Syntheses, characterisation and photophysical studies of novel biological labelling reagents derived from luminescent iridium(III) terpyridine complexes

Article abstract of DOI:10.1039/b107163g

A series of new luminescent iridium(III) terpyridine complexes functionalised with an isothiocyanate group, [Ir(tpy-R)(tpy-C₆H₄-NCS-p)](PF₆)₃ [R = H (1), C₆H₅ (2), C₆H₄-CH₃-p (3), C₆H₄-Cl-p (4)] has been synthesised, characterised and their photophysical properties studied; the X-ray crystal structure of one of the intermediate complexes, [Ir(tpy-C₆H₄-Cl-p)(CF₃SO₃) ₃] (4b), has also been determined; complex 1 has been used as a luminescent label for proteins.

Full text of DOI:10.1039/b107163g

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Syntheses, characterisation and photophysical studies of novel
biological labelling reagents derived from luminescent iridium(III)
terpyridine complexes
Kenneth Kam-Wing Lo,*a Chi-Keung Chung,a Dominic Chun-Ming Nga and Nianyong Zhub
a
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue,
Kowloon, Hong Kong, P. R. China. E-mail:bhkenlo @cityu.edu.hk
Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China
b
Received (in Montpellier, France) 7th August 2001, Accepted 19th September 2001
First published as an Advance Article on the web
A series of new luminescent iridium(III) terpyridine complexes functionalised with an isothiocyanate group,
[Ir(tpy-R)(tpy-C6H4-NCS-p)](PF6)3 [R ¼ H (1), C6H5 (2), C6H4-CH3-p (3), C6H4-Cl-p (4)] has been synthesised,
characterised and their photophysical properties studied; the X-ray crystal structure of one of the intermediate
complexes, [Ir(tpy-C6H4-Cl-p)(CF3SO3)3] (4b), has also been determined; complex 1 has been used as a
luminescent label for proteins.
The design of transition metal complexes that can bind and=or
react at specific locations of biomolecules has been arousing
much interest.1 By virtue of their flexible coordination geometry
and rich photophysical and electrochemical properties, many
transition metal complexes have been covalently linked to
nucleoside phosphoramidites for solid-phase DNA synthesis, as
well as other biological molecules such as nucleic acids, peptides
and proteins for a wide range of mechanistic and analytical
investigations.2–18 While the design of many luminescent bio-
logical probes has relied on ruthenium(II),3a–c,6a,7,8a,b,9–12,15–18
and recently osmium(II)6d,8d,17 and rhenium(I)3b,d,8c,e com-
Scheme 1
plexes, the possibility of using the isoelectronic iridium(III)
complexes as luminescent biological labelling reagents has not
been explored.
The photoluminescence properties of iridium(III) complexes
have been known for many years.19–27 Their photophysical
properties have also been shown to provide unique advantages
over their d6 counterparts. In terms of molecular structures,
iridium(III) shows a higher variety, including its remarkable
ability to form mono-, bis- and triscyclometallated complexes.
The high structural variety allows better control of the excited-
state nature and emission properties of these complexes. With
different polypyridine ligands19,21,25,27 and=or cyclometallating
ligands,20,22–24,26 luminescent iridium(III) complexes can offer a
wider range of emission energy and, in many cases, longer
emission lifetimes compared to the ruthenium(II) analogues.
Recently, Williams and co-workers described the utilisation
of a series of interesting luminescent iridium(III) terpyridine
complexes as pH27a and chloride ion27b probes. With this in
mind, we believe that luminescent iridium(III) polypyridine
complexes are promising candidates for various bioanalytical
applications. It is also anticipated that these complexes can
offer additional advantages over traditional organic fluoro-
phores28 in biological labelling in consideration of their long-
lived and intense luminescence, large Stokes’ shifts and high
photostability. In this paper, we report the syntheses, char-
acterisation and photophysical properties of a series of new
luminescent iridium(III) terpyridine complexes functionalised
with an isothiocyanate moiety, [Ir(tpy-R)(tpy-C6H4-NCS-p)]-
(PF6)3 [tpy-R ¼ 40-substituted 2,20:60,200-terpyridine, where
R ¼ H (1), C6H5 (2), C6H4-CH3-p (3), C6H4-Cl-p (4);
tpy-C6H4-NCS-p ¼ 40-(4-isothiocyanatophenyl)-2,20:60,200-ter-
pyridine] (Scheme 1). The incorporation of the isothiocyanate
group allows these complexes to react with the primary amine
groups of biological substrates to form bioconjugates with
stable thiourea linkages.28,29 On the other hand, the syntheses
of 2–4 have involved the use of a series of new precursor
complexes, [Ir(tpy-R)(CF3SO3)3] [R ¼ C6H5 (2b), C6H4-CH3-p
(3b), C6H4-Cl-p (4b)]. The X-ray crystal structure of one of
these intermediates, 4b, has also been studied.
Experimental
Materials and reagents
All solvents were of analytical reagent grade. IrCl3 Á 3H2O, 2,20:
60,200-terpyridine and thiophosgene were purchased from
Aldrich and were used without purification. Human serum
albumin (HSA) fraction V and bovine serum albumin (BSA)
were obtained from Calbiochem and were used as received. All
buffer components were of molecular biology grade and used
without purification. The 40-aryl-substituted 2,20:60,200-ter-
pyridine derivatives tpy-R (R ¼ C6H5 , C6H4-CH3-p, C6H4-Cl-p,
C6H4-NO2-p) were synthesised from the reactions of 2-acetyl-
pyridine, ammonium acetate, acetamide and the corresponding
benzaldehydes according to reported procedures.30 The ligand
DOI: 10.1039/b107163g
New J. Chem., 2002, 26, 81–88
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tpy-C6H4-NH2-p was prepared from the reduction of tpy-
C6H4-NO2-p by hydrazine monohydrate and palladium on
charcoal in refluxing ethanol based on a related synthesis.31
The trichloroiridium(III) terpyridine complexes [Ir(tpy-R)Cl3]
(R ¼ H, C6H5 , C6H4-CH3-p, C6H4-Cl-p) were synthesised
from the reactions of IrCl3 Á 3H2O and the corresponding ter-
pyridines in degassed ethylene glycol at 160 ꢀC for 20 min.19b,27
Ho), 7.56(d, 2H, J ¼ 8.0 Hz, Hm), 2.54 (s, 3H, CH3). Positive-
ion ESI-MS: m=z 813 {[Ir(tpy-C6H4-CH3-p)(CF3SO3)2]}þ. IR
(KBr) n=cmÀ1: 1340 (s, CF3SO3), 1252 (s, CF3SO3), 1196(s,
CF3SO3), 1019 (m, CF3SO3), 975 (s, CF3SO3). Anal. calcd for
C25H17N3F9S3O9Ir: C, 31.19; H, 1.78; N, 4.36; found C, 31.01;
H, 1.50; N, 4.14.
[Ir(tpy-C6H4-Cl-p)(CF3SO3)3] (4b). The synthesis was simi-
lar to that for 2b except that [Ir(tpy-C6H4-Cl-p)Cl3] (306mg,
0.48 mmol) was used instead of [Ir(tpy-C6H5)Cl3]. Complex 4b
was isolated as orange-yellow crystals. Yield: 358 mg (76%).
1H NMR (300 MHz, acetone-d6 , 298 K, relative to TMS): d
9.33 (d, 2H, J ¼ 6.1 Hz, H6 and H600), 9.29 (s, 2H, H30 and
H50), 9.05 (d, 2H, J ¼ 7.4 Hz, H3 and H300), 8.57 (dt, 2H,
J ¼ 8.0 and 1.4 Hz, H4 and H400), 8.35–8.28 (m, 4H, Ho , H5
and H500), 7.78 (d, 2H, J ¼ 8.5 Hz, Hm). Positive-ion ESI-MS:
Syntheses
[Ir(tpy-H)(tpy-C6H4-NH2-p)](PF6)3 (1a). A mixture of
[Ir(tpy-H)Cl3] (90 mg, 0.17 mmol) and tpy-C6H4-NH2-p (55
mg, 0.17 mmol) in degassed ethylene glycol (10 ml) was heated
at 160 ꢀC for 20 min under an inert atmosphere of nitrogen in
the dark. The mixture was then cooled to room temperature
and a saturated aqueous solution of NH4PF6 was added to
precipitate an orange-red solid. The solid was washed with
cold water and then a mixture of methanol and ether, and then
dried in vacuo. Subsequent recrystallisation of the complex
from acetone–diethyl ether afforded 1a as air-stable orange-red
m=z 833 {[Ir(tpy-C6H4-Cl-p)(CF3SO3)2]}þ. IR (KBr) n/cmÀ1
:
1347 (s, CF3SO3), 1233 (s, CF3SO3), 1202 (s, CF3SO3), 1018
(m, CF3SO3), 974 (s, CF3SO3). Anal. calcd for
C24H14N3F9S3O9ClIr: C, 29.32; H, 1.44; N, 4.27; found C,
29.36; H, 1.38; N, 4.09.
1
crystals. Yield: 100 mg (50%). H NMR (300 MHz, acetone-
d6 , 298 K, relative to TMS): d 9.44 (s, 2H, H30 and H50 of tpy-
C6H4-NH2-p), 9.27 (d, 2H, J ¼ 8.5 Hz, H6and H6 00 of tpy-H),
9.17 (d, 2H, J ¼ 7.3 Hz, H6and H6 00 of tpy-C6H4-NH2-p), 9.00
(t, 1H, J ¼ 7.9 Hz, H40 of tpy-H), 8.99 (d, 2H, J ¼ 6.7 Hz, H30
and H50 of tpy-H), 8.43–8.37 (m, 4H, H4 and H400 of tpy-H,
H4 and H400 of tpy-C6H4-NH2-p), 8.33 (d, 2H, J ¼ 5.9 Hz, H3
and H300 of tpy-H), 8.22 (d, 2H, J ¼ 8.8 Hz, Ho of tpy-C6H4-
[Ir(tpy-C6H5)(tpy-C6H4-NH2-p)](PF6)3 (2a). A mixture of
2b (180 mg, 0.19 mmol) and tpy-C6H4-NH2-p (62 mg, 0.19
mmol) in degassed ethylene glycol (10 ml) was heated at 160 ꢀC
for 20 min under an inert atmosphere of nitrogen in the dark.
The mixture was then cooled to room temperature and a
saturated aqueous solution of NH4PF6 was added to pre-
cipitate an orange-red solid. The solid was washed with cold
water and then a mixture of methanol and ether, and then
dried in vacuo. The solid was then purified by column chro-
matography (silica gel) using gradient elution from CH3CN to
CH3CN–H2O–saturated aqueous KNO3À(70 : 27.5 : 2.5). The
product was then converted to the PF6 salt by metathesis.
Subsequent recrystallisation of the complex from acetone–
diethyl ether afforded 2a as air-stable orange-red crystals.
Yield: 122 mg (51%). 1H NMR (300 MHz, acetone-d6 , 298 K,
relative to TMS): d 9.47 (s, 2H, H30 and H50 of tpy-C6H5), 9.33
(s, 2H, H30 and H50 of tpy-C6H4-NH2-p), 9.11 (d, 2H, J ¼ 8.8
Hz, H6and H6 00 of tpy-C6H5), 9.08 (d, 2H, J ¼ 9.1 Hz, H6and
H600 of tpy-C6H4-NH2-p), 8.36–8.28 (m, 6H, H4, H400 and Ho
of tpy-C6H5 , H4 and H400 of tpy-C6H4-NH2-p), 8.25 (d, 2H,
J ¼ 4.7 Hz, H3 and H300 of tpy-C6H5), 8.22 (d, 2H, J ¼ 8.8 Hz,
Ho of tpy-C6H4-NH2-p), 8.15 (d, 2H, J ¼ 5.0 Hz, H3 and H300
of tpy-C6H4-NH2-p), 7.79–7.73 (m, 3H, Hm and Hp of tpy-
C6H5), 7.64–7.54 (m, 4H, H5 and H500 of tpy-C6H5 , H5 and
H500 of tpy-C6H4-NH2-p), 7.00 (d, 2H, J ¼ 8.8 Hz, Hm of tpy-
C6H4-NH2-p), 5.65 (s, 2H, NH2). Positive-ion ESI-MS: m=z
00
NH2-p), 8.16(d, 2H, J ¼ 5.6Hz, H3 and H3 of tpy-C6H4-
NH2-p), 7.70–7.60 (m, 4H, H5 and H500 of tpy-H, H5 and H500
of tpy-C6H4-NH2-p), 7.02 (d, 2H, J ¼ 8.8 Hz, Hm of tpy-C6H4-
NH2-p), 5.84 (s, 2H, NH2). Positive-ion ESI-MS: m=z 374
{[Ir(tpy-H)(tpy-C6H4-NH2-p)]3þ þ eÀ}2þ. IR (KBr) n/cmÀ1
:
838 (s, PF6À). Anal. calcd for C36H27N7P3F18Ir: C, 36.50; H,
2.30; N, 8.28; found C, 36.61; H, 2.37; N, 8.25.
[Ir(tpy-C6H5)(CF3SO3)3] (2b). A mixture of [Ir(tpy-
C6H5)Cl3] (290 mg, 0.48 mmol) and trifluoromethanesulfonic
acid (2.1 ml, 23.65 mmol) was refluxed in 1,2-dichlorobenzene
(25 ml) under an inert atmosphere of nitrogen in the dark for
12 h. The mixture was then cooled to room temperature. The
solvent and the excess acid were carefully removed by decan-
tation. The brownish yellow semi-solid left was washed with a
copious amount of petroleum ether and then dissolved in
CH2Cl2 (5 ml) and loaded onto a chromatographic column.
Alumina was used as the stationary phase and CH2Cl2 as the
eluent. The first yellow band was collected and evaporated to
dryness. Subsequent recrystallisation from acetone–petroleum
ether afforded [Ir(tpy-C6H5)(CF3SO3)3] as orange-yellow
485 {[Ir(tpy-C6H5)(tpy-C6H4-NH2-p)](PF6)}2þ
.
IR (KBr)
1
crystals. Yield: 360 mg (80%). H NMR (300 MHz, acetone-
d6 , 298 K, relative to TMS): d 9.33 (d, 2H, J ¼ 5.6Hz, H6and
n=cmÀ1: 840 (s, PF6À). Anal. calcd for C42H31N7P3F18Ir: C,
40.01; H, 2.48; N, 7.78; found C, 40.15; H, 2.54; N, 8.04.
H600), 9.26(s, 2H, H3 and H50), 9.07 (d, 2H, J ¼ 7.9 Hz, H3
0
and H300), 8.57 (dt, 2H, J ¼ 7.9 and 1.5 Hz, H4 and H400), 8.33–
8.27 (m, 4H, Ho , H5 and H50), 7.74 (t, 2H, J ¼ 7.0 Hz, Hm),
7.68–7.63 (m, 1H, Hp). Positive-ion ESI-MS: m=z 799 {[Ir(tpy-
C6H5)(CF3SO3)2]}þ. IR (KBr) n=cmÀ1: 1348 (s, CF3SO3), 1237
(s, CF3SO3), 1197 (s, CF3SO3), 1019 (s, CF3SO3), 974 (s,
CF3SO3). Anal. calcd for C24H15N3F9S3O9Ir: C, 30.38; H,
1.59; N, 4.43; found C, 30.51; H, 1.50; N, 4.13.
[Ir(tpy-C6H4-CH3-p)(tpy-C6H4-NH2-p)](PF6)3 (3a). Th e s yn -
thesis was similar to that for 2a except that 3b (183 mg, 0.19
mmol) was used instead of 2b. Complex 3a was isolated as
1
orange-red crystals. Yield: 99 mg (41%). H NMR (300 MHz,
acetone-d6 , 298 K, relative to TMS): d 9.47 (s, 2H, H30 and
H50 of tpy-C6H4-CH3-p), 9.34 (s, 2H, H30 and H50 of tpy-
00
C6H4-NH2-p), 9.12 (d, 2H, J ¼ 7.9 Hz, H6and H6 of00tpy-
[Ir(tpy-C6H4-CH3-p)(CF3SO3)3] (3b). The synthesis was
similar to that for 2b except that [Ir(tpy-C6H4-CH3-p)Cl3] (297
mg, 0.48 mmol) was used instead of [Ir(tpy-C6H5)Cl3]. Com-
plex 3b was isolated as orange-yellow crystals. Yield: 290 mg
(63%). 1H NMR (300 MHz, acetone-d6 , 298 K, relative to
C6H4-CH3-p), 9.09 (d, 2H, J ¼ 8.5 Hz, H6and H6
of
tpy-C6H4-NH2-p), 8.36–8.25 (m, 8H, H4, H400, H3, H300 and Ho
of tpy-C6H4-CH3-p, H4 and H400 of tpy-C6H4-NH2-p), 8.22 (d,
2H, J ¼ 8.8 Hz, Ho of tpy-C6H4-NH2-p), 8.17 (d, 2H, J ¼ 5.3 Hz,
H3 and H300 of tpy-C6H4-NH2-p), 7.64–7.55 (m, 6H, H5, H500
and Hm of tpy-C6H4-CH3-p, H5 and H500 of tpy-C6H4-NH2-p),
7.00 (d, 2H, J ¼ 8.8 Hz, Hm of tpy-C6H4-NH2-p), 5.68 (s, 2H,
NH2), 2.55 (s, 3H, CH3). Positive-ion ESI-MS: m=z 492 {[Ir(tpy-
C6H4-CH3-p)(tpy-C6H4-NH2-p)](PF6)}2þ.IR(KBr)n=cmÀ1:840(s,
00
TMS): d 9.33 (dd, 2H, J ¼ 5.5 and 1.1 Hz, H6and H6 ), 9.23
(s, 2H, H30 and H50), 9.06(d, 2H, J ¼ 7.7 Hz, H3 and H300),
8.56(dt, 2H, J ¼ 5.8 and 1.4 Hz, H4 and H400), 8.29 (ddd, 2H,
J ¼ 7.7, 5.8 and 1.4 Hz, H5 and H500), 8.20 (d, 2H, J ¼ 8.2 Hz,
82
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PF6À). Anal. calcd for C43H33N7P3F18Ir: C, 40.51; H, 2.61; N,
7.69; found C, 40.49; H, 2.40; N, 7.54.
Hz, Ho of tpy-C6H4-NCS-p), 8.42–8.36(m, 4H, H4 and H4 00 of
tpy-C6H4-NCS-p, H4 and H400 of tpy-C6H4-CH3-p), 8.28–8.21
(m, 6H, Ho , H3 and H300 of tpy-C6H4-CH3-p, H3 and H300 of
tpy-C6H4-NCS-p), 7.78 (d, 2H, J ¼ 8.2 Hz, Hm of tpy-C6H4-
NCS-p), 7.66–7.59 (m, 6H, Hm , H5 and H500 of tpy-C6H4-
CH3-p, H5 and H500 of tpy-C6H4-NCS-p), 2.55 (m, 3H, CH3).
Positive-ion ESI-MS: m=z 513 {[Ir(tpy-C6H4-CH3-p)(tpy-
C6H4-NCS-p)](PF6)}2þ. IR (KBr) n=cmÀ1: 2093 (m, NCS), 838
(s, PF6À). Anal. calcd for C44H31N7P3F18SIr: C, 40.13; H, 2.37;
N, 7.45; found C, 39.95; H, 2.29; N, 7.31.
[Ir(tpy-C6H4-Cl-p)(tpy-C6H4-NH2-p)](PF6)3 (4a). The synth-
esis was similar to that for 2a except that 4b (187 mg, 0.19 mmol)
was used instead of 2b. Complex 4a was isolated as orange-red
crystals. Yield: 115 mg (47%). 1H NMR (300 MHz, acetone-d6 ,
298 K, relative to TMS): d 9.45 (s, 2H, H30 and H50 of tpy-
C6H4-Cl-p), 9.31 (s, 2H, H30 and H50 of tpy-C6H4-NH2-p),
9.07 (d, 4H, J ¼ 7.9 Hz, H6and H6 00 of tpy-C6H4-Cl-p, H6 and
H600 of tpy-C6H4-NH2-p), 8.37–8.27 (m, 6H, H4, H400 and Ho of
tpy-C6H4-Cl-p, H4 and H400 of tpy-C6H4-NH2-p), 8.23 (d, 2H,
J ¼ 4.7 Hz, H3 and H300 of tpy-C6H4-Cl-p), 8.21 (d, 2H, J ¼ 8.2
Hz, Ho of tpy-C6H4-NH2-p), 8.11 (d, 2H, 5.0 Hz, H3 and H300 of
tpy-C6H4-NH2-p), 7.78 (d, 2H, J ¼ 8.8 Hz, Hm of tpy-C6H4-Cl-
p), 7.64–7.53 (m, 4H, H5 and H500 of tpy-C6H4-Cl-p, H5 and H500
of tpy-C6H4-NH2-p), 7.00 (d, 2H, J ¼ 8.5 Hz, Hm of tpy-C6H4-
NH2-p), 5.61 (s, 2H, NH2). Positive-ion ESI-MS: m=z 429
[Ir(tpy-C6H4-Cl-p)(tpy-C6H4-NCS-p)](PF6)3 (4). The synth-
esis was similar to that for 1 except that 4a (109 mg, 84.4 mmol)
was used instead of 1a. Complex 4 was isolated as yellow
crystals. Yield: 45 mg (40%). 1H NMR (300 MHz, acetone-d6 ,
298 K, relative to TMS): d 9.58 (s, 2H, H30 and H50 of tpy-
C6H4-NCS-p), 9.57 (s, 2H, H30 and H50 of tpy-C6H4-Cl-p),
00
9.17–9.14 (m, 4H, H6and H6 of tpy-C6H4-NCS-p, H6and
{[Ir(tpy-C6H4-Cl-p)(tpy-C6H4-NH2-p)]3þ þ eÀ}2þ
.
IR (KBr)
H600 of tpy-C6H4-Cl-p), 8.42–8.35 (m, 8H, Ho , H4 and H400 of
tpy-C6H4-NCS-p, Ho , H4 and H400 of tpy-C6H4-Cl-p), 8.24–
8.22 (m, 4H, H3 and H300 of tpy-C6H4-NCS-p, H3 and H300 of
tpy-C6H4-Cl-p), 7.82 (d, 2H, J ¼ 8.21 Hz, Hm of tpy-C6H4-
NCS-p), 7.78 (d, 2H, J ¼ 8.80 Hz, Hm of tpy-C6H4-Cl-p), 7.66–
7.60 (m, 4H, H5 and H500 of tpy-C6H4-NCS-p, H5 and H500 of
tpy-C6H4-Cl-p). Positive-ion ESI-MS: m=z 523 {[Ir(tpy-C6H4-
Cl-p)(tpy-C6H4-NCS-p)](PF6)}2þ. IR (KBr) n=cmÀ1: 2089 (w,
NCS), 842 (s, PF6À). Anal. calcd for C43H28N7P3F18SClIr: C,
38.62; H, 2.11; N, 7.33; found C, 38.69; H, 2.39; N, 7.27.
n=cmÀ1: 841 (s, PF6À). Anal. calcd for C42H30N7P3F18ClIr: C,
38.95; H, 2.33; N, 7.57; found C, 38.87; H, 2.40; N, 7.67.
[Ir(tpy-H)(tpy-C6H4-NCS-p)](PF6)3 (1). To a mixture of 1a
(100 mg, 84.4 mmol) and CaCO3 (34 mg, 339.7 mmol) in 8 ml of
dry acetone was added CSCl2 (13 ml, 170.5 mmol). After being
stirred for 2 h in the dark under nitrogen, the suspension was
filtered and evaporated to dryness to yield an orange-yellow
solid. Recrystallisation from acetone–petroleum ether afforded
1 as orange-yellow crystals. Yield: 60 mg (58%). 1H NMR (300
MHz, acetone-d6 , 298 K, relative to TMS): d 9.64 (s, 2H, H30
and H50 of tpy-C6H4-NCS-p), 9.28 (d, 2H, J ¼ 8.2 Hz, H6and
Labelling of HSA and BSA with complex 1
00
H600 of tpy-H), 9.19 (d, 2H, J ¼ 8.0 Hz, H6and H6 of tpy-
C6H4-NCS-p), 9.00 (t, 1H, J ¼ 9.3 Hz, H40 of tpy-H), 8.99 (d,
2H, J ¼ 8.2 Hz, H30 and H50 of tpy-H), 8.45–8.37 (m, 6H, Ho ,
H4 and H400 of tpy-C6H4-NCS-p, H4 and H400 of tpy-H), 8.25
(d, 2H, J ¼ 4.7 Hz, H3 and H300 of tpy-H), 8.19 (d, 2H, J ¼ 5.2
Hz, H3 and H300 of tpy-C6H4-NCS-p), 7.80 (d, 2H, J ¼ 8.5 Hz,
In a typical labelling reaction, complex 1 (3.0 mg, 2.45 mmol) in
20 ml anhydrous DMSO was added to HSA (3.0 mg, 45.5
nmol) or BSA (3.0 mg, 45.5 nmol) dissolved in 180 ml of 50
mM carbonate buffer pH 9.1. The suspension was stirred
slowly in the dark at room temperature for 48 h. The solid
residue was then removed by centrifugation. The supernatant
was diluted to 1.0 ml with 50 mM Tris-HCl pH 7.4 and loaded
onto a PD-10 column (Pharmacia) that had been equilibrated
with the same buffer. The first yellow band that came out of
the column was collected and the solution was concentrated
with a YM-30 centricon (Amicon). The labelled protein was
further purified by HPLC equipped with a size-exclusion col-
umn (Waters, Protein Pak, 8.0 Â 300 mm). The mobile phase
H
m of tpy-C6H4-NCS-p), 7.65 (br s, 4H, H5 and H500 of tpy-H,
H5 and H500 of tpy-C6H4-NCS-p). Positive-ion ESI-MS: m=z
468 {[Ir(tpy-H)(tpy-C6H4-NCS-p)](PF6)}2þ. IR (KBr) n=cmÀ1
:
2095 (m, NCS), 840 (s, PF6À). Anal. calcd for
C37H25N7P3F18SIr: C, 36.22; H, 2.05; N, 7.99; found C, 36.23;
H, 2.10; N, 7.86.
[Ir(tpy-C6H5)(tpy-C6H4-NCS-p)](PF6)3 (2). The synthesis
was similar to that for 1 except that 2a (106mg, 84.4 mmol) was
used instead of 1a. Complex 2 was isolated as yellow crystals.
was 50 mM Tris-HCl pH 7.4 at a flow rate of 0.75 ml minÀ1
.
The retention times of the labelled protein and free labels were
ca. 10.8 and 19.9 min, respectively.
1
Yield: 65 mg (59%). H NMR (300 MHz, acetone-d6 , 298 K,
relative to TMS): d 09.58 (s, 2H, H30 and H50 of tpy-C6H4-NCS-
p), 9.56(s, 2H, H3 and H50 of tpy-C6H5), 9.19–9.14 (m, 4H,
Physical measurements and instrumentation
00
00
H6and H6 of tpy-C6H4-NCS-p, H6and H6 of tpy-C6H5),
8.45–8.32 (m, 8H, Ho , H4 and H400 of tpy-C6H4-NCS-p, Ho ,
H4 and H400 of tpy-C6H5), 8.25–8.21 (m, 4H, H3 and H300 of
tpy-C6H4-NCS-p, H3 and H300 of tpy-C6H5), 7.81–7.74 (m, 5H,
Hm of tpy-C6H4-NCS-p, Hm and Hp of tpy-C6H5), 7.66–7.60
(m, 4H, H5 and H500 of tpy-C6H4-NCS-p, H5 and H500 of tpy-
C6H5). Positive-ion ESI-MS: m=z 506{[Ir(tpy-C 6H5)(tpy-
C6H4-NCS-p)](PF6)}2þ. IR (KBr) n=cmÀ1: 2089 (w, NCS), 841
(s, PF6À). Anal. calcd for C43H29N7P3F18SIr: C, 39.64; H, 2.24;
N, 7.53; found C, 39.69; H, 2.00; N, 7.49.
1H NMR spectra were recorded on a Varian Mercury 300
MHz NMR spectrometer at 298 K. Positive-ion ESI mass
spectra were recorded on a Perkin Elmer Sciex API 365 mass
spectrometer. IR spectra were recorded on a Perkin Elmer
1600 series FT-IR spectrophotometer. Elemental analyses were
carried out on an Elementar Analysensysteme GmbH Vario
EL elemental analyser. Electronic absorption and steady-state
emission/excitation spectra were recorded on a Hewlett–
Packard 8452A diode array spectrophotometer and a Spex
Fluorolog-2 Model F 111 fluorescence spectrophotometer,
respectively. Unless specified otherwise, all solutions for pho-
tophysical studies were degassed with no fewer than four
successive freeze-pump-thaw cycles and stored in a 10 cm3
round bottomed flask equipped with a side-arm 1 cm fluores-
cence cuvette and sealed from the atmosphere by a Rotaflo
HP6/6 quick-release Teflon stopper. Luminescence quantum
yields were measured by the optical dilute method32 using an
aerated aqueous solution of [Ru(bpy)3]Cl2 (F ¼ 0.028)33 as the
[Ir(tpy-C6H4-CH3-p)(tpy-C6H4-NCS-p)](PF6)3 (3). The syn-
thesis was similar to that for 1 except that 3a (108 mg, 84.4
mmmol) was used instead of 1a. Complex 3 was isolated as
yellow crystals. Yield: 81 mg (73%). 1H NMR (300 MHz,
acetone-d6 , 298 K, relative to TMS): d 9.58 (s, 2H, H30 and
H50 of tpy-C6H4-NCS-p), 9.54 (s, 2H, H30 and H50 of tpy-
00
C6H4-CH3-p), 9.18–9.15 (m, 4H, H6and H6 of tpy-C6H4-
00
NCS-p, H6and H6 of tpy-C6H4-CH3-p), 8.43 (d, 2H, J ¼ 8.8
New J. Chem., 2002, 26, 81–88
83

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standard solution. The excitation source for emission lifetime
measurements was the 355 nm output (third harmonic) of a
Quanta-Ray Q-switched GCR-150-10 pulsed Nd-YAG laser.
Luminescence decay signals from a Hamamatsu R928 photo-
multiplier tube were converted to potential changes by a 50 O
load resistor and then recorded on a Tektronix Model TDS
620A digital oscilloscope.
Results and discussion
Synthesis
Owing to the chemical inertness of the coordination sphere of
iridium(III), harsh reaction conditions are usually required for
coordination of polypyridine and cyclometallating ligands. In
this work, all four trichloroiridium(III) terpyridine complexes
[Ir(tpy-R)Cl3] (R ¼ H, C6H5 , C6H4-CH3-p, C6H4-Cl-p) were
synthesised from reactions of IrCl3 Á 3H2O and the corre-
sponding terpyridines in ethylene glycol at 160 ꢀC for 20
min.19b,27 The amine-containing precursor complexes [Ir(tpy-
R)(tpy-C6H4-NH2-p)](PF6)3 [R ¼ H (1a), C6H5 (2a), C6H4-
CH3-p (3a), C6H4-Cl-p (4a)] were, however, prepared by two
different methods. Complex 1a was obtained, in a moderate
yield, from the reaction of [Ir(tpy-H)Cl3] and tpy-C6H4-NH2-p
in ethylene glycol at 160 ꢀC for 20 min, followed by metathesis
with NH4PF6 . However, we found that direct reactions of
[Ir(tpy-R)Cl3] (R ¼ C6H5 , C6H4-CH3-p, C6H4-Cl-p) and tpy-
C6H4-NH2-p under similar conditions gave many side-pro-
ducts and the desired complexes were isolated in very low
yields. Therefore, an alternative synthetic procedure for the
amine-containing complexes 2a–4a was sought. In view of the
Crystal structure determination
Crystal data for 4b. [C24.75H17Cl2.50F9IrN3O9.50S3]; M ¼
ꢀ
1056.42, triclinic, P1, a ¼ 14.376(3), b ¼ 15.528(3), c ¼18.926(4)
+
+
A, a ¼ 75.08(3), b ¼ 69.97(3), g ¼ 65.96(3)ꢀ, U ¼ 3590.6(12) A3,
Z ¼ 4, m(Mo-Ka) ¼ 4.180 mmÀ1. A crystal of dimensions
0.5 Â 0.3 Â 0.15 mm mounted in a glass capillary was used for
data collection at 28 ꢀC on a MAR diffractometer with a 300
mm image plate detector using graphite monochromated Mo-
+
Ka radiation (l ¼ 0.71073 A). Data collection was made with a
2ꢀ oscillation step of j, 300 s exposure time and scanner dis-
tance at 120 mm. One hundred images were collected. The
images were interpreted and intensities integrated using the
program DENZO.34 The structure was solved by direct
methods (SIR-97).35 Almost all atoms were located according
to the direct methods and the successive least-squares Fourier
cycles. The positions of the other non-hydrogen atoms were
found after successful refinement by full-matrix least-squares
refinement (SHELXL -97).36 One water molecule was located;
one CH2Cl2 solvent molecule was also located. Another posi-
tion was found to be occupied by another CH2Cl2 solvent
molecule. However, due to high thermal parameters of the
atoms, the occupancies were set to half (a free refinement
of the occupancy gave rise to a similar value); meanwhile
fact that [Ir(bpy)3]2þ
,
cis-[Ir(bpy)2(PPh3)H]2þ and cis-
[Ir(bpy)2H2]þ can be conveniently synthesised from the pre-
cursor complex cis-[Ir(bpy)2(CF3SO3)2](CF3SO3),37 we
attempted a similar synthesis and successfully isolated a series
of new intermediate complexes [Ir(tpy-R)(CF3SO3)3],
[R ¼ C6H5 (2b), C6H4-CH3-p (3b), C6H4-Cl-p (4b)] from the
reactions of [Ir(tpy-R)Cl3] (R ¼ C6H5 , C6H4-CH3-p, C6H4-
Cl-p) and trifluoromethanesulfonic acid in refluxing 1,2-
dichlorobenzene. The amine-containing complexes 2a–4a were
then obtained by heating a mixture of 2b–4b and the ligand
tpy-C6H4-NH2-p in ethylene glycol at 160 ꢀC for 20 min,
followed by anion exchange, column purification and recrys-
tallisation from acetone–diethyl ether. We found that using
[Ir(tpy-R)(CF3SO3)3] instead of [Ir(tpy-R)Cl3] as the precursor
complexes for 2a–4a can give the desired products in higher
yields. It appears that tris(trifluoromethanesulfonato)iridium
(III) complexes of this kind are versatile starting materials for
syntheses of heteroleptic iridium(III) terpyridines.
restraints were applied to assume similar C–Cl bond lengths
within a range from 1.68 to 1.72 A. Two Ir complex molecules
+
were located in one asymmetric unit and each contains one
disordered CF3SO3À . One molecule has F atoms disordered as
rotated along the S–C bond; the other molecule has disordered
S (S5 and S50), O (O15 and O150) and F (F13, F130, F14, F140,
F15 and F150) atoms. For convergence of refinements, S50–
+
O150 was assumed to be near 1.38(2) A and C(47)–F bonds
were assumed to be similar. All 12465 independent reflections
The target complexes [Ir(tpy-R)(tpy-C6H4-NCS-p)](PF6)3
[R ¼ H (1), C6H4-CH3-p (2), C6H5 (3), C6H4-Cl-p (4)] were
prepared from reactions of the amine-containing complexes
1a–4a and CSCl2 in the presence of CaCO3 in acetone. Similar
procedures have been adopted for the syntheses of ruthe-
nium(II)7,8a and osmium(II)8d isothiocyanate complexes
[M(N-N)2(phen-5-NCS)]2þ [M ¼ Ru(II), Os(II); N-N 2,20-
bipyridine, 1,10-phenanthroline; phen-5-NCS ¼ 5-isothiocyanato-
1,10-phenanthroline]. The conversion of the amine moieties of
1a–4a to the isothiocyanate groups of 1–4 was associated with
a downfield shift of the NMR resonance signals of two Hm
protons of tpy-C6H4-NH2-p (from ca. d 7.0 in tpy-C6H4-NH2-p
to d 7.8 in tpy-C6H4-NCS-p) and the observation of an IR
absorption peak at ca. 2089–2095 cmÀ1, typical of nNCS
stretching. All the new complexes were characterised by 1H
NMR, positive-ion ESI-MS and IR, and gave satisfactory
elemental analyses.
P
P
2
{Rint ¼ 0.0368
(Rint
¼
jFo ÀFo2(mean)j= [Fo2]),
9210
reflections larger than 4s(Fo)} from a total 26033 reflections
participated in the full-matrix least-squares refinement against
F2. One crystallographic asymmetric unit consists of two for-
mula units, including one water molecule, and one and a half
CH2Cl2 solvent molecules. In the final stage of least-squares
refinement, the disordered atoms and atoms of the second
CH2Cl2 molecule (half occupancy) were refined isotropically,
the other non-H atoms were refined anisotropically. H atoms
were generated by the program SHELXL -97. The positions of
H atoms were calculated based on a riding mode with thermal
parameters equal to 1.2 times that of the associated C atoms,
and participated in the calculation of final R indices. Since the
structure refinements are against F2, R indices based on F2 are
larger than (more than double) those based on F. For com-
parison with older refinements based on F and an OMIT
threshold, a conventional index R1 based on observed F
values larger than 4s(Fo) is also given [corresponding to
X-Ray crystal structure determination
P
P
2 2
2
intensity ꢁ 2s(I)]: wR2 ¼ { [w(Fo À Fc ) ]= [w(Fo2)2]}1=2
,
P
P
R1 ¼ jj Foj À jFcjj= jFoj; w ¼ 1=[s2(Fo2) þ (aP)2 þ bP],
Compared to organometallic iridium(III) systems, crystal
structures of iridium(III) terpyridine complexes are very
rare.19b,25a Single crystals of 4b were obtained by layering a
concentrated dichloromethane solution of the complex with
petroleum ether. The perspective views of the two independent
molecules of 4b with the atomic numbering scheme are shown
in Fig. 1. Selected bond distances and angles are listed in
Table 1. As a consequence of the geometric constraints
imposed by the tpy-C6H4-Cl-p ligand, the iridium(III) centre is
where P is [2Fc þ Max(Fo2 ,0)]=3. Convergence [(D=s)max
¼
2
À 0.001, ave. 0.001) for 926variable parameters by full-matrix
least-squares refinement on F2 was reached at R1 ¼ 0.0473 and
wR2 ¼ 0.1337 with the parameters a and b being 0.0995 and
0.0, respectively.
CCDC reference number 175721. See http:==www.rsc.org=
suppdata=nj=b1=b107163g for crystallographic data in CIF or
other electronic format.
84
New J. Chem., 2002, 26, 81–88

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+
À
Table 1 Selected bond distances (A) and angles (ꢀ) for complex 4b
in a distorted octahedral geometry, with the CF3SO3 ligands
occupying three meridionally arranged coordination sites as
+
expected. The average Ir–N bond distances (ca. 1.938 A for the
+
Ir(1)–N(1)
Ir(1)–N(2)
Ir(1)–N(3)
Ir(1)–O(1)
Ir(1)–O(4)
Ir(1)–O(7)
2.056(7)
1.938(6)
2.052(7)
2.055(6)
2.094(6)
2.059(6)
Ir(2)–N(4)
Ir(2)–N(5)
Ir(2)–N(6)
Ir(2)–O(10)
Ir(2)–O(13)
Ir(2)–O(16)
2.070(6)
1.938(6)
2.041(6)
2.048(5)
2.039(6)
2.086(5)
central pyridine ring and 2.055 A for the peripheral ones) and
the N–Ir–N bite angles (80.0–81.3ꢀ) are in excellent agreement
with those observed in the related complexes [Ir(tpy-H)2]3þ
+
(1.978–2.058 A, 80.0–80.3ꢀ)19b and [Ir{2,3,5,6-tetrakis(2-py-
+
ridyl)pyrazine}Cl3] (1.917–2.032 A, 80.8ꢀ).25a As a result of the
N(1)–Ir(1)–N(2)
N(1)–Ir(1)–N(3)
N(1)–Ir(1)–O(1)
N(1)–Ir(1)–O(4)
N(1)–Ir(1)–O(7)
N(2)–Ir(1)–N(3)
N(2)–Ir(1)–O(1)
N(2)–Ir(1)–O(4)
N(2)–Ir(1)–O(7)
N(3)–Ir(1)–O(1)
N(3)–Ir(1)–O(4)
N(3)–Ir(1)–O(7)
O(1)–Ir(1)–O(4)
O(1)–Ir(1)–O(7)
O(4)–Ir(1)–O(7)
80.2(3)
161.4(3)
94.1(2)
98.0(3)
95.5(3)
81.3(2)
93.2(2)
177.0(3)
94.9(3)
88.0(3)
100.5(3)
85.0(3)
89.2(3)
168.4(2)
83.0(3)
N(4)–Ir(2)–N(5)
N(4)–Ir(2)–N(6)
N(4)–Ir(2)–O(16)
N(4)–Ir(2)–O(10)
N(4)–Ir(2)–O(13)
N(5)–Ir(2)–N(6)
N(5)–Ir(2)–O(10)
N(5)–Ir(2)–O(13)
N(5)–Ir(2)–O(16)
N(6)–Ir(2)–O(10)
N(6)–Ir(2)–O(13)
N(6)–Ir(2)–O(16)
O(10)–Ir(2)–O(13)
O(10)–Ir(2)–O(16)
O(13)–Ir(2)–O(16)
80.0(2)
161.0(2)
105.5(2)
95.6(2)
90.1(3)
81.3(2)
93.3(2)
94.3(3)
174.3(2)
88.2(2)
88.6(3)
93.2(2)
171.2(2)
87.8(2)
84.2(2)
steric hindrance between the meta protons of the central py-
ridine ring and the ortho protons of the phenyl ring, these two
rings are twisted about the interannular C–C bonds, resulting
in dihedral angles of ca. 16.0 and 17.8ꢀ in the two independent
molecules. These values are similar to those observed in
[Ni(tpy-C6H5)2]Cl2 (16.7 and 17.8ꢀ)38 but noticeably smaller
than those of [Pt(tpy-C6H5)Cl] (33.4ꢀ)39 and [Cu{40-(p-
1,4,7-triazacyclonon-1-ylmethylphenyl)-2,20:60,200-terpyridine}-
(H2O)2]2þ (24.2ꢀ).40
Electronic absorption and emission properties
All the new iridium(III) terpyridine isothiocyanate complexes
are soluble in acetonitrile and acetone, giving yellow solutions.
The electronic absorption spectral data for 1–4 are summarised
in Table 2. As examples, the absorption spectra of 1 and 2 are
shown in Fig. 2. For all four complexes, the intense absorption
peaks and shoulders at ca. 252–372 nm with extinction coeffi-
cients of the order of 104 dm3 molÀ1 cmÀ1 are assigned to
intraligand (IL) transitions. Due to the lack of 40-aryl sub-
stituents on the tpy-H ligand of 1, the extinction coefficients of
the absorption bands are smaller than those of 2–4 (Fig. 2). On
the other hand, all the complexes show weaker absorptions
tailing into the lower energy region. It is likely that these
absorption features are partially due to spin-allowed and
spin-forbidden metal-to-ligand charge-transfer (MLCT)
[dp(Ir) ! p*(terpyridines)] transitions. The possible observa-
tion of the latter is a result of the heavy atom effect.41
Upon photoexcitation, complexes 1–4 exhibit intense and
long-lived yellow emission in CH3CN at 298 K and green
emission in low-temperature alcohol glass. The photophysical
data are listed in Table 3. As an example, the emission and
excitation spectra of 3 in CH3CN at 298 K and EtOH–MeOH
glass at 77 K are shown in Fig. 3(a) and 3(b), respectively. The
room-temperature solution emission spectra of all the com-
plexes exhibit structured features, with an emission maximum
occurring at ca. 527–530 nm and a shoulder at ca. 555–558 nm.
The emission lifetimes fall on the microsecond timescale in
both degassed and aerated CH3CN solutions (Table 3). This
finding, together with the large Stokes’ shifts (ca. 0.93–1.07 eV),
suggests a phosphorescence nature of the emission. In view
of the structured emission solution spectra, long emission
lifetimes and small radiative decay rate constants (kr), the
emission of the complexes is assigned to an 3IL
[p ! p*(terpyridines)] emissive state, which is mixed with some
3MLCT [dp(Ir) ! p*(terpyridines)] character. The involve-
ment of the 3MLCT character is supported by the blue shifts of
the emission maxima in the low-temperature glass [Table 3,
3
Fig. 3(a) and 3(b)], since a similar shift is absent for pure IL
emitters such as [Ir(tpy-H)2]3þ 19b
.
Table 2 Electronic absorption spectral data for complexes 1–4 in
CH3CN at 298 K
l
abs=nm (e=dm3 molÀ1 cmÀ1
)
1
2
3
4
252 (44,245), 280 (41,950), 312 (29,640), 322 (29,835),
338 sh (24,660), 352 (24,140), 372 (18,835)
252 (48,915), 282 (47,415), 300 sh (43,615), 320 sh (38,725),
344 sh (29,980), 370 sh (23,740)
252 (47,665), 280 (45,295), 308 (40,620), 318 sh (39,295),
344 sh (30,625), 370 sh (25,270)
Fig. 1 Perspective drawings of the two independent molecules of 4b
with the atomic numbering scheme. Hydrogen atoms have been
omitted for clarity. Thermal ellipsoids are shown at the 20% prob-
ability level.
252 (46,450), 282 (43,190), 302 sh (40,885), 322 sh (37,300),
344 sh (30,335), 370 sh (22,960)
New J. Chem., 2002, 26, 81–88
85

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Fig. 2 Electronic absorption spectra of 1 (——) and 2 (------) in
CH3CN at 298 K.
It is likely that the tpy-C6H4-NCS-p ligand plays an
3
important role in the IL=3MLCT states of 1 and probably in
2–4 as well, based on the high similarity in emission energy
among the complexes (especially between 1 and 2–4), although
the involvement of the ‘‘ancillary’’ terpyridine ligands tpy-R
(R ¼ C6H5 , C6H4-CH3-p, C6H4-Cl-p) cannot be totally
neglected. On the other hand, the emission band shapes of the
complexes, as well as the long emission lifetimes, resemble
those of a class of related homoleptic and heteroleptic iridium-
(III) terpyridine complexes, [Ir(tpy-C6H3-tBu2-m)2]3þ, [Ir(tpy-
C6H4-CH3-p)2]3þ and [Ir(tpy-CH3)(tpy-C6H4-CH3-p)]3þ 19b
for
,
which a mixed IL=3MLCT excited state has been suggested,
except that the emission maxima for the latter complexes occur
at higher energy (ca. 506nm in CH 3CN at room temperature).
The lower emission energy of the complexes in the current
work is attributable to a higher degree of p-conjugation in the
tpy-C6H4-NCS-p ligand and the electron-withdrawing effects
of the isothiocyanate group, which are both expected to sta-
3
Fig. 3 Emission (——) and excitation (------) spectra of 3 in (a)
CH3CN at 298 K and (b) EtOH–MeOH (4 : 1 v=v) at 77 K.
bilise the IL=3MLCT states.
3
originate from an un-equilibrated 3IL=3MLCT excited state
associated essentially with the ‘‘ancillary’’ terpyridine ligands.
This assignment is based on the finding that [Ir(tpy-C6H4-CH3-
p)2]3þ, a structural analogue of 3, emits at 494 nm with a
lifetime of 39 ms in 77 K glass.19b The absence of a shorter-lived
component for 1 is probably a result of the higher energy
of the 3IL and 3MLCT states involving the tpy-H ligand,
given that the 77 K glass emission of [Ir(tpy-H)2]3þ occurs at
458 nm with a lifetime of 26 ms.19b It is noteworthy that dual
luminescence decays have been reported in other heteroleptic
iridium(III) terpyridine complexes in fluid solutions at room
temperature.27b
The emission lifetimes of the complexes in CH3CN at 298 K
are remarkably long (Table 3). The reason for the observation
that the excited state of 3 is the longest-lived (16.6 ms) while
that of 4 the shortest (8.4 ms) is not fully understood. One
possible explanation, however, is that the degree of 3MLCT
character of the excited state of 3 is lower than that of 4.
Mixing of 3MLCT character into the 3IL excited states of Ir(III)
terpyridine complexes has been shown to shorten the emission
lifetimes and lower the luminescence quantum yields con-
siderably.27 On the other hand, it is interesting to note that
while the 77 K emission lifetime of 1 follows a single expo-
nential (ca. 144 ms), 2–4 display double-exponential decays,
with longer- and shorter-lived components of ca. 123–109 ms
and 25–19 ms, respectively. We assign the longer-lived com-
ponent, which is approximately an order of magnitude longer
than the solution emission lifetimes at room temperature, to a
mixed 3IL=3MLCT excited state involving mainly the tpy-
C6H4-NCS-p ligand. The shorter-lived component appears to
Labelling of proteins
Since the isothiocyanate moiety can react with primary amine
groups of biomolecules to form stable thiourea,28,29 it is
our intention to incorporate an isothiocyanate moiety into
Table 3 Photophysical data for complexes 1–4
lema=nm
toa=ms
Fa
kra=sÀ1
knra=sÀ1
tb=ms
lemc=nm
toc=ms
1
2
3
4
530, 555 sh
528, 555 sh
527, 558 sh
528, 558 sh
13.2
0.025
1.9 Â 103
 103
7.4 Â 104
7.7 Â 104
5.6 Â 104
1.2 Â 105
1.4
1.3
1.5
1.4
500, 534, 578 sh
500, 534, 578 sh
504, 530, 576sh
502, 534, 578 sh
144.0
12.60.031
16.6
8.4
2.5
0.064
0.030
123.4 (74%), 24.2 (26%)
108.9 (28%), 25.3 (72%)
117.3 (48%), 18.8 (52%)
3.9 Â 103
3.6 Â 103
a
b
c
In degassed CH3CN at 298 K. In aerated CH3CN at 298 K. In EtOH–MeOH (4 : 1 v=v) at 77 K.
86
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Table 4 Photophysical data for conjugates 1-HSA and 1-BSA in 50
mM Tris-HCl pH 7.4 at 298 K
isothiocyanate complexes display intense and long-lived emis-
sion in degassed and aerated acetonitrile solutions at 298 K
and in low-temperature glass. The emission is assigned to
lema=nm
toa=ms
tb=ms
3
Conjugate
originate from a predominant IL [p ! p*(terpyridines)] exci-
3
ted state mixed with some MLCT [dp(Ir) ! p*(terpyridines)]
1-HSA
1-BSA
530
530
1.52 (10%)
0.17 (90%)
0.99 (19%)
0.11 (81%)
1.32 (16%)
0.21 (84%)
0.85 (23%)
0.15 (77%)
character. Two proteins, HSA and BSA, have been labelled
with 1. The bioconjugates exhibit intense and long-lived
emission in aqueous buffers under ambient conditions. The
potential use of these luminescent iridium(III) labelling reagents
in different bioanalytical applications is currently under
investigation.
a
b
In degassed buffer. In aerated buffer.
luminescent iridium(III) terpyridine complexes so that they can
be used as covalent labels for biological substrates. In this
work, the proteins HSA and BSA have both been labelled with
complex 1. The bioconjugates 1-HSA and 1-BSA were purified
by size-exclusion chromatography to remove the unreacted
complex. The electronic absorption spectra of the conjugates
display intense absorptions in the visible region, ascribed to the
absorption properties of the iridium(III) chromophores of the
labels. The iridium : protein ratios have been estimated based
on the absorption spectral data according to the following
equation:
Acknowledgements
We thank the Hong Kong Research Grants Council (project
no. CityU 1116=00P) and the City University of Hong Kong
for financial support. C.K.C. acknowledges the receipt of a
postgraduate studentship and a Research Tuition Scholarship,
both administered by the City University of Hong Kong. We
are grateful to Dr Vicky W. M. Lee and Dr Y. L. Pui for
syntheses of some of the ligands and Prof. Vivian W. W. Yam
of The University of Hong Kong for access to the equipment
for photophysical measurements and for helpful discussions.
½IrŠ
A380e280p
¼
½proteinŠ A280e380i ꢀ A380e280i
where A380 and A280 are the absorbance values of the bio-
conjugates at 380 and 280 nm, respectively; e280p is the
extinction coefficient of the protein at 280 nm; e380i and e280i
are those of the iridium complex at 380 and 280 nm, respec-
tively. Iridium : protein ratios of 2.7 and 1.4 have been
determined for 1-HSA and 1-BSA, respectively. These values
are similar to related systems with other transition metal
complexes as labels.7,8b,d,e,14
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4
kq ¼ 2.9 Â 108 dm3 molÀ1
s
À1; 1-BSA, kq ¼ 1.3 Â 109 dm3
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s
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8
We have synthesised and characterised a series of new
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functional group. The syntheses of these complexes have
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