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DOI: 10.1039/C6CC10066J
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8·
PF , 15·PF and 16·PF ) (Figures S6-S13 and Table 2, ESI†). sections) collected from subjects with clinically diagnosed AD
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The emission spectra for these compounds are strongly and tissue and from an age-matched control were pre-
influenced by the nature of the conjugated amyloid binding treatment with bovine serum albumin (BSA) to prevent non-
moieties (
to , emission maxima occurred within the range of λem = 428 – with either 8a·PF
54 nm, while for the complexes linked to II, emission was red- examined by epi-fluorescence microscopy (λex = 359 nm, λem
I
and II). In the case of the Re(I) complexes coupled selective binding and the tissue sections were then treated
I
6
or 15·PF and the treated tissue was
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=
shifted to λem = 434 – 467 nm.
461 nm). The Re(I) complex treated sections were then
Ligand challenge experiments with L-histidine and L-cysteine compared to contiguous sections that were immune-stained
were conducted to evaluate the stability of the Re(I) with an anti-amyloid β peptide antibody 1E8 to identify Aβ
complexes 8a·PF 8b·PF and 16·PF . These Re(I) complexes plaques (Figure and b).
were chosen as they represent examples of the two different
isomeric forms produced by the acyclic ligand (8a·PF and
b·PF ) and a complex of the macrocyclic ligand (16·PF ). The
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,
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a
Fig. 4. (a) Epi-fluorescence microscopy image of AD affected frontal cortex brain
tissue treated with the Re(I) complex 15·PF (λex = 359 nm, λem = 461 nm) and (b)
microscopy image of the contiguous section immune-stained with an anti-
amyloid β peptide antibody 1E8 showing the positions of the amyloid plaques.
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reactions between the Re(I) complexes and the amino acids
were monitored by HPLC in combination with ESI-MS and the
results of these studies are shown in Figures S14 – S17 (ESI†).
The Re(I) complexes bearing the bis(NHC)-amine ligand
The Re(I) complex 8a·PF
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(benzothiazole moiety) showed no
showed
evidence of Aβ plaque binding but complex 15·PF
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excellent apparent co-locatisation with the immunostaining
(Figure 4a and b) and importantly, the epi-fluorescent image
coordination mode (8a·PF6 and 16·PF ) showed no sign of
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decomposition over a 24 h period in the presence of either of
these amino acids, while complex 8b·PF6 with the
NHC/amine/amidate ligand coordination mode was unstable
and decomposed within 4 h of mixing (results not shown).
Thioflavin T (ThT) is widely used for the detection of amyloid
and for evaluating interactions between molecules of interest
(
Figure S18, ESI†) for the age-matched control showed no
evidence of non-specific binding of complex 15·PF . The reason
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for the lack of amyloid binding displayed by 8a·PF is unknown
but it may be the associated with the inefficient binding of the
benzothiazole moiety in the presence of the bulky metal
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complex. In addition, the benzothiazole moiety of 8a·PF
1
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1
7 10, 18, 19
1
1
and amyloid fibrils.
The binding of Re(I) complexes
lacks the –OH group present on [ C]PiB (Figure 1), which may
also alter its capacity to bind to amyloid.
7a·PF6, 8a·PF6, 15·PF6 and 16·PF6 to fibrils formed from
synthetic Aβ1–42 peptide was evaluated in a ThT fluorescence
assay. The formation of fibrils from Aβ1-42 was confirmed by
increasing fluorescence intensity of ThT at λ = 485 nm in a
freshly prepared solution of Aβ1-42 over a period of 50 h
In conclusion, a series of Re(I) tricarbonyl complexes of acyclic
and macrocyclic tridentate NHC ligands coupled to stilbene- or
benzothiazole-based amyloid binding groups have been
synthesized. Two linkage isomeric forms were obtained for the
complexes of the acyclic ligand, while only the desired
coordination mode (bis-NHC-amine) was observed for the
macrocyclic ligands. The capacity of these compounds to bind
to amyloid plaques in human AD brain tissue was evaluated
(
control, Figure 3, black line). In contrast the addition of the
Re(I) complexes (20 M) resulted in little or no increase in
fluorescence intensity of ThT in the presence of Aβ1-42 (Figure
). These results suggest that the chosen Re(I) complexes bind
ꢀ
3
competitively with ThT to Aβ1-42 fibrils or that the Re
complexes inhibit fibril formation.
and complex 15·PF bound selectively to the Aβ plaques with
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low background fluorescence and no evidence of non-specific
binding. These results demonstrate the potential for
appropriately designed NHC ligands to be incorporated into
diagnostic imaging agents for AD. Radiochemical studies are
currently being undertaken to allow these bis(NHC)-amine
ligands to be labelled with Tc-99m. We note at this stage that
the cationic nature of these complexes may result in
undesirable biodistribution as certain lipophilic cationic Tc-
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9m complexes e.g. Tc-99m sestamibi (Cardiolite) and Tc-99m
tetrofosmin (Myoview) accumulate in myocardial cell
mitochondria as a result of increased membrane potentials
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0-22
and are used clinically as heart perfusion imaging agents.
This work has been supported by the Australian Research
Council (DP150102741) and the La Trobe University
Understanding Disease Research Focus Area.
Fig. 3. Time dependent changes in Thioflavin T (ThT) fluorescence over a period
of 52 h for solutions of ThT (40
ꢀ
M) and freshly prepared Aβ(1-42) (10
ꢀ
M) at 37
(Pink), 8a·PF (Green),
°
1
C in the presence of: no addition (control, black), 7a·PF
5·PF (Red) and 16·PF (Blue) (20 M in each case).
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Notes and references
The capacity complexes 8a·PF
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(conjugated II) and 15·PF
(
conjugated to I) to bind to the Aβ plaques in human AD brain
1
.
J. R. Jensen, K. Cisek, N. S. Honson and J. Kuret, Biorg.
Med. Chem., 2011, 19, 5147-5154.
tissues was investigated. Frontal cortex brain tissue (7 µm
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