Four phosphorescent rhenium(I) polypyridine amine complexes appended
with a poly(ethylene glycol) (PEG) pendant
[Re(N^N)(CO)3(py-PEG-NH2)](PF6) (py-PEG-NH2 =
3-amino-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine,
MWPEG = 5000 Da, PDIPEG < 1.08; N^N = 1,10-phenanthroline (phen),
3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen),
4,7-diphenyl-1,10-phenanthroline (Ph2-phen)) and
[Re(bpy-PEG)(CO)3(py-NH2)](PF6) (bpy-PEG =
4-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)-4'-methyl-2,2'-
bipyridine; py-NH2 = 3-aminopyridine) and their PEG-free counterparts
[Re(N^N)(CO)3(py-Et-NH2)](PF6) (py-Et-NH2 =
3-amino-5-(N-(ethyl)aminocarbonyl)pyridine; N^N = phen, Me4-phen,
Ph2-phen) and [Re(bpy-Et)(CO)3(py-NH2)](PF6) (bpy-Et =
4-(N-(ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine) were synthesized and
characterized and their biological properties were examined. Upon
photoexcitation, all the complexes displayed intense and long-lived green to
orange triplet metal-to-ligand charge-transfer (3MLCT) (dπ(Re) → π*(N^N))
emission. Interestingly, while the PEG pendants did not significantly perturb
their emission energy, the emission quantum yields of the PEG-amine
complexes were lower than those of their PEG-free counterparts. The PEG
complexes exhibited significantly higher water solubility and lower cytotoxicity
than their PEG-free counterparts, indicating that the covalent attachment of a
PEG pendant to rhenium(I) polypyridine complexes is an effective way to
increase their biocompatibility. All the complexes were found to be
effectively internalized into HeLa cells. Upon internalization, they were
diffusely distributed in the cytoplasm with enriched staining in mitochondria.
The PEG-amine complexes were activated with thiophosgene to yield the
isothiocyanate complexes [Re(N^N)(CO)3(py-PEG-NCS)](PF6) (py-PEG-NCS
= 3-isothiocyanato-5-(N-(2-(ω-methoxypoly(1-oxapropyl))-
ethyl)aminocarbonyl)pyridine; N^N = phen, Me4-phen, Ph2-phen) and
[Re(bpy-PEG)(CO)3(py-NCS)](PF6) (py-NCS = 3-isothiocyanatopyridine) and
their potential application as a new class of PEGylation reagents was
examined. These complexes were reacted with a model substrate
n-butylamine to yield the thiourea complexes
[Re(N^N)(CO)3(py-PEG-Bu)](PF6) (py-PEG-Bu = 3-n-butylthioureidyl-5-(N-
(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N^N = phen,
Me4-phen, Ph2-phen) and [Re(bpy-PEG)(CO)3(py-Bu)](PF6) (py-Bu =
3-n-butylthioureidylpyridine). Additionally, the isothiocyanate complexes
were used to label a protein bovine serum albumin (BSA) and a polymer
poly(ethyleneimine) (PEI). Upon irradiation, all the PEGylated BSA and PEI
conjugates exhibited intense and long-lived emission in aqueous buffer under
ambient conditions. The DNA-binding and polyplex-formation properties of
one of the PEGylated PEI conjugates were studied and compared with those
of unmodified PEI. As revealed from the results of agarose gel
electrophoresis and luciferase assays, the transfection properties of PEI were
retained after being PEGylated by the PEG-isothiocyanate complexes. The
in vivo toxicity of the one of the PEG-amine complexes was investigated
using zebrafish embryos as an animal model. Similar to the results of the
cellular experiments, the PEG complex exhibited higher LD50 values than its
PEG-free counterpart, indicating that modification of the complex with a PEG
unit can not only reduce its cytotoxicity, but also its in vivo toxicity.
A series of rhenium(I) polypyridine complexes functionalized with a
diaminoaromatic ligand [Re(N^N)(CO)3(py-DA)](CF3SO3) (py-DA =
3,4-diaminopyridine; N^N = phen, Me4-phen, Ph2-phen,
2,9-dimethyl-1,10-phenanthroline (Me2-phen)) was developed for
phosphorescent sensing of intracellular nitric oxide (NO). Since the
reactivity of the diaminoaromatic complexes toward NO is determined by the
electron density of the diaminoaromatic moiety, complexes equipped with an
electron-rich diaminoaromatic ligand [Re(N^N)(CO)3(py-DA-OCH3)](CF3SO3)
(py-DA-OCH3 = 3-(N-(2-amino-5-methoxyphenyl)aminomethyl)pyridine; N^N
= phen, Me4-phen, Ph2-phen) were designed as reactive phosphorogenic
sensors for NO. These complexes were only weakly emissive due to the
diaminoaromatic moiety that quenches the 3MLCT (dπ(Re) → π*(N^N))
emission by photoinduced electron transfer (PET). However, in the
presence of NO, these weakly-emssive diamine complexes were converted to
the strongly-emissive triazole derivatives
[Re(N^N)(CO)3(py-triazole)](CF3SO3) (py-triazole =
1H-1,2,3-triazolo[4,5,c]pyridine; N^N = phen, Me4-phen, Ph2-phen, Me2-phen)
and [Re(N^N)(CO)3(py-triazole-OCH3)](CF3SO3) (py-triazole-OCH3 =
3-((6-methoxybenzotriazol-1-yl)methyl)pyridine; N^N = phen, Me4-phen,
Ph2-phen), respectively. Upon addition of an NO-releasing reagent
3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7)
to a solution of the diamine complex, significant emission enhancement (I/Io =
ca. 31 − 62) was observed. The emission enhancement was attributed to
the conversion of the diamine complex to its triazole counterpart, which was
subsequently confirmed by ESI-MS. These complexes displayed high
reaction selectivity toward NO, and their emission intensity was found to be
independent on pH under neutral and basic conditions (pH > 6). Although
both series of complexes were effectively internalized into HeLa cells, they
displayed very different intracellular NO-sensing properties. Confocal
microscopy images show that the py-DA complexes could not be converted to
their triazole derivatives inside living cells. In contrast, the py-DA-OCH3
complexes can not only detect NO produced exogenously in HeLa cells by
NOC-7, but also NO that was generated endogenously by murine
macrophage RAW264.7 cells upon treatment with lipopolysaccharide (LPS).
These results highlighted that the rich electron density of the diaminoaromatic
ligand is crucial to the intracellular NO-sensing capability of the complexes.
Three phosphorescent rhenium(I) polypyridine dibenzocyclooctyne
(DIBO) complexes [Re(N^N)(CO)3(py-C6-DIBO)](CF3SO3) (py-C6-DIBO =
3-(N-(6-(3,4:7,8-dibenzocyclooctyne-5-oxycarbonylamino)hexyl)aminocarbonyl)
pyridine; N^N = phen, Me4-phen, Ph2-phen) and their DIBO-free
counterparts [Re(N^N)(CO)3(py-C6-BOC)](CF3SO3) (py-C6-BOC =
3-(N-(6-(tert-butoxycarbonylamino)hexyl)aminocarbonyl)pyridine; N^N = phen,
Me4-phen, Ph2-phen) were synthesized and characterized. Upon
photoexcitation, all the complexes displayed intense and long-lived yellow
3MLCT (dπ(Re) → π*(N^N)) emission. The second-order rate constants (k2)
for the reactions of the DIBO complexes toward benzyl azide were
determined to range from 0.077 − 0.091 M–1 s–1, which are similar to those of
other DIBO compounds such as 4-dibenzocyclooctynol. This indicates that
modification of the DIBO unit with a positively-charged rhenium(I) center did
not significantly perturb its reactivity toward azide. As revealed from the
SDS-PAGE analysis, the DIBO complexes can be selectively coupled to
azide-modified proteins including BSA, human serum albumin (HSA), and
apotransferrin (aTf) and the resulting bioconjugates displayed strong
phosphorescence upon photoexcitation. The DIBO complexes were found
to accumulate in CHO cells with moderate cytotoxicity. Upon incubation of
CHO cells with these complexes, relatively weak intracellular emission was
observed. In contrast, upon pretreatment of the cells with
1,3,4,6-tetra-O-acetyl-N-azidoacetyl-D-mannosamine (Ac4ManNAz), intense
emission from the cell membrane and some internal compartments were
observed. All these results indicated that the DIBO complexes are possible
reagents for the imaging of azide-labeled biomolecules.
Apart from the strained alkyne-azide couple, tetrazine and dienophile
is another commonly used reaction couple for bioorthogonal applications.
Four mononuclear rhenium(I) tetrazine complexes
[Re(N^N)(CO)3(py-3-Tz)](CF3SO3) (py-3-Tz =
6-methyl-3-pyrid-3-yl-1,2,4,5-tetrazine; N^N = Me2-phen, Ph2-phen) and
[Re(N^N)(CO)3(py-4-Tz)](CF3SO3) (py-4-Tz =
6-methyl-3-pyrid-4-yl-1,2,4,5-tetrazine; N^N = Me2-phen, Ph2-phen) and two
binuclear rhenium(I) tetrazine complexes
[{Re(N^N)(CO)3}2(μ-py-Tz-py)](CF3SO3)2 (py-Tz-py =
3,6-bis(pyrid-3-yl)-1,2,4,5-tetrazine; N^N = Me2-phen, Ph2-phen) were
synthesized and characterized. Upon photoexcitation, all the complexes
exhibited 3MLCT (dπ(Re) → π*(N^N)) emission. These complexes were only
weakly emissive due to the tetrazine moiety that quenches the 3MLCT (dπ(Re)
→ π*(N^N)) emission by fluorescence resonance-energy transfer (FRET).
The reactivity of the tetrazine moiety toward dienophiles including
5-norbornen-2-ol (NBO) and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol
(BCN) was significantly enhanced after being attached to the
positively-charged rhenium(I) polypyridine unit. Additionally, the reactivity of
the binuclear complexes was substantially higher than that of their
mononuclear counterparts. Upon addition of the dienophile to a solution of
the tetrazine complex, emission enhancement was observed. Interestingly,
the emission enhancement factors for the reaction with BCN (from 8.0 to
181.1) were significantly larger than those with NBO (from 1.1 to 8.0),
indicating that the reaction products play an important role in determining the
phosphorogenic properties of the complexes. As revealed from SDS-PAGE
analysis, the tetrazine complexes can be selectively coupled to BCN-modified
proteins and the resulting bioconjugates displayed strong phosphorescence
upon photoexcitation. Although the tetrazine complexes were effectively
internalized into HeLa cells, no significant intracellular emission was observed
upon incubation of the cells with these complexes. In contrast, after
pretreatment of HeLa cells with the BSA-BCN conjugate, punctate
cytoplasmic staining was observed, indicating that the tetrazine complex can
function as a versatile bioorthogonal probe to trace BCN-tagged biomolecules
in an intracellular environment.
| Date of Award | 16 Feb 2015 |
|---|
| Original language | English |
|---|
| Awarding Institution | - City University of Hong Kong
|
|---|
| Supervisor | Kam Wing Kenneth LO (Supervisor) |
|---|