A spiropyran with enhanced fluorescence: A bright, photostable and red-emitting calcium sensor

Article abstract of DOI:10.1016/j.tet.2017.11.020

A rationally designed, pyrene-spiropyran hybrid Ca²⁺ sensor (Py-1) with enhanced fluorescence intensity compared to a standalone spiropyran analogue is presented. Importantly, Py-1 retains the characteristic red emission profile of the spiropyran, while fibre-based photostability studies show the sensor is stable after multiple cycles of photoswitching, without any sign of photodegradation. Such properties are of real advantage for cell-based sensing applications. An interesting observation is that, Py-1 presents with two excitation options; direct green excitation (532 nm) of the photoswitch for a red emission, and UV excitation (344 nm) of the component pyrene, which gives rise to distinct blue and red emissions. This proof-of-concept hybrid sensing system presents as a more general approach to brighter spiropyran-based sensors.

Full text of DOI:10.1016/j.tet.2017.11.020

Accepted Manuscript
A spiropyran with enhanced fluorescence: A bright, photostable and red-emitting
calcium sensor
Georgina M. Sylvia, Sabrina Heng, Akash Bachhuka, Heike Ebendorff-Heidepriem,
Andrew D. Abell
PII:
S0040-4020(17)31156-0
10.1016/j.tet.2017.11.020
TET 29100
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 29 August 2017
Revised Date: 5 November 2017
Accepted Date: 7 November 2017
Please cite this article as: Sylvia GM, Heng S, Bachhuka A, Ebendorff-Heidepriem H, Abell AD,
A spiropyran with enhanced fluorescence: A bright, photostable and red-emitting calcium sensor,
Tetrahedron (2017), doi: 10.1016/j.tet.2017.11.020.
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A spiropyran with enhanced fluorescence:
a bright, photostable and red-emitting
calcium sensor
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Georgina M. Sylvia, Sabrina Heng, Akash Bachhuka, Heike Ebendorff-Heidepriem and Andrew D. Abell
ARC Centre of Excellence for Nanoscale BioPhotonics, Institute for Photonics and Advanced Sensing and
Department of Chemistry, The University of Adelaide, South Australia, Australia 5005.

1
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Tetrahedron
journal homepage: www.elsevier.com
A spiropyran with enhanced fluorescence: a bright, photostable and red-emitting
calcium sensor
a
a
a
a
Georgina M. Sylvia , Sabrina Heng *, Akash Bachhuka , Heike Ebendorff-Heidepriem and Andrew D.
Abell *
a
a
ARC Centre of Excellence for Nanoscale BioPhotonics, Institute for Photonics and Advanced Sensing and Department of Chemistry, The University of
Adelaide, South Australia, Australia 5005.
ARTICLE INFO
ABSTRACT
2
+
Article history:
Received
Received in revised form
Accepted
A rationally designed, pyrene-spiropyran hybrid Ca sensor (Py-1) with enhanced fluorescence
intensity compared to a standalone spiropyran analogue is presented. Importantly, Py-1 retains
the characteristic red emission profile of the spiropyran, while fibre-based photostability studies
show the sensor is stable after multiple cycles of photoswitching, without any sign of
photodegradation. Such properties are of real advantage for cell-based sensing applications. An
interesting observation is that, Py-1 presents with two excitation options; direct green excitation
Available online
(
532 nm) of the photoswitch for a red emission, and UV excitation (344 nm) of the component
Keywords:
Spiropyran
Merocyanine
pyrene, which gives rise to distinct blue and red emissions. This proof-of-concept hybrid sensing
system presents as a more general approach to brighter spiropyran-based sensors.
Fluorescence enhancement
Calcium sensor
Photoswitchable
2
009 Elsevier Ltd. All rights reserved.
1
7-20
1
. Introduction
Calcium ions (Ca ) act as a ubiquitous chemical signaller in
autofluorescence.
Finally, these sensors can be prepared in a
modular fashion to allow the incorporation of a range of metal-
specific ionophores to target a given metal ion.
2+
22-24
cell proliferation, differentiation and death; and as such are
essential for cellular function. It is thus important to sense and
quantify Ca in cells and this is typically achieved using small
molecule fluorescence-based sensors, in combination with
fluorescence microscopy.
1-4
While many spiropyran-based, photoswitchable metal ion
sensors have been reported, these are somewhat limited by an
inherent lack of brightness. An ability to enhance the
fluorescence signal of spiropyran would thus extend the range of
sensing capability for live-cell imaging using confocal
microscopy. One way to address this limitation is to couple a
traditional fluorophore to a spiropyran, in order to combine the
beneficial properties of both types of sensors, while overcoming
the limitations of each in isolation. A few such coupled
2+
5-7
These fluorescent sensors are
8-10
commonly based on fluorescein (e.g. Calcein, Fluo-4)
and
which present a high
fluorescence emission intensity (i.e. brightness) when bound to
11,12
coumarin (e.g. Calcein Blue, BTC)
2+
Ca . However, most of these sensors suffer from photo
instability
absence of the ion. In addition, the high excitation energies (i.e.
UV/blue) of these fluorophores are damaging to cells,
cause autofluorescence, which 17,o18verlaps with the desired
fluorescence signal of the sensors.
13,14
and high background fluorescence signal in the
25-29
molecules have been described,
stationary state of spiropyran as an ‘on/off’ switch to control the
fluorescence output of the fluorophore through Förster resonance
however these use the photo-
15,16
and
On the other hand, ion sensors based on photochromic
19,20
molecules, such as spiropyrans,
are known to exhibit
21
excellent photostability. A spiropyran undergoes photochromic
con-version between a ring-opened, polar merocyanine (MC)
isomer and the ring-closed, non-polar spiropyran (SP), on
irradiation with UV (or dark) and white light, respectively (see
Figure 1). In addition, the MC state is highly fluorescent when
chelating to an appropriate analyte compared to the SP state,
resulting in a low background signal in the absence of the
analyte. Furthermore, the green excitation and red emission
profiles typical of the highly fluorescent MC isomer are not
damaging to cells, and do not overlap with cellular
Figure 1. Structures of Py-1 as the ring-closed, weakly fluorescent spiropyran
(
SP, ‘off’) isomer, and the ring-opened, highly fluorescent merocyanine (MC-
2+
Ca , ‘on’) complex.

2
Tetrahedron
ies MANUSCRIPT
30,31
electron transfer (FRET).
As such, the foc
A
us
C
of
C
th
E
es
P
e
TED
stu
d
has been on photocontrol of a non-photoswitchable core
fluorophore, rather than potential enhancement of the spiropyran
in isolation.
In this work, we demonstrate that coupling of a traditional
2+
fluorophore (pyrene) to a photoswitch (spiropyran) with Ca -
selective ionophore (Py-1, Figure 1), gives rise to an 80-fold
2+
brighter fluorescence emission in the presence of Ca , when
compared to a non-coupled spiropyran analogue. Importantly,
Py-1 retains the photostability, red emission profile and low
background fluorescence signal typical of spiropyran-based
sensors. Pyrene was chosen as the traditional fluorophore in this
proof-of-concept study for ease of synthesis, and given that it has
32
a distinct fluorescence emission profile to that of spiropyran.
2+
We present a detailed characterisation of this novel Ca sensor,
Py-1, and propose that the incorporation of a suitable traditional
fluorophore may be a general method for improving the
brightness of spiropyran-based sensors.
2
. Results/Discussion
2
.1. Design
The pyrene-modified spiropyran sensor, Py-1, was designed to
2+
be selective for Ca with the incorporation of a 1-aza-15-crown-
-ether ionophore at C8’ of the benzopyran ring (see Figure 1).
5
The phenolic oxygen of the MC isomer also serves to stabilize
metal ion binding in the ionophore (as shown by the dotted line
Scheme 1. Synthesis of Py-1.
33
in Figure 1), to define the ion-binding domain. The pyrene
fluorophore was attached at the N1 indolic nitrogen (labelled in
Figure 1) through a short alkyl tether. Previous work from our
group has shown that in general, substitution at this position does
34,35
not affect photoswitching of the spiropyran.
An electron
withdrawing C6’-NO2 group was incorporated to promote
2
+
formation of the MC(Py-1)-Ca complex, and hence improve the
2+
36
Ca -responsive fluorescence signal.
.2. Synthesis
Py-1 was prepared as outlined in Scheme 1. Commercially
2
available 1-pyrenemethanol was reacted with thionyl chloride in
toluene to give chloromethylpyrene 4. 2,3,3-Trimethylindolenine
was then reacted with 4 in the presence of sodium iodide,
followed by treatment with aqueous sodium hydroxide to give
the indoline 5. 1-Aza-15-crown-5-ether was alkylated with
chloride 6 to give the benzaldehyde 7. Finally, a condensation
reaction between the aldehyde 7 and indoline 5 in refluxing
ethanol, followed by purification of the product by normal-phase
column chromatography, gave the pyrene-modified spiropyran
Py-1 in 16 % yield over four steps. The non-pyrene modified
sensor 2 was prepared as a control, as previously described in the
Figure 2. Absorbance of Py-1 (50 µM, black line) and in the presence of
2+
a 2-fold excess of Ca (100 µM, red line).
2
+
isomerization of Py-1 to the more coloured MC(Py-1)-Ca
complex.
22-24
2
.4. Fluorescence Characterisation
37
literature.
2
.4.1. Excitation at 532 nm.
2
.3. Absorbance Characterisation
The fluorescence emission spectra of Py-1 were next
measured in order to characterise the emission profile and
determine the effect of the pyrene moiety on sensor brightness.
Sensor 2 was included for comparative studies, as this azacrown-
bearing spiropyran does not contain the N1 indolic pyrene moiety
Absorbance spectra of Py-1 were first measured in the
2+
absence and presence of excess Ca , in order to characterise
metal binding-induced SP to MC isomerization. First, the
absorbance of Py-1 (50 µM in acetonitrile) was measured.
Results displayed in Figure 2 (black line), show strong
absorbance maxima for the pyrene fluorophore (329 nm and 344
(
see Scheme 1). Results displayed in Figure 3a show that, in the
2+
presence of excess Ca , Py-1 gives a strong, red emission
maximum (605 nm), 8-fold higher than that observed in the
absence of the ion (635 nm). Similar emission maxima were
observed for sensor 2 (Figure 3b) in the presence of Ca (600
nm), but with a higher background fluorescence in the absence of
the ion (625 nm). Despite both sensors sharing a common aza-
32
nm) with weaker absorbances for the SP (405 nm) and MC (554
19
2
+
nm) isomers. The presence of these two, distinct absorption
bands allows for excitation of the sensor at 344 nm and 532 nm.
Next, the absorbance of Py-1 (50 µM) in the presence of excess
2+
2+
Ca (100 µM) was measured. Binding of Py-1 to Ca (red line)
caused a 4-fold increased intensity and hypsochromic shift of the
MC absorbance to 520 nm. Thus, metal ion binding induces
crown ether-based Ca -binding domain, Py-1 shows enhanced
selectivity for Ca compared to 2. This suggests that the pyrene
2+
moiety may stabilize the ring-opened MC isomer, which we

3
know binds metal ions. Importantly, Py-1
A
wa
C
s
C
fo
E
un
P
d
T
toEDbe MAN
T
U
he
S
f
C
luo
R
re
I
s
P
ce
T
nce emission of Py-1 with excitation at the
2+
approx. 80-fold brighter than sensor 2 in the presence of Ca .
Other information, such as quantum yield calculations, can be
found in the supplementary information.
pyrene absorbance maximum (344 nm) was next investigated,
2
+
with results displayed in Figure 4a. In the absence of Ca , two
distinct maxima are observed for the pyrene fluorophore (390
nm) and merocyanine (635 nm) isomer, respectively, with a 2:1
Next, the ion selectivity profiles of Py-1 and 2 were
investigated in order to determine the effect of the pyrene
substitution on metal ion binding. The fluorescence emission
2+
emission intensity ratio. Binding of Py-1 to Ca caused a
hypsochromic shift of the MC emission to 605 nm, and a change
in the intensity ratio of the two emissions to approx. 1:3. These
separate emission maxima with a single excitation may be
explained by Förster resonance electron transfer (FRET)
occurring within the Py-1 sensor, where pyrene acts as the FRET
‘donor’ and MC the ‘acceptor’ (see Figure 4a, inset). In support
of this explanation, excitation of literature sensor 2 at 344 nm,
which lacks the pyrene moiety, gives no such emission (Figure
S3). Additionally, the resulting change in emission intensity of
2+
spectra in the presence of excess of Ca and other biologically
+
+
+
+
2+
2+
2+
2+
relevant ions (Li , Na , K , Cs , Mg , Mn , Cu and Zn ) were
measured, with results displayed in Figure 3c. The sensors
showed a similar selectivity profile with 2 to 4-fold affinity
2+
measured for Ca
over all other ions. Critically, these
fluorescence results demonstrate that the pyrene-spiropyran
sensor, Py-1, is brighter than the non-modified sensor 2 with an
improved signal-to-noise ratio, while retaining the red emission
profile and metal-ion selectivity, in agreement with our design
strategy.
2+
the two maxima in response to Ca suggests that the efficiency
of FRET between the pyrene and MC is affected by metal
chelation to Py-1. The absorbance spectra from Figure 2 shows a
hypsochromic shift and increase in the MC absorbance of Py-1 in
2
.4.2. Excitation at 344 nm.
2+
the presence of Ca . This ion-induced change is likely causing
better overlap with the fluorescence emission of pyrene, and
hence allowing for greater FRET transfer to occur between donor
and acceptor fluorophores. Interestingly, the FRET-induced MC
emission profile of Py-1 is identical to that observed with
excitation at 532 nm, albeit of a lower intensity.
The fluorescence intensity ratio of MC/pyrene emissions in
the presence of various metal ions was next measured, to further
Figure 4. (A) Fluorescence emission spectrum of Py-1 (black) and
2+
in the presence of Ca (red) with excitation at 344 nm. Inset: Proposed
Figure 3. Fluorescence spectra of (A) Py-1 and (B) 2 (black lines)
2+
mechanism of fluorescence emission of MC(Py-1)-Ca complex. (B)
2+
and in the presence of Ca (red lines). Excitation was at 532 nm. (C)
Fluorescence emission selectivity of Py-1 (black) and 2 (red) in the
presence of a 2-fold excess of biologically relevant metal ions.
Selectivity of Py-1 with excitation at 344 nm showing ratio of the
MC/Py fluorescence emission intensities in the presence of biologically
relevant metal ions.

4
Tetrahedron
MAth
investigate the effect of ionophore selectiv
A
ity
C
o
C
n
E
th
P
e
T
F
E
RE
D
T
N
at
U
Py
S
-1
C
c
R
an
I
photoswitch without photobleaching, compared to
P
T
efficiency. From the results in Figure 4b, the greatest transfer
the commercially available sensor.
2+
efficiency is seen in the presence of Ca , with a MC/pyrene ratio
-fold greater than that of sensor in the absence of metal ion.
3
. Conclusion
8
Importantly, this trend in fluorescence ratio is consistent with the
selectivity of the azacrown ionophore with excitation at 532nm
established earlier.
In this work, we present a new rationally designed,
2+
photostable, red-emitting Ca sensor. This hybrid pyrene-
spiropyran sensor (Py-1) has enhanced fluorescence intensity
compared to a non-coupled analogue, while retaining the
characteristic red emission profile of the spiropyran. It presents
two other excitation options; direct green excitation (532 nm) of
the photoswitch for a red emission and UV excitation (344 nm)
of the pyrene moiety, which gives rise to distinct blue and red
emissions. Fibre-based photostability studies show that Py-1 is
stable after multiple cycles of photoswitching, without any sign
of photobleaching. Importantly, this new pyrene-spiropyran
coupled system presents an opportunity for a general method
towards the fluorescence enhancement of spiropyran-based
sensors.
2
.5. Photostability Characterisation
In the final part of this study, photoswitching between the
highly fluorescent MC and weakly fluorescent SP isomers of Py-
was characterised using an in-house fabricated silica-based
1
suspended core microstructured optical fibre (SCF), in order to
assess the photostability of the sensor. Microstructured optical
fibers offer the advantage that interaction between the light and
chemical species can be extended along the entire length of the
fiber, while maintaining the integrity of the device. In SCFs, the
glass core is suspended in air by thin struts, allowing a portion of
the guided light to extend outside the fiber core into the
surrounding holes which serve as low-volume sample chambers
4. Experimental Section
38,39
(see supplementary information, Figure S5).
13
1
General. All C NMR and H NMR spectra were recorded on
The SCF was immersed in a solution of Py-1 sensor (500 µM
an Agilent Technologies 500 MHz NMR with DD2 console in
in acetonitrile), with Py-1 taken up into the holes of the fibre by
capillary action. Fluorescence measurement in the SCF revealed
the highly fluorescent MC form of Py-1 (λem max ~ 635 nm),
presented as the first point in Figure 5. Next, irradiation with the
DMSO-d (Cambridge Isotope Laboratories, Cambridge, MA).
6
Chemical shifts (δ) are reported in ppm. Chemical shifts of
DMSO-d (δ = 39.52 ppm), DMSO-d (δ = 2.50 ppm) or TMS
6
C
6
H
1
3
(δ = 0.0 ppm) were used as internal standards in all C NMR
and H NMR experiments respectively. High resolution mass
H
1
5
32 nm laser light to photoswitch to the weakly fluorescent SP
isomer resulted in a 6-fold decreased fluorescence intensity. The
second switching cycle in the dark allowed the sensor to
photoswitch back to the highly fluorescent MC form, with the
fluorescence intensity returned to pre-irradiation levels. In all
cases, excitation was achieved with 5 x15 ms pulses of the 532
nm green laser. This process was repeated over 10 cycles,
demonstrating the photo-reversibility of the system. For
comparison, the fluorescence emission of Rhodamine B (100 µM
in acetonitrile) was recorded under the same experimental
conditions, with results displayed in Figure 5 (red line).
Rhodamine B suffers from considerable photobleaching, with a
spectrometry was performed on the Agilent 6230 TOF LC-MS.
HPLC grade acetonitrile was used in all related experiments. All
metal ions used in this work were in the form of perchlorate salt,
except for Cs SO . Absorbance and fluorescence measurements
were performed at 25 °C using a BioTek Synergy H4 Hybrid
Multi-Mode Microplate Reader. Fluorescence quantum yield
2
4
(Φ ) of Py-1 (in acetonitrile) was calibrated with Rhodamine B
F
40
(in water) as a standard. Calcein was purchased from TCI
America. All other reagents were purchased from Sigma-Aldrich
3
7
25
and used without further purification. Compounds 2, 5, and
41
7
were synthesized according to literature procedures.
6
-fold decrease in the normalized fluorescence intensity by the
8
-((1,4,7,10-Tetraoxa-13-azacyclopentadecan-13-yl)methyl)-
eighth photoswitching cycle. These results clearly demonstrate
3
',3'-dimethyl-6-nitro-1'-(pyren-1-ylmethyl)spiro [chromene-2,2'-
indoline] (Py-1). To a solution of 5 (196 mg, 0.5 mmol) in dry
ethanol (15 mL) was added 7 (209 mg, 0.5 mmol) and the
reaction heated at reflux for 18 hrs. The solvent was removed in
vacuuo to give a dark red solid (209 mg). A sample (100 mg) of
the crude material was purified on silica by gradient column
chromatography using 0-10 % methanol in dichloromethane to
1
give Py-1 as a purple-red solid (10 mg, 10 %). H NMR (500
MHz, DMSO-d ) δ 8.34 – 8.32 (m, 1H, ArH), 8.30 (m, 2H, ArH),
6
8
.21 (m, 1H, ArH), 8.19 (m, 1H, ArH), 8.15 (m, 2H, ArH), 8.10
(
(
m, 1H, ArH), 8.03 – 8.01 (m, 2H, ArH), 7.99 (m, 1H, ArH), 7.23
d, 1H, ArH, J = 7.0 Hz), 7.01 - 6.98 (m, 2H, ArH), 6.83 (t, 1H,
ArH, J = 7.5 Hz), 6.36 (d, 1H, ArH, J = 7.5 Hz), 5.97 (d, 1H,
ArH, J = 10.0 Hz), 5.14 (d, 1H, CHH, J = 16.5 Hz), 5.04 (d, 1H,
CHH, J = 17.0 Hz), 3.58- 3.42 (m, 20H, 10 x CH ), 2.58 – 2.46
2
1
3
(
m, 2H, CH ), 1.30 (s, 3H CH ), 1.22 (s, 2H, CH ) ppm;
C
2
3
3
Figure 5. Photostability of Py-1 (black) within an optical fibre over 10
cycles of photoswitching, where each cycle denotes photoconversion from the
highly fluorescent MC isomer to the weakly fluorescent SP isomer upon
irradiation with 532 nm green laser light for 2 min. Incubation in the dark for 5
min results in reversion to the MC state. Excitation for fluorescence
measurements was at 532 nm, with 5 x 15 ms pulses at 1.5 µW power. The
photostability of Rhodamine B (red) was investigated under the same
irradiation conditions.
NMR (126 MHz, DMSO-d ) δ 157.1, 147.1, 140.2, 132.0, 130.9,
6
1
1
5
2
27.3, 127.1, 126.6, 125.5, 125.4, 124.2, 124.1, 121.5, 119.9,
18.7, 72.5, 70.5, 70.0, 69.9, 69.8, 69.9, 69.7, 69.2, 60.4, 54.1,
2.5, 35.3, 31.5, 31.4, 29.2, 29.0, 28.9, 28.8, 26.8, 26.1, 25.3,
2.3, 19.9, 15.7, 14.2 ppm; HRMS-ESI (m/z) calculated for
+
+
C H N O [M + H] 754.3487, found 754.3480, and [M + Na]
7
46
47
3
7
76.3306, found 776.3305.

5
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The authors would like to acknowledge funding support from
the Centre for Nanoscale BioPhotonics (CNBP), through the
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