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
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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-
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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.
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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