Chemistry - A European Journal
10.1002/chem.201603630
COMMUNICATION
stained by Ir(ppy)2bpy after 7 days has also been shown in
Figure 3, which indicates these iridium(III) staining agents have
good stabilities.
agents have equivalent or even better performance than the
commercial agent of SYPRO Ruby both in terms of LOD and
time cost. It’s the first time to thoroughly reveal the relationships
between chemical structure of luminescent iridium(III)
complexes and the performance of protein staining. Furthermore,
this work gives the first experimental evidence of the potential
applications of charged-neutral complexes in protein staining,
which opens an avenue to exploit novel protein staining agents
in the future.
Moreover, commercial staining agent of SYPRO Ruby has
also been performed as positive standard to evaluate the
properties of these iridium(III) agents. The image of protein
bands after SDS-PAGE and the LOD are shown in Figure S1
and listed in Table S2, respectively. Accordingly, we can safely
assumed that these iridium(III) staining agents are equivalent or
even better than the commercial agent of SYPRO Ruby in terms
of LOD. Beyond LOD, the staining protocol of these iridium(III)
agents is also simpler than that of SYPRO Ruby because there
is no troublesome of de-staining step for these iridium(III)
staining agents.
Last but not the least, after carefully investigating the
properties of these iridium(III) staining agents, including LOD
and protein to protein variations, it’s interesting to find that
staining agents containing 2-phenypyridine (ppy), i.e. cationic
complex 1, Ir(ppy)2bpy and charged-neutral complex Ir(ppy)3
outperformed to other iridium(III) staining agents. Increasing
conjugation of coordination ligand, either main ligand (from
complex 1 to complex 3) or ancillary ligand (Ir(ppy)2bpy to
complex 1) would not obviously improve the performance of
staining agents. Even the charged-neutral complex, for example
Ir(ppy)3, also have excellent performance of protein staining.
Though the cationic staining agents is better than its
corresponding charged-neutral form in the point of LOD (for
example, comparing 1 with Ir(ppy)3 or comparing 3 with Ir(pq)3),
we can safely make a conclusion that the electrostatic forces
may play some effect in the staining process, but not the
decisive factor. As for the reasons of staining mechanism for
these agents, as we know, these iridium(III) complexes are
hydrophobic[39] and the proteins in the gel after SDS-PAGE are
also hydrophobic,[23] which may be the direction of explaining
their binding affinities.
Supporting Information
The synthetic routes and characterization of iridium(III) complexes;
experimental details for protein staining, living cell imaging and
cytotoxicity assay; the summarized photophysical properties of 1-4; the
protein staining results from commercial agent of SYPRO Ruby; The
limits of detection (LOD) of iridium complexes and SYPRO Ruby for
different proteins; Fluorescence images of HeLa cells incubated with
complexes 1-4 for 1 hour at 37 °C; Cytotoxicity of complexes 1-4 and
NMR spectra.
Acknowledgements
This work was supported by National Natural Science
Foundation of China (NSFC No. 21505097, 21302213); NSF of
Jiangsu Province (BK20150283); University Scientific Research
Project of Jiangsu Province (15KJB150027); the Priority
Academic Program Development of Jiangsu Higher Education
Institutions (PAPD); Excellent Innovation Team in Science and
Technology of University in Jiangsu Province and Jiangsu
Collaborative Innovation Center of Technology and Material for
Water Treatment.
Beyond protein staining, we have also extended the
potential applications of cationic luminescent complexes 1-4 into
living cell imaging. The luminescent images of HeLa cells co-
incubated with 20 M iridium(III) complexes for 1 hours at 37 °C
has been shown in Figure S2 in Supporting Information, which
shows that the intracellular distribution of complexes 1-4 with
DIP ligand is mainly in the cytoplasm, rather than nucleus and
membrane. Furthermore, the cytotoxicity assays of complexes
1-4 towards three cancer cell lines (HeLa, A549, MCF-7) are
performed and the IC50 data has also been listed in Table S3 in
Supporting Information. The lower IC50s of complexes 1-4
toward cancer cell lines indicate complexes 1-4 have anticancer
activities as well. All these results demonstrated these cationic
iridium(III) complexes with DIP ligand have potential applications
in bio-imaging or emerging cancer therapeutics beyond protein
staining.
In conclusion, seven luminescent iridium(III) complexes have
been synthesized and investigated their performances in protein
staining and living cell imaging. Most importantly, beside the
effect of main ligand, the conjugation effect of ancillary ligand
and charge effect of iridium(III) complexes on protein staining
have also been studied by carrying out experiments using three
prominent control compounds named Ir(ppy)2bpy, Ir(ppy)3 and
Ir(pq)3 as protein staining agents. These iridium(III) staining
Keywords: iridium complex • protein staining • neutral •
luminescent • organometallic
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