A 18F radiolabelled Zn(II) sensing fluorescent probe

Article abstract of DOI:10.1039/c8cc00687c

A selective fluorescent probe for Zn(ii), AQA-F, has been synthesized. AQA-F exhibits a ratiometric shift in emission of up to 80 nm upon binding Zn(ii) ([AQA-F] = 0.1 mM, [Zn(ii)Cl₂] = 0-300 μM). An enhancement of quantum yield from Φ = 4.2% t

Full text of DOI:10.1039/c8cc00687c

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Reactivity differences of Sc
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18
A F Radiolabelled Zn(II) Sensing Fluorescent Probe
a‡
a‡
b
a
c
Thomas W. Price, George Firth, Charlotte J. Eling, Michelle Kinnon, Nicholas J. Long, Justin
a
a
Received 00th January 20xx,
Accepted 00th January 20xx
Sturge, and Graeme J. Stasiuk *.
DOI: 10.1039/x0xx00000x
www.rsc.org/
1
0
A
selective fluorescent probe for Zn(II), AQA-F, has been gland (total Zn(II) concentration = 800-1500
µM) where
synthesized. AQA-F exhibits a ratiometric shift in emission of up to uptake from extracellular fluids is controlled by the Zrt-like Irt-
7
8
0 nm upon binding Zn(II) ([AQA-F] = 0.1 mM, [Zn(II)Cl
µM). An enhancement of quantum yield from Φ = 4.2% to Φ = suppressor gene SLC39A1 (encoding ZIP1) downregulates ZIP1
5% is also observed. AQA-F has a binding constant, K = 15.2 µM protein levels in PCa tissue, leading to 62–75% lower levels of
2
= 0-300 like Protein 1 (ZIP1) transporter. Mutation of the tumor
3
d
9
with Zn(II). This probe has been shown to respond to endogenous Zn(II) accumulation. This significant decrease in malignant
Zn(II) levels in vitro in prostate and prostate cancer cell lines. tissue allows for the potential diagnosis of prostate cancer
1
8
[
F]AQA-F has been synthesized with a radiochemical yield of through measurement of the Zn(II) concentration in the
.6% and a radiochemical purity of 97% in 88 minutes. AQA-F prostate.
8
shows the potential for a dual modal PET/fluorescence imaging Over the last two decades, research into probes which give an
probe for Zn(II).
indication of disease progression by responding to different
concentrations of Zn(II) has become key area of
a
1
1,12,13
Prostate cancer (PCa) is the 2nd most common cancer
interest.
developed;
A plethora of optical probes for Zn(II) have been
typically by modifying organic fluorophores to
1
4–23
worldwide for males, with >161,000 new cases in the US in
1
2017 (19% of male cancer cases; 10% of total cancer cases).
include Zn(II) sensing units. Modification of fluorescein by
addition of dipicolylamine (DPA) units produced a high affinity
Diagnosis and active surveillance of PCa involves measurement
of serum levels of prostate specific antigen (PSA), with invasive
biopsies used for determination of the prognostic Gleason
Score. Novel, simple diagnostic tests are needed to catch the
disease early and this may be achieved by assessing
endogenous Zn(II) levels.
2
4
fluorescence based probe, ZP1. Further development led to
ZPP1 which demonstrates a ratiometric based detection of zinc
2
with a 10 nm shift in emission. Compartmentalisation of zinc
5
2
6
in diseases has been highlighted by similar probes.
Zn(II) has been identified as a key metal in a range of biological
,3
2
functions; its homeostasis is maintained by a number of
proteins including Zrt-like Irt-like proteins (ZIP) and cation
4
diffusion facilitators (CDF). Overabundance of Zn(II) is
5
implicated in a number of diseases (e.g. Alzheimer’s disease,
6
7
diabetes and PCa ). Zn(II) has emerged as a promising
diagnostic target in PCa progression as the homeostasis of
8
,9
Zn(II) is disrupted in diseased tissue.
The largest concentration of Zn(II) is found in the prostate
Scheme 1: Synthesis of AQA-F
Combining these optical probes with an additional imaging
2
7,28
modality can improve the properties of the probe.
particular, the pairing of PET and optical imaging can allow for
In
2
7,29
direct cellular validation of the PET probe.
Whilst
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9–33
radiolabelling of optical probes is well reported,
few addition of one equivalent; following this, the ratio began to
examples of “smart” dual modal optical-PET probes are plateau. This indicates that AQA-F binds Zn(II) in a 1:1 molar
3
1,34,35
present in the literature.
The quinoline-based AQZ probe developed by Zhang et al. is A decrease in fluorescence intensity was observed at acidic pH
an organic fluorophore and Zn(II) chelator showing excellent due to protonation of the secondary amine (pK = 6.62, Figure
d
ratio with a K of 15.2 µM.
3
6
a
ratiometric fluorescence properties upon Zn(II) binding. AQZ 3). This protonation also limits Zn(II) binding; there is no
has recently been shown attached to a MRI chelate as a dual- increase in fluorescence intensity in the presence of Zn(II) at
3
7
modal probe for diagnosis of diabetes. The Zn(II) binding acidic pH. The fluorescence intensity begins to increase at pH 6
properties of AQZ were retained after functionalization (pK = 6.86), with the maximum intensity observed in the
a
through the terminal alcohol group.
physiological pH window which is promising for biological
Figure 1. Fluorescence spectra of AQA-F (0.1 mM, λex = 320
nm) in HEPES buffer (10 mM, pH 7.68) solution in the presence
of increasing concentrations of Zn(II) (0-3 equivalents, 2.5-300
Figure 2. Absorption spectra of AQA-F (0.1 mM) in HEPES
buffer (10 mM, pH 7.68) in the presence of increasing
concentrations of Zn(II) (0-3 equivalents, 2.5-300 µM, n = 3).
µ
M, n = 3).
applications. Above pH 8.5 a reduction in fluorescence
We herein report the development of a fluorinated fluorescent
probe, AQA-F, which upon binding Zn(II) exhibits a change in
emission profile allowing endogenous Zn(II) levels to be
monitored. Furthermore, we have undertaken preliminary
radiolabeling studies to demonstrate the feasibility of
intensity of probe bound to Zn(II) was also observed (pKa=
9
.68). This decrease could be a result of a deprotonation of a
39
water molecule bound to the metal centre.
The fluorescent response of AQA-F demonstrates specificity
for Zn(II) over other divalent metal ions, especially those found
in biological environments (Figure 4). A small enhancement in
the emission at 500 nm of AQA-F is seen in the presence of
Ca(II). A significant increase in fluorescence emission was
observed for Zn(II). It must be noted that AQA-F has the
1
8
producing [ F]AQA-F. This Zn(II) sensor is designed to set the
foundation for a dual modal PET/fluorescence probe for
prostate cancer diagnosis.
AQA-F was synthesized in 4 steps from 2-fluoroethanol
(
Scheme 1). The fluorinated chain 2-(2-fluoroethoxy)ethan-1-
amine hydrochloride ( ) was prepared by condensation of tert-
butyl (2-hydroxyethyl)carbamate ( ) with 2-fluoroethyl 4-
methylbenzenesulfonate ( ) under basic conditions to yield
followed by deprotection under acidic conditions.
Conjugation of to 2-chloro-N-(quinol-8-yl)-acetamide (
6
3
4
5
3
8
6
2
),
3
6
under the same conditions reported for the synthesis of AQZ,
yielded AQA-F in an 80% yield.
AQA-F demonstrated an excitation maximum of 320 nm and
an emission maximum of 420 nm (Figure S4) with quantum
yield, Φ = 4.2% (Table S4). Upon Zn(II) binding an 8-fold
enhancement in fluorescence intensity was observed (Figure
1). This fluorescence enhancement (Φ = 35%) was also
accompanied by a red shift in the emission maxima of 80 nm
to 500 nm.
highest affinity for Zn(II) compared to all of these metals (Table
Three isosbestic points at 243, 278 and 323 nm were observed S3, Figure S1).
in the absorption spectra for AQA-F upon addition of Zn(II) Figure 3. Fluorescence intensity of AQA-F (0.1 mM, λex = 320
(Figure 2). The ratio of the absorbance at 260 and 278 nm nm, λem = 500 nm) in KCl (0.1 M) solution with variation of
(Inset, Figure 2) highlighted the binding of Zn(II) until the
2
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solution pH. Black = AQA-F, Red = AQA-F + 1 equivalent ZnCl
n = 3)
2
.
anhydrous MeCN (Figure S12). Following this, hydrolysis of the
1
Boc protecting group under acidic conditions gave [ F]AQA-F
8
(
with a 54.3% RCC (Figure S13). The radiochemical purity after
To validate AQA-F in vitro it was incubated with a healthy purification by semi-preparative HPLC is recorded at 97%,
epithelial prostate cell line (RWPE-1) and a grade IV prostate (Figure S10), with an isolated radiochemical yield of 8.6% with
adenocarcinoma cell line (PC-3) (Figure 5, Figure S3). AQA-F is a total synthesis time of 88 minutes, thus showing the
internalized by cells following a 30 minute incubation period.
The change in the emission profile of AQA-F upon binding
Zn(II) could be used to assess the relative level of Zn(II) in cells.
The ratio of emission at λem = 500 nm to λem = 420 nm (shown
in Figure 5) is directly related to the Zn(II) concentration
possibilities for a PET tracer for Zn(II).
(Figure 1). This ratio is greater in the healthy prostate cells
than in the PC-3 cell line (Table S5), indicating a greater Zn(II)
concentration in the RWPE-1 cell line. This reflects the reduced
Zn(II) uptake of prostate cancer cells due to the
7
,9,40
downregulation of the ZIP-1 Zn(II) transporter.
In both cell
lines, the ratio increases when incubated with additional Zn(II)
Figure 5. A collection of micrographs showing RWPE-1 and PC-
3
cells that have been incubated with 100
µM AQA-F and
Rhodamine Concanavalin A. Scale bar = 50 µm.
(Table S5).
AQA-F demonstrates a red shift of 80 nm from 420 nm to 500
nm when binding Zn(II). This novel probe has been shown to
bind Zn(II) in a 1:1 ratio with the highest affinity of all the
Figure 4. Metal binding assay showing the fluorescence
response of AQA-F (0.1 mM, λex = 320 nm, λem = 500 nm) in
HEPES buffer (10 mM, pH = 7.68) upon addition of 1 equivalent
of metal chloride. (n = 3)
d
metals tested, possessing a K of 15.2 µM. AQA-F exhibits a
fluorescence maximum at physiological pH and has been
shown to be internalized by healthy and cancerous prostate
cells within 30 minutes. The shift from 420 nm to 500 nm
when binding Zn(II) allows for differentiation of free probe and
1
8
Zn(II)-bound probe through fluorescent microscopy. [ F]AQA-
F
was radiolabelled in 8.6% RCY with radiochemical purity of
7%, thus showing the potential for dual modal
9
a
PET/fluorescence imaging probe for Zn(II). The fluorescent
probe, AQA-F, which has the potential for the in vitro
determination of endogenous Zn(II) levels has been
synthesized as both a cold standard and radiolabelled
1
8
1
8
Scheme 2. Synthesis of [ F]AQA-F
analogue [ F]AQA-F.
1
8
A preliminary study to produce [ F]AQA-F (Scheme 2) was
performed in a two-step process from . The first step
Conflicts of interest
8
1
8
There are no conflicts to declare.
introduces the F isotope through nucleophilic substitution at
the terminal tosylate leaving group, with a radiochemical
o
conversion (RCC) of 82.6%, in 15 minutes at 110 C in
Notes and references
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The authors would like to thank the British Heart Foundation for the
research grant FS/16/62/32220.
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