Recent advances in laser confocal and multi-photon microscopy have greatly facilitated in-depth studies of many cellular and biomedical relevant biological / biochemical processes. Multi-photon techniques offer a series of special advantageous features for bio-imaging, such as the reduction of autofluorescence and photo-damage to biological samples, and the capability of deep tissue penetration, that allow unambiguous observation of the various sub-cellular structures within live cells at different stages of their cell cycle. They also enable the reveal of real-time and near-real-time information about the formation, transportation, utilization and degradation of targeted biomolecules within live cells and tissues. Multi-photon techniques are also beginning to find their uses in in vivo imaging for biomedical studies as well as in clinical diagnostics. However, up to now, there are still not Recent advances in laser confocal and multi-photon microscopy have greatly facilitated in-depth studies of many cellular and biomedical relevant biological / biochemical processes. Multi-photon techniques offer a series of special advantageous features for bio-imaging, such as the reduction of autofluorescence and photo-damage to biological samples, and the capability of deep tissue penetration, that allow unambiguous observation of the various sub-cellular structures within live cells at different stages of their cell cycle. They also enable the reveal of real-time and near-real-time information about the formation, transportation, utilization and degradation of targeted biomolecules within live cells and tissues. Multi-photon techniques are also beginning to find their uses in in vivo imaging for biomedical studies as well as in clinical diagnostics. However, up to now, there are still not sufficient effective bio-compatible multi-photon luminophores that are available for the construction of useful imaging probes. There is a great and urgent need for the development of new multi-photon luminophores to meet the demand of live cell and biomedical imaging.
Cyclometalated platinum(II) complexes are known to possess rich photophysical and photochemical properties. On the other hand, the application of this class of organometallic complexes as two-photon cell imaging probes is scarce. This thesis describe the design, synthesis, photophysical properties and potentials in live cell imaging applications of two new types of cyclometalated platinum(II) complexes that contain 1,3-azolyl cyclometalating ligands (HL2 and HL3), both possess a Cphenyl, a Npyridyl and a Nimidazolyl donor moiety. The only difference between these two ligand systems is the orientation of the imidazole ring with respect to the central pyridyl donor. We demonstrated that the use of heterocyclic imidazolyl donors enabled easy modification of the cyclometalating ligands for the fine-tuning of the photophysical properties of the resulting cyclometalated platinum(II) complexes.
A series of cycloplatinated complexes of HL2 and HL3, with different ancillary ligands (e.g. Cl, PPh3, dppm) have been prepared and their photophysical properties examined. All of these complexes were found to be emissive in solutions at room
temperature. In acetonitrile, emission maxima of [Pt(L2)Cl] and [Pt(L3)Cl] were
centered at 521 nm (φlum = 0.18, τo = 1.28 μs) and 503 nm (φlum = 0.10 τo = 2.36 μs)
respectively. Origins of these emissions were assigned the triplet metal-to-ligand
charge transfer (3MLCT) transition, probably mixed with some triplet intraligand (3IL)
character. Upon replacing the chloride ancillary ligand with triphenylphosphine, the
cationic complexes [Pt(L2)PPh3]+ (λmax = 510 nm, φlum = 0.22, τo = 6.08 μs) and
[Pt(L3)PPh3]+ (λmax = 491nm, φlum = 0.23, τo = 6.66 μs) were found to have their
emission maxima blue shifted, together with a slight increase in emission quantum
yield and emission lifetime. Upon deprotonation of the 1-imidazolyl-NH on their
cyclometalating ligands, a gradual red-shift in emission maxima together with a
decrease in emission intensity were observed for both complexes. The acid
dissociation constants, pKa, of the two complexes in 2:1(v/v) DMF / aqueous buffer
were found to be ca. 4.3 and 5.0 respectively. By replacing the chloride ligand with a
bridging ligand, 1,1-bis(diphenylphosphino)methane (μ-dppm), cycloplatinated
dimers were formed. The two resulting complexes {[Pt(L2)]2(μ-dppm)}2+ (λmax = 621
nm, φlum = 0.19, τo = 2.06 μs) and {[Pt(L3)]2(μ-dppm)}2+ (λmax = 574 nm, φlum = 0.21,
τo = 2.10 μs) were found to have their room temperature emission maxima in solution
red shifted compared to their corresponding PPh3 mononers. This is attributable to the staining specifically. For other cycloplatinated complexes, they were mainly found in
the cytoplasm after live cell internalization. Cell images taken with linear (λex = 405
nm) and two-photon (λex = 750 nm) excitation overlap well with each other. This
further confirms that the newly developed cyclometalated Pt(II) complexes are good
two-photon luminophores and probes for cell imaging.
Finally, the 1-imidazolyl-NH sites of the new cycloplatinated complexes were
utilized for the functionalization of the two-photon luminophores with reactive
moieties for the bio-labelling purposes. Functional groups such as succinimide and
maleimide, which are reactive towards amines and thiols moieties commonly found in
biomolecules such as proteins, antibodies and small peptides, were incorporated into
the cyclometalated Pt(II) luminophores via a carbon chain linker attached to the
1-imidazolyl-N site of the ligands. Abilities of the resulting complexes to label
biomolecules under mild and bio-compatible conditions were demonstrated with BSA,
a common serum albumin protein. Multiple labeling of the protein with the
luminophores was easily achieved without self-quenching. This demonstrates one of
the key advantages of coordination and organometallic luminophores over commonly
used organic-based luminophores in fluoro-tagging of biomolecules. Furthermore,
gradual red-shift in the emission of the cyclometalated Pt(II) bio-labeling agents were observed upon increased loading of the cycloplatinated fluoro-tags on the protein. Controlled unfolding of the labeled protein confirmed that this red-shift in the emission maxima was resulted from the formation of Pt-Pt interactions among the cyclometalated Pt(II) bio-labels on the protein that were in close proximity to each other.
| Date of Award | 3 Oct 2012 |
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| Original language | English |
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| Awarding Institution | - City University of Hong Kong
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| Supervisor | Hon Wah Michael LAM (Supervisor) |
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