properties have
nicant broad 1MLCT absorption bands at ca. 450 nm, been studied by HABA assays, luminescence titrations and

Fig. 6. Bright eld (a), uorescent (b) and overlaid (c) images of a buer solution containing avidin-modied microspheres and complex 1a in the absence
of biotin. (d)(f) Conditions are the same as those of (a)(c), respectively, except that the avidin molecules used were blocked by excess biotin.
 Kenneth K.-W. Lo, Terence K.-M. Lee / Inorganica Chimica Acta 360 (2007) 293302
kinetics experiments. The most important feature of these
systems is that they displayed extremely weak emission in
aqueous solution under ambient conditions but showed
large emission enhancement and excited-state lifetime
extension upon binding to avidin. The confocal microscopy
studies revealed that they can function as probes for avidin
immobilised on microspheres. We believe that these results
can lead to the development of more sensitive luminescent
probes for avidin.
4. Experimental
4.1. Materials and reagents
All solvents were of analytical reagent grade. Ruthe-
nium(III) chloride hydrate (Arcos), bpy (BDH), phen
(Acros), Ph2-phen (Aldrich), 1,2-diaminopropane (IL), bio-
tin (Acros), N-hydroxysuccinimide (NHS) (Acros), N,N 0 -
dicyclohexylcarbodiimide (Acros), 1,6-hexanediamine
(Acros), HABA (Sigma), 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide hydrochloride (EDC) (Acros) and avidin
(Calbiochem) were used as received. Carboxyl-functional-
ised microspheres (diameter: 10.14 lm, concentration:
1.09 g cm3) were purchased from Bangs Laboratories. Eth-
ylenediamine (Acros) was distilled over KOH before use.
cis-[Ru(NN)2Cl2] 2H2O [11], 2-(methoxycarbonyl)dipyr-
ido[3,2-f:2 0 ,3 0 -h]quinoxaline [12], biotinyl-N-hydroxysucc-
inimidyl ester [20] and dpq-C2-B [5h] were prepared by
reported methods.
4.2. Physical measurements and instrumentation
Equipment for characterisation, electrochemical and
photophysical studies has been described previously [5d].
Luminescence quantum yields were measured using the
optically dilute method [21] with an aerated aqueous solu-
tion of [Ru(bpy)3]Cl2 (Uem = 0.028, kex = 455 nm) [22] as
the standard solution.
4.3. Synthesis of 2-((6-amino)hexyl)amidodipyrido[3,2-
f:2 0 ,3 0 -h]quinoxaline (dpq-C6-NH2)
A mixture of 2-(methoxycarbonyl)dipyrido[3,2-f:2 0 ,3 0 -
h]quinoxaline (0.53 g, 1.83 mmol) and 1,6-hexanediamine
(21.2 g, 183 mmol) in MeOH (150 mL) was stirred at room
temperature for 48 h. The yellow solution was then evapo-
rated to dryness under vacuum. The residual yellow oil was
mixed with 50 mL of CH2Cl2 and the solution was then
washed with water (3 200 mL). The organic layer was col-
lected, dried over MgSO4, and then evaporated to dryness
to give dpq-C6-NH2 s a yellow solid. Yield: 0.31 g (45%).
Positive-ion ESI-MS ion clusters at m/z 375 {M+H+}+.
4.4. Synthesis of dpq-C6-B
A mixture of dpq-C6-NH2 (0.31 g, 0.83 mmol) and bio-
tinyl-N-hydroxysuccinimidyl ester (0.31 g, 0.91 mmol) in
299
DMF (30 mL) was stirred at room temperature for 12 h.
The yellow solution was then evaporated to dryness under
vacuum. The crude product was washed with MeOH and
diethyl ether to give dpq-C6-B as a pale brown solid. Yield:
0.23 g (47%). 1H NMR (300 MHz, DMSO-d6, 298 K,
TMS): d 9.909.76 (dd, 1H, J = 8.6, 1.4 Hz, Hc), 9.56 (s,
1H, Hd), 9.50 (br, 1H, dpq-CONH), 9.449.33 (dd, 1H,
J = 8.5, 1.4 Hz, He), 9.269.18 (m, 2H, Ha and Hg), 8.02
7.86 (m, 2H, Hb and Hf), 7.77 (br, 1H, NH-biotin), 6.48
6.19 (m, 2H, NH of biotin), 4.314.20 (m, 1H, NCH of bio-
tin), 4.144.01 (m, 1H, NCH of biotin), 3.483.37 (m, 2H,
dpq-CONHCH2), 3.182.91 (m, 3H, COC4H8CHS of bio-
tin and CH2NH-biotin), 2.872.69 (m, 1H, SCH of biotin),
2.53 (d, 1H, Jgem = 11.2 Hz, SCH of biotin), 2.111.92 (m,
2H, COCH2C3H6 of biotin), 1.711.13 (m, 14H,
COCH2C3H6 of biotin and dpq-CONHCH2C4H8); IR
(KBr) (m/cm1): 3319 (br, NH), 1701 (s, C@O); positive-
ion ESI-MS ion clusters at m/z 602 {M+H+}+.
4.5. Synthesis of [Ru(bpy)2(dpq-C2-B)](PF6)2 (1a)
A mixture of cis-[Ru(bpy)2Cl2] 2H2O (57 mg,
0.11 mmol) and dpq-C2-B (71 mg, 0.13 mmol) in 20 mL
of 50% aqueous ethanol was heated at reux for 12 h.
The colour of the solution turned from purple to deep
red. The volume of the mixture was reduced to ca. 10 mL
and the solution was then ltered. Excess KPF6 was added
to the solution to precipitate a deep red solid. The solid was
collected by ltration, washed with water and a small
amount of cold MeOH, and diethyl ether, and then recrys-
tallised from acetone/diethyl ether to give complex 1a as
red crystals. Yield: 73 mg (53%). 1H NMR (300 MHz, ace-
tone-d6, 298 K, TMS): d 10.24 (d, 1H, J = 7.6 Hz, Hc of
dpq-C2-B), 9.77 (s, 1H, Hd of dpq-C2-B), 9.719.61 (m,
2H, dpq-CONH and He of dpq-C2-B), 8.928.79 (m, 4H,
H3 and H3 0 of bpy), 8.658.53 (m, 2H, Ha and Hg of
dpq-C2-B), 8.27 (t, 2H, J = 7.9 Hz, Hb and Hf of dpq-
C2-B), 8.228.00 (m, 8H, H4, H4 0 , H6 and H6 0 of bpy),
7.72 (br, 1H, NH-biotin), 7.65 (t, 2H, J = 6.6 Hz, H5 of
bpy), 7.467.34 (m, 2H, H5 of bpy), 5.685.49 (m, 2H,
NH of biotin), 4.394.23 (m, 1H, NCH of biotin), 4.15
4.05 (m, 1H, NCH of biotin), 3.783.49 (m, 4H, dpq-
CONHC2H4), 2.992.88 (m, 1H, COC4H8CHS of biotin),
2.732.65 (m, 1H, SCH of biotin), 2.512.42 (m, 1H,
SCH of biotin), 2.362.22 (m, 2H, COCH2C3H6 of biotin),
1.721.22 (m, 6H, COCH2C3H6 of biotin); IR (KBr) (m/
cm1): 3437 (br, NH), 1696 (s, C@O), 846 (s, PF); posi-
tive-ion ESI-MS ion clusters at m/z 1103 {MPF6  }+, 479
{M2PF6  }2+. Anal. Calc. for RuC47H44N12O3SP2-
F12 (CH3)2CO 2H2O: C, 44.75; H, 4.06; N, 12.52. Found:
C, 44.83; H, 4.25; N, 12.80%.
4.6. Synthesis of [Ru(bpy)2(dpq-C6-B)](PF6)2 (1b)
The procedure was similar to that of complex 1a, except
that dpq-C6-B (78 mg, 0.13 mmol) was used instead of
dpq-C2-B. The crude product was recrystallised from
300 Kenneth K.-W. Lo, Terence K.-M. Lee / Inorganica Chimica Acta 360 (2007) 293302
acetone/diethyl ether to give complex 1b as red crystals.
Yield: 88 mg (61%). 1H NMR (300 MHz, acetone-d6,
298 K, TMS): d 9.97 (d, 1H, J = 8.2 Hz, Hc of dpq-C6-
B), 9.80 (s, 1H, Hd of dpq-C6-B), 9.66 (d, 1H,
J = 8.5 Hz, He of dpq-C6-B), 9.24 (t, 1H, J = 6.1 Hz,
dpq-CONH), 8.898.81 (m, 4H, H3 and H3 0 of bpy),
8.61 (d, 1H, J = 5.3 Hz, Ha of dpq-C6-B), 8.58 (d, 1H,
J = 5.3 Hz, Hg of dpq-C6-B), 8.27 (t, 2H, J = 7.9 Hz, Hb
and Hf of dpq-C6-B), 8.218.00 (m, 8H, H4, H4 0 , H6
and H6 0 of bpy), 7.65 (t, 2H, J = 6.6 Hz, H5 of bpy),
7.437.37 (m, 2H, H5 of bpy), 7.12 (t, 1H, J = 4.8 Hz,
NH-biotin), 5.89 (br, 1H, NH of biotin), 5.68 (br, 1H,
NH of biotin), 4.494.40 (m, 1H, NCH of biotin), 4.35
4.26 (m, 1H, NCH of biotin), 3.603.47 (m, 2H, dpq-
CONHCH2), 3.233.12 (m, 3H, COC4H8CHS of biotin
and CH2NH-biotin), 2.852.73 (m, 1H, SCH of biotin),
2.65 (d, 1H, Jgem = 12.3 Hz, SCH of biotin), 2.362.22
(m, 2H, COCH2C3H6 of biotin), 1.781.29 (m, 14H,
COCH2C3H6 of biotin and dpq-CONHCH2C4H8); IR
(KBr) (m/cm1): 3431 (br, NH), 1692 (s, C@O), 851 (s,
PF); positive-ion ESI-MS ion clusters at m/z 1159
{MPF6  }+, 507 {M2PF6  }2+. Anal. Calc. for
RuC51H52N12O3SP2F12 H2O: C, 46.33; H, 4.12; N,
12.71. Found: C, 46.52; H, 4.28; N, 12.64%.
4.7. Synthesis of [Ru(phen)2(dpq-C2-B)](PF6)2 (2a)
The procedure was similar to that of complex 1a, except
that cis-[Ru(phen)2Cl2] (58 mg, 0.11 mmol) was used
instead of cis-[Ru(bpy)2Cl2] 2H2O. The crude product
was recrystallised from acetone/diethyl ether to give com-
plex 2a as deep red crystals. Yield: 64 mg (45%). 1H
NMR (300 MHz, DMSO-d6, 298 K, TMS): d 10.04 (d,
1H, J = 7.6 Hz, Hc of dpq-C2-B), 9.80 (s, 1H, Hd of dpq-
C2-B), 9.74 (m, 1H, dpq-CONH), 9.51 (d, 1H,
J = 7.0 Hz, He of dpq-C6-B), 8.848.71 (m, 4H, H4 and
H7 of phen), 8.39 (s, 4H, H5 and H6 of phen), 8.268.12
(m, 5H, NH-biotin and H2 and H9 of phen), 8.088.02
(m, 2H, Ha and Hg of dpq-C2-B), 7.987.84 (m, 2H, Hb
and Hf of dpq-C2-B), 7.827.71 (m, 4H, H3 and H8 of
phen), 6.386.27 (m, 2H, NH of biotin), 4.224.10 (m,
1H, NCH of biotin), 4.043.96 (m, 1H, NCH of biotin),
3.563.38 (m, 4H, dpq-CONHC2H4), 2.982.87 (m, 1H,
COC4H8CHS of biotin), 2.692.58 (m, 1H, SCH of biotin),
2.482.35 (m, 1H, SCH of biotin), 2.172.04 (m, 2H,
COCH2C3H6 of biotin), 1.631.13 (m, 6H, COCH2C3H6
of biotin); IR (KBr) (m/cm1): 3477 (br, NH), 1653 (s,
C@O), 839 (s, PF); positive-ion ESI-MS ion clusters at
m/z 1151 {MPF6  }+, 503 {M2PF6  }2+. Anal. Calc.
for RuC51H44N12O3SP2F12 2H2O: C, 45.99; H, 3.63; N,
12.62. Found: C, 45.74; H, 3.58; N, 12.83%.
4.8. Synthesis of [Ru(phen)2(dpq-C6-B)](PF6)2 (2b)
The procedure was similar to that of complex 1b, except
that cis-[Ru(phen)2Cl2] (58 mg, 0.11 mmol) was used
instead of cis-[Ru(bpy)2Cl2] 2H2O. The crude product
was recrystallised from acetone/diethyl ether to give com-
plex 2b as red crystals. Yield: 69 mg (47%). 1H NMR
(300 MHz, acetone-d6, 298 K, TMS): d 9.93 (d, 1H,
J = 7.9 Hz, Hc of dpq-C6-B), 9.80 (s, 1H, Hd of dpq-C6-
B), 9.62 (d, 1H, J = 8.2 Hz, He of dpq-C6-B), (t, 1H,
J = 4.9 Hz, dpq-CONH), 8.82 (d, 4H, J = 8.5 Hz, H4 and
H7 of phen), 8.39 (s, 4H, H5 and H6 of phen), 8.618.52
(m, 4H, Ha and Hg of dpq-C6-B and H2 of phen), 8.48
8.38 (m, 6H, H5, H6 and H9 of phen), 8.027.92 (m, 2H,
Hb and Hf of dpq-C6-B), 7.897.78 (m, 4H, H3 and H8
of phen), 7.21 (t, 1H, J = 4.8 Hz, NH-biotin), 5.95 (br,
1H, NH of biotin), 5.72 (br, 1H, NH of biotin), 4.53
4.40 (m, 1H, NCH of biotin), 4.374.28 (m, 1H, NCH of
biotin), 3.593.42 (m, 2H, dpq-CONHCH2), 3.223.11
(m, 3H, COC4H8CHS of biotin and CH2NH-biotin),
2.882.81 (m, 1H, SCH of biotin), 2.62 (d, 1H,
Jgem = 12.1 Hz, SCH of biotin), 2.172.22 (m, 2H,
COCH2C3H6 of biotin), 1.791.31 (m, 14H, COCH2C3H6
of biotin and dpq-CONHCH2C4H8); IR (KBr) (m/cm1):
3437 (br, NH), 1691 (s, C@O), 846 (s, PF); positive-ion
ESI-MS ion clusters at m/z 1207 {MPF6  }+, 531
{M2PF6  }2+. Anal. Calc. for RuC55H52N12O3SP2F12:
C, 48.86; H, 3.88; N, 12.43. Found: C, 48.80; H, 4.24; N,
12.60%.
4.9. Synthesis of [Ru(Ph2-phen)2(dpq-C2-B)](PF6)2 (3a)
The procedure was similar to that of complex 1a, except
that cis-[Ru(Ph2-phen)2Cl2] 2H2O (96 mg, 0.11 mmol) was
used instead of cis-[Ru(bpy)2Cl2] 2H2O. The crude prod-
uct was recrystallised from acetone/diethyl ether to give
complex 3a as red crystals. Yield: 72 mg (41%). 1H NMR
(300 MHz, acetone-d6, 298 K, TMS): d 10.25 (dd, 1H,
J = 11.3 and 8.7 Hz, Hc of dpq-C2-B), 9.74 (s, 1H, Hd of
dpq-C2-B), 9.719.60 (m, 2H, He of dpq-C2-B and dpq-
CONH), 8.818.62 (m, 6H, Ha and Hg of dpq-C2-B and
H2 and H9 of Ph2-phen), 8.35 (s, 4H, H5 and H6 of Ph2-
phen), 8.127.98 (m, 2H, Hb and Hf of dpq-C2-B), 7.88
7.77 (m, 4H, H3 and H8 of Ph2-phen), 7.767.56 (m,
21H, Ph of Ph2-phen and NH-biotin), 5.605.48 (m, 2H,
NH of biotin), 4.354.20 (m, 1H, NCH of biotin), 4.15
4.01 (m, 1H, NCH of biotin), 3.773.48 (m, 4H, dpq-
CONHC2H4), 2.982.89 (m, 1H, COC4H8CHS of biotin),
2.712.60 (m, 1H, SCH of biotin), 2.41 (d, 1H,
Jgem = 14.7 Hz, SCH of biotin), 2.362.21 (m, 2H,
COCH2C3H6 of biotin), 1.741.21 (m, 6H, COCH2C3H6
of biotin); IR (KBr) (m/cm1): 3437 (br, NH), 1670 (s,
C@O), 840 (s, PF); positive-ion ESI-MS ion clusters at
m/z 1455 {MPF6  }+, 655 {M2PF6  }2+. Anal. Calc.
for RuC75H60N12O3SP2F12 2H2O: C, 55.05; H, 3.94; N,
10.27. Found: C, 55.28; H, 4.24; N, 10.54%.
4.10. Synthesis of [Ru(Ph2-phen)2(dpq-C6-B)](PF6)2
(3b)
The procedure was similar to that of complex 1b,
except that cis-[Ru(Ph2-phen)2Cl2] 2H2O (96 mg,
 Kenneth K.-W. Lo, Terence K.-M. Lee / Inorganica Chimica Acta 360 (2007) 293302
0.11 mmol) was used instead of cis-[Ru(bpy)2Cl2] 2H2O.
The crude product was recrystallised from acetone/diethyl
ether to give complex 3b as red crystals. Yield: 84 mg
(46%). 1H NMR (300 MHz, acetone-d6, 298 K, TMS): d
9.98 (d, 1H, J = 8.2 Hz, Hc of dpq-C6-B), 9.80 (s, 1H,
Hd of dpq-C6-B), 9.66 (d, 1H, J = 8.2 Hz, He of dpq-
C6-B), 9.23 (t, 1H, J = 4.9 Hz, dpq-C6-NH), 8.748.61
(m, 6H, Ha and Hg of dpq-C6-B and H2 and H9 of
Ph2-phen), 8.35 (s, 4H, H5 and H6 of Ph2-phen), 8.08
7.97 (m, 2H, Hb and Hf of dpq-C6-B), 7.847.76 (m,
4H, H3 and H8 of Ph2-phen), 7.747.55 (m, 20H, Ph of
Ph2-phen), 7.12 (t, 1H, J = 4.6 Hz, NH-biotin), 5.84 (br,
1H, NH of biotin), 5.61 (br, 1H, NH of biotin), 4.48
4.40 (m, 1H, NCH of biotin), 4.324.25 (m, 1H, NCH
of biotin), 3.593.45 (m, 2H, dpq-CONHCH2), 3.22
3.10 (m, 3H, COC4H8CHS of biotin and CH2NH-biotin),
2.892.79 (m, 1H, SCH of biotin), 2.61 (d, 1H,
Jgem = 12.3 Hz, SCH of biotin), 2.15 (t, 2H, J = 6.9 Hz,
COCH2C3H6 of biotin), 1.771.30 (m, 14H, COCH2C3H6
of biotin and dpq-CONHCH2C4H8); IR (KBr) (m/cm1):
3437 (br, NH), 1696 (s, C@O), 846 (s, PF); positive-
ion ESI-MS ion clusters at m/z 1511 {MPF6  }+, 683
{M2PF6  }2+. Anal. Calc. for RuC79H68N12O3SP2F12:
C, 57.28; H, 4.14; N, 10.15. Found: C, 57.30; H, 4.29;
N, 10.44%.
4.11. HABA assays and determination of Kd
Details of the assays and procedures for determination
of Kd have been described previously [5d,g].
4.12. Luminescence titrations
Aliquots (5 lL) of an avidin solution (25 lM) in 50 mM
potassium phosphate buer at pH 7.4 at 298 K were added
to the ruthenium(II) biotin complex (2.8 lM) in 2 mL of
50 mM potassium phosphate buer at pH 7.4/DMSO
(9:1, v/v) at 1-min intervals. The emission spectrum of
the solution was then measured. The titration results were
compared to control titrations in which an avidin solution
saturated with excess biotin (ca. 2.5 mM) was used as the
titrant.
4.13. Preparation of avidin-coated microspheres
A 50-lL portion of carboxyl-functionalised micro-
spheres (diameter: 10.14 lm, concentration: 1.09 g cm3)
was centrifuged and the solid was washed with deionised
water (1 mL 4). After the microspheres were resuspended
in 200 lL of deionised water, EDC (1 mg) and NHS (1 mg)
were added. The mixture was stirred at room temperature
for 15 min. Then, avidin (4 mg) dissolved in 800 lL of
50 mM carbonate buer at pH 9.3 was added and the sus-
pension was incubated at room temperature for 12 h. The
avidin-coated microspheres were collected by centrifuga-
tion, washed with 50 mM potassium phosphate buer at
pH 7.4 (1 mL 4), and resuspended in 500 lL of the same
301
buer to give a microsphere stock solution. A 20-lL por-
tion of the stock solution was added to complex 1a
(11 lM) in a mixture of 500 lL of phosphate buer/DMSO
(9:1, v/v). The mixture was incubated at room temperature
for 30 min. The microspheres were then collected by centri-
fugation, resuspended in 100 lL of phosphate buer, and a
20-lL portion was loaded onto a glass slide. The sample
was then imaged by a confocal microscope (Carl Zeiss,
LSM510) with an excitation wavelength of 488 nm and
an emission wavelength >560 nm. In the control experi-
ment, the reaction mixture contained excess biotin
(11 mM).
Acknowledgement
We thank the City University of Hong Kong (Project
No. 7001985) for nancial support.
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