"Turn-on" FRET-based luminescent iridium(iii) probes for the detection of cysteine and homocysteine

Article abstract of DOI:10.1039/c0cc04288a

"Turn-on" FRET-based luminescent probes containing iridium(iii) complexes for the detection of Cys and Hcy have been designed and synthesized. The relationship between the steric bulk of the probe and the corresponding emission intensity enhancement towards detection of Cys and Hcy has been studied.

Full text of DOI:10.1039/c0cc04288a

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‘‘Turn-on’’ FRET-based luminescent iridium(III) probes for the detection
of cysteine and homocysteinew
Hoi-Yan Shiu,a Man-Kin Wong*b and Chi-Ming Che*a
Received 8th October 2010, Accepted 22nd February 2011
DOI: 10.1039/c0cc04288a
‘‘Turn-on’’ FRET-based luminescent probes containing
iridium(III) complexes for the detection of Cys and Hcy have been
designed and synthesized. The relationship between the steric bulk
of the probe and the corresponding emission intensity enhancement
towards detection of Cys and Hcy has been studied.
these shortcomings, new FRET systems using transition metal
complexes, such as that of ruthenium(II),15 osmium(III)16 and
rhenium(I),17 have been reported. Studies on FRET systems based
on luminescent iridium(III) complexes are sparse.18 In this work,
we explored the use of luminescent iridium(III) complexes in the
design of FRET-based probes for the detection of Cys and Hcy.
Along with the development of a ‘‘turn-on’’ FRET-based
fluorescent probe for the detection of Cys and Hcy,19 we used
iridium(III) complexes to replace the organic fluorophore in
the design of the FRET system. The iridium(III) complex,
[Ir(ppy)2(phen-NHCOCH2I)]+, displays a comparatively red-
shifted emission maximum at 590 nm and with a high emission
quantum yield of 0.23 in CH3CN upon excitation at 360 nm.7
On the basis of a thiol-assisted cleavage of vinyl sulfide linkage
from an alkynone-modified cysteine-containing peptide,20 the
luminophore [Ir(ppy)2(phen-NHCO–)]+ was incorporated in
the design of FRET-based probes for the detection of Cys and
Hcy.19 The synthesis and characterization of the iridium(III)
probes are given in ESI.w
Probe 1 is weakly emissive (FF = 0.0028) with a spectral
overlap between the emission spectrum of the iridium(III)
luminophore and the absorption spectrum of quencher.
Excitation at 360 nm would therefore result in diminished
emission at wavelength 590 nm and shortened lifetime (ESIw).
Attack on 1 by thiol separates the metal luminophore from the
quencher. As a result, an enhanced emission at 590 nm from
the dye 1M could be detected (Scheme 1).
Luminescent transition metal complexes continue to receive a
surge of interest in the design of biological probes and labels1–4
owing to their attractive photophysical properties such as long
emission lifetimes (100 ns to ms), tunable emission wavelengths,
high photostability and large Stokes shifts (hundreds of nm).5,6
In this regard, iridium(III) complexes are appealing for the
design of biological probes. Iridium(III) complexes containing
cyclometalated and polypyridine ligands display rich photo-
physical properties and a broad range of iridium(III) complexes
with tunable excitation and emission maxima can be prepared.6,7
Li and co-workers had demonstrated a neutral iridium(III)
complex [Ir(pba)2(acac)]8 and a cationic iridium(III) complex
[Ir(pba)2(bpy)]+ 9 as phosphorescent chemodosimeters for the
detection of cysteine (Cys) and homocysteine (Hcy); both Cys
and Hcy are well known to play significant roles in human
health.10–12 However, these iridium(III) probes were reported
to have a long response time of 24 h. Therefore, it remains a
challenging task to develop selective and sensitive luminescent
metal-based probes having a short response time for the
detection of Cys and Hcy.
Fluorescence resonance energy transfer (FRET) has been
extensively used as an operational principle in the arena of
molecular imaging and bioanalysis, because FRET systems are
sensitive to subtle changes in the separation distance between the
donor and acceptor.13,14 However, most FRET systems are
constructed by connecting two organic chromophores, suffer
from small Stokes shifts (tens of nm) and short emission lifetimes
(1–20 ns), and are susceptible to photo-bleaching. To circumvent
The time course of the reaction between probe 1 and Hcy
was studied by monitoring the emission intensity of the
reaction mixture at 590 nm (Fig. 1). Upon treatment with
Hcy (1.25 mM) in pH 8.1 PBS buffer/CH3CN (1/3) at room
temperature, a more than 32-fold increase in the emission
intensity at 590 nm was detected within the first 5 min. This
increase in emission intensity signifies a high initial reaction
rate of the cleavage of vinyl sulfide linkage of 1 by the
nucleophilic Hcy. After 15 min, the increase in emission
intensity leveled off. Therefore, an assay time of 15 min was
used in the evaluation of the performance of 1 in the detection
of thiols in subsequent studies. Up to a 46-fold increase in
emission intensity was observed when 1 (25 mM) was allowed
to react with Hcy (1.25 mM) and in a molar ratio of 1 : 50.
Up to a 33-fold increase in emission intensity was observed
when 1 at 25 mM reacted with Cys (1.25 mM) in a molar ratio
of 1 : 50.
a Department of Chemistry, State Key Laboratory on Synthetic
Chemistry and Open Laboratory of Chemical Biology of the Institute
of Molecular Technology for Drug Discovery and Synthesis,
The University of Hong Kong, Pokfulam Road, Hong Kong, China.
E-mail: cmche@hku.hk; Fax: +852 2857-1586
b Department of Applied Biology and Chemical Technology,
The Hong Kong Polytechnic University, Hung Hom, Kowloon,
Hong Kong, China. E-mail: bcmkwong@inet.polyu.edu.hk;
Fax: +852 2364-9932
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c0cc04288a
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4367–4369 4367

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emission intensity at 590 nm was obtained when the concentra-
tion ratio of [Cys]/[1] was increased from 0 to 100 (ESIw).
To examine the selectivity of the FRET-based iridium(III)
probes towards thiols, probe 1 (25 mM) was treated with a panel
of twenty essential amino acids, Hcy and GSH. Significant
enhancement in emission intensity has been found upon addi-
tion of Hcy or Cys to probe 1 in solution. On the other hand,
treatment of up to 50 equivalents of GSH or the other 19 amino
acids (Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys,
Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val) with 1 did not result in
emission enhancement (Fig. 3). Probe 1 was found to be
selective towards Hcy and Cys. The intensity of the emission
signals from the three biological thiols are in the order of
Hcy > Cys c GSH. In the literature, the steric bulk of these
three thiols are in the ascending order: Hcy o Cys { GSH.21
The thiol group residing on Hcy is relatively less bulky than
that on Cys.21 This difference in steric bulk accounts for the
difference in emission intensity enhancements in the detection
of Hcy and that of Cys by probe 1. The ratio of emission
intensity enhancements upon the addition of Hcy and Cys is
1.4 : 1. Attempts were made to further increase the ratio of
emission intensity enhancements. A series of alkyl groups
(methyl 2a, isopropyl 2b, piperidinyl 2c and tert-butyl 2d)
were incorporated into the design of FRET-based iridium(III)
probes (Scheme 2). The alkyl group increases the steric bulk of
the environment around the vinyl sulfide linkage so that only
the less bulky Hcy is able to cleave the FRET-based probe,
leading to an enhancement in emission intensity. The synthesis
and characterization of probes 2a–d are given in ESI.w
Scheme 1 Working principle of 1.
Fig. 1 Respective time course of the reaction between 1 and Hcy and Cys.
Upon treatment of 1 (25 mM) with Hcy at different concentra-
tions in pH 8.1 PBS buffer/CH3CN (1/3), the emission intensity
at 590 nm was measured after 15 min (Fig. 2). When the emission
intensity of the mixture containing 1 and Hcy at 590 nm was
plotted against Hcy concentration in the range of 0–2.5 mM,
a calibration curve revealing a good linearity (correlation
coefficient R = 0.99, detection limit = 0.13 mM, ESIw) was
obtained. The addition of Hcy resulted in up to a 88-fold increase
in emission intensity at 590 nm when the concentration ratio of
[Hcy]/[1] was increased from 0 to 100. Up to a 65-fold increase in
Fig. 3 Emission enhancement in the detection of different analytes by 1.
Fig. 2 Emission spectral traces of 1 in the presence of Hcy (0–2.5 mM).
4368 Chem. Commun., 2011, 47, 4367–4369
Scheme 2 Structure of probes 2a–d.
c
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To compare the ratios of emission intensity enhancements, each
of the probes 2a–d at a concentration of 25 mM was treated with
Hcy or Cys (1.75 mM) in pH 8.1 PBS buffer/CH3CN (1/3), the
corresponding emission intensity at 590 nm was measured after
15 min. It was found that the ratio increased with the steric bulk of
the group in close proximity of the vinyl sulfide linkage (Fig. 4).
Probes 1 and 2a were found to differentiate Hcy from Cys with a
selectivity ratio of around 1.5 : 1. This ratio was increased to 2 : 1
and to 3 : 1 by an isopropyl group in 2b and a piperidinyl group in
2c respectively. Among the 5 probes prepared, the tert-butyl group
in 2d was found to lead to the greatest differentiation between Hcy
and Cys with a selectivity ratio of 5 : 1. Therefore, 2d was chosen
for the follow-up experiments.
Polytechnic University (PolyU Departmental General Research
Funds and Competitive Research Grants for Newly Recruited
Junior Academic Staff) and the Areas of Excellence Scheme
established under the University Grants Committee of the Hong
Kong Special Administrative Region, China (AoE/P-10/01).
Notes and references
1 (a) P. Wu, E. L.-M. Wong, D.-L. Ma, G. S.-M. Tong, K.-M. Ng
and C.-M. Che, Chem.–Eur. J., 2009, 15, 3652–3656; (b) P. K.-M.
Siu, D.-L. Ma and C.-M. Che, Chem. Commun., 2005, 1025–1027;
(c) C.-M. Che, J.-L. Zhang and L.-R. Lin, Chem. Commun., 2002,
2556–2557.
2 (a) K. K.-W. Lo, K. Y. Zhang, S.-K. Leung and M.-C. Tang,
Angew. Chem., Int. Ed., 2008, 47, 2213–2216; (b) K. K.-W. Lo,
W.-K. Hui, C.-K. Chung, K. H.-K. Tsang, D. C.-M. Ng, N. Zhu
and K.-K. Cheung, Coord. Chem. Rev., 2005, 249, 1434–1450.
3 K. M.-C. Wong, W.-S. Tang, B. W.-K. Chu, N. Zhu and
V. W.-W. Yam, Organometallics, 2004, 23, 3459–3465.
4 D.-L. Ma, W.-L. Wong, W.-H. Chung, F.-Y. Chan, P.-K. So,
T.-S. Lai, Z.-Y. Zhou, Y.-C. Leung and K.-Y. Wong, Angew.
Chem., Int. Ed., 2008, 47, 3735–3739.
Probe 2d was examined for the detection of Hcy and Cys in
commercial human blood plasma. The conversion of disulfides
to free thiols was conducted by treating the plasma sample
with a reducing agent, triphenylphosphine, according to the
literature procedure.22 For quantitative measurement, a standard
addition method with Hcy as the standard was employed to
estimate the concentrations of Hcy and Cys (ESIw). The total
concentration of Hcy and Cys in the plasma was found to be
0.31 mM; this value is well above the detection limit of probe
2d, i.e. 0.12 mM and is within the range of reported con-
centrations of Hcy and Cys found in normal human blood
plasma, i.e. 0.25–0.38 mM.23
5 V. Fernandez-Moreira, F. L. Thorp-Greenwood and M. P. Coogan,
´
Chem. Commun., 2010, 46, 186–202.
6 K. K.-W. Lo, Struct. Bonding, 2007, 123, 205–245.
7 K. K.-W. Lo, C.-K. Chung, T. K.-M. Lee, L.-H. Lui, K. H.-K.
Tsang and N. Zhu, Inorg. Chem., 2003, 42, 6886–6897.
8 H. Chen, Q. Zhao, Y. Wu, F. Li, H. Yang, T. Yi and C. Huang,
Inorg. Chem., 2007, 46, 11075–11081.
9 L. Xiong, Q. Zhao, H. Chen, Y. Wu, Z. Dong, Z. Zhou and F. Li,
Inorg. Chem., 2010, 49, 6402–6408.
´
10 (a) R. Hong, G. Han, J. M. Fernandez, B.-J. Kim, N. S. Forbes
In summary, we have developed FRET-based iridium(III)
complexes for the detection of Hcy and Cys on the basis of
conjugate addition of thiols to the vinyl sulfide linkage followed
by elimination. The iridium(III) probes display advantages of a
significantly low background emission signal and large Stokes
shift between excitation and emission energies. The probes
feature high selectivity for Hcy and Cys over other amino
acids and GSH under physiological conditions. The relation-
ship between the steric bulk of the vinyl sulfide linkage and
emission intensity enhancement by Hcy or Cys has also been
studied. The iridium(III) probe with a tert-butyl group is able
to differentiate Hcy from Cys in a ratio of 5 : 1. This probe
gave a fast signal response time and a good linearity range
(R = 0.99) for quantification. In addition, it could be used for
the detection of Hcy and Cys in human blood plasma.
This work was supported by The University of Hong Kong
(University Development Fund), Hong Kong Research Grants
Council (PolyU 7052/07P; HKU 1/CRF/08), The Hong Kong
and V. M. Rotello, J. Am. Chem. Soc., 2006, 128, 1078–1079;
(b) N. F. Zakharchuk, N. S. Borisova and T. V. Titova, J. Anal.
Chem., 2008, 63, 171–179; (c) M. Kemp, Y.-M. Go and
D. P. Jones, Free Radical Biol. Med., 2008, 44, 921–937.
11 S. Shahrokhian, Anal. Chem., 2001, 73, 5972–5978.
12 (a) S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques, I. H. Rosenberg,
R. B. D’ Agostino, P. W. F. Wilson and P. A. Wolf, N. Engl. J. Med.,
2002, 346, 476–483; (b) H. Refsum, P. M. Ueland, O. Nygard and
S. E. Vollset, Annu. Rev. Med., 1998, 49, 31–62; (c) O. Nekrassova,
N. S. Lawrence and R. G. Compton, Talanta, 2003, 60, 1085–1095.
13 K. E. Sapsford, L. Berti and I. L. Medintz, Angew. Chem., Int. Ed.,
2006, 45, 4562–4588.
14 M. J. Hangauer and C. R. Bertozzi, Angew. Chem., Int. Ed., 2008,
47, 2394–2397.
´
15 (a) J. Serin, X. Schultze, A. Adronov and J. M. J. Frechet,
Macromolecules, 2002, 35, 5396–5404; (b) E. K. Kainmuller,
´
¨
E. P. Olle and W. Bannwarth, Chem. Commun., 2005,
5459–5461; (c) J. M. Haider, R. M. Williams, L. De Cola and
Z. Pikramenou, Angew. Chem., Int. Ed., 2003, 42, 1830–1833.
16 (a) D. J. Hurley and Y. Tor, J. Am. Chem. Soc., 2002, 124,
13231–13241; (b) E. V. Bichenkova, X. Yu, P. Bhadra,
H. Heissigerova, S. J. A. Pope, B. J. Coe, S. Faulkner and
K. T. Douglas, Inorg. Chem., 2005, 44, 4112–4114.
17 (a) M.-J. Li, W.-M. Kwok, W. H. Lam, C.-H. Tao, V. W.-W.
Yam and D. L. Phillips, Organometallics, 2009, 28, 1620–1630;
(b) V. W.-W. Yam, H.-O. Song, S. T.-W. Chan, N. Zhu,
C.-H. Tao, K. M.-C. Wong and L.-X. Wu, J. Phys. Chem. C,
2009, 113, 11674–11682.
18 K. K.-W. Lo, M.-W. Louie and K.-Y. Zhang, Coord. Chem. Rev.,
2010, 254, 2603–2622.
19 H.-Y. Shiu, H.-C. Chong, Y.-C. Leung, M.-K. Wong and
C.-M. Che, Chem.–Eur. J., 2010, 16, 3308–3313.
20 H.-Y. Shiu, T.-C. Chan, C.-M. Ho, Y. Liu, M.-K. Wong and
C.-M. Che, Chem.–Eur. J., 2009, 15, 3839–3850.
21 S. Zhang, J. E. I. Wright, G. Bansal, P. Cho and H. Uludag,
Biomacromolecules, 2005, 6, 2800–2808.
22 W. Wang, J. O. Escobedo, C. M. Lawrence and R. M. Strongin,
J. Am. Chem. Soc., 2004, 126, 3400–3401.
23 O. Rusin, N. N. S. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang,
I. M. Warner, F. B. Dawan, K. Lian and R. M. Strongin, J. Am.
Chem. Soc., 2004, 126, 438–439.
Fig. 4 Emission enhancement in the detection of Hcy/Cys by 1, 2a–d.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4367–4369 4369