Maximizing the thiol-activated photodynamic and fluorescence imaging functionalities of theranostic reagents by modularization of Bodipy-based dyad triplet photosensitizers

Article abstract of DOI:10.1039/c5tb01857a

To maximize both the activatable singlet oxygen (¹O₂) production and fluorescence of theranostic photodynamic (PDT) reagents, herein we propose a modularized molecular structural profile, i.e. the intersystem crossing (ISC) and fluorescence functionalities were accomplished with different modules in a dyad, thus enabling the activated ¹O₂ production yield (Φ_Δ, PDT) and the fluorescence yield (Φ_F) to both approach 100%. The PDT and the fluorescence were caged with a thiol-cleavable disulfide bond (-S-S-) linker and an electron trap (2,4-dinitrobenzenesulfide, DNBS). This new molecular structural profile is different from that of conventional theranostic PDT reagents, which are based on a single chromophore for both PDT and fluorescence; thus, the limitation of Φ_Δ + Φ_F = 100% exists for only half of our new molecular profile. To this end, six Bodipy dyads were prepared. The photophysical properties of the dyads were studied with steady state absorption, fluorescence and nanosecond transient absorption spectroscopy. The dyads show weak PDT and luminescence, due to the caging effect. In the presence of thiols (GSH or Cys), cleavage of the disulfide linker and DNBS occurs, and the PDT and fluorescence modules are activated simultaneously (Φ_F: 1.3% → 47.6%; Φ_Δ: 16.7% → 71.5%). These results are useful in designing activatable PDT/fluorescence imaging theranostic reagents.

Full text of DOI:10.1039/c5tb01857a

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DOI: 10.1039/C5TB01857A
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PAPER
Maximizing the Thiol-Activated Photodynamic and Fluorescence
Imaging Functionalities of Theranostic Reagents by Modularization of
the Bodipy-based Dyad Triplet Photosensitizers
a,
a
a
b
b,
Jianzhang Zhao, * Ling Huang, Xiaoneng Cui, Shujing Li, and Huijian Wu *
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
1
To maximize both the activatable singlet oxygen ( O
2
) production and fluorescence of theranostic
photodynamic (PDT) reagents, herein we propose a modularized molecular structural profile, i.e. the
intersystem crossing (ISC) and the fluorescence functionalities were accomplished with different modules
1
1
1
2
0
5
0
in a dyad, thus the activated O production yield (Φ , PDT) and the fluorescence yield (Φ ) can both
2
Δ
F
approach 100%. The PDT and the fluorescence were caged with thiol-cleavable disulfide bond (−S−S−)
linker and electron trap (2,4-dinitrobenzenesulfide, DNBS). This new molecular structural profile is
different from the conventional theranostic PDT reagents, which are based on single chromophore for
both PDT and fluorescence, thus the limitation of Φ + Φ = 100% exists, only half of our new molecular
profile. To this end six Bodipy dyads were prepared. The photophysical properties of the dyads were
studied with steady state absorption/fluorescence spectroscopies and nanosecond transient absorption
spectroscopy. The dyads show weak PDT and luminescence, due to the caging effect. In the presence of
thiols (GSH or Cys), cleavage of disulfide linker and DNBS occurred, the PDT and fluorescence modules
were activated simultaneously (Φ : 1.3%→47.6%; Φ : 16.7%→71.5%). These results are useful for
Δ
F
F
Δ
designing of activatable PDT/fluorescence imaging theranostic reagents.
based on a single chromophore profile, as a result, the efficient
45 ISC required for producing of triplet excited state will inevitably
quench the fluorescence of the same chromophore, and the
maximal sum of the yield of the two competing processes of ISC
and fluorescence will be less than 100%. In order to address this
challenge and to maximize the luminescence efficiency, as well
Introduction
Photodynamic therapy (PDT) reagents are in particular interest
due to its ability of production of cytotoxic singlet oxygen ( O )
upon photoiiradiation.
1
2
1
−6
Among the other desired properties,
2
3
3
4
5
0
5
0
strong absorption of visible or near-IR light, high triplet excited
state yield, low dark toxicity are crucial for PDT reagents.
Porphyrin derivatives were used as the first generation PDT
5
5
6
6
0
5
0
5
as the PDT effect, the two functionalities can be accomplished by
separate chromophores, for example in a dyad, which are able to
be simultaneously activated by cancer-related biomarkers, such as
high concentration of biological thiols (GSH and Cys).
3
reagents. Recently, activatable PDT reagents were developed,
4
,7−10
such as acid-activatable iodo-azaBodipy derivatives,
porphyrin compounds,
PDT reagents based on fluorescence-resonance-energy-transfer
(FRET) mechanism have been reported, e.g. photodynamic
1
1−13
14−16
PDT molecular beacons,
production with
or
1
photoswitched
O
2
photochromic
16
20
molecular beacon, or the photosensitizer aptamer switch. In
these activatable PDT reagents, usually a photosensitizer and a
quencher, such as black hole quencher, was linked by cleavable
bonds or aptamers. Targeted cleavage of the linker or binding to
specific enzyme or biomarkers for disease (small molecules) will
activate the PDT reagents. Porphyrin and Rhodamine was used to
construct a caspase-specific luminescent imaging reagents with
the ability of induce cell death and at the same time, to monitor
1
7
chromophores. However, switching of the triplet excited states
of organic chromophores was not studied in detail with
1
8
nanosecond time-resolved transient absorption spectroscopy. In-
depth investigation into the variation of the triplet excited state
with time-resolved spectroscopy will be useful for future design
of the activatable PDT reagents.
Moreover, it is highly desired to develop PDT reagents which
1
give activated O photosensitizing and luminescence at the same
12
2
the cell death by ratiometric fluorescence. However, in that case
time, so that treatment/monitoring bifunctional theranostic
reagents can be attained. However, investigations on these PDT
compounds are rare. Actually there is a dilemma for these
bifunctional PDT compounds since these reagents are usually
the PDT effect was not caged. Recently phthalocyanine complex
was caged with 2,4-dinitrobenzenesulfide, the phthalocyanine
1
9
1
9
19a
chromophore acts as PDT as well as the fluorescence module.
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

Journal of Materials Chemistry B
Page 2 of 18
In this case the single chromophore play both the fluorescence
and triplet state production roles. A Bodipy dyad was reported to
show thiol activatable fluorescence, but the PDT effect was not 15 the O production or the fluorescence can be maximized, which
absorption spectroscopy. The activated PDT and the fluorescence
was achieved with separate modules in a caged dyad, thus either
1
View Article Online
DOI: 10.1039/C5TB01857A
2
1
9b
caged.
is different from the conventional theranostic PDT materials
1
9a
5
In order to address the above challenges, herein we prepared
Bodipy-iodoBodipy dyads (Scheme 1) to achieve the following
goals: (a) to attain simultaneous thiol-activated fluorescence and
which contain a single chromophore. The caging of the PDT
property and the fluorescence was achieved with different
approaches, such as FRET and electron transfer. Thiol-cleavable
PDT effect with different chromophores in a dyad, for which the 20 disulfide bond (−S−S−) was used as the linker between the PDT
2
1−27
PDT and the fluorescence was caged; (b) feasibly tuneable
fluorescence emission wavelength, which offer the feasibility for
multi-colour luminescent imaging; (c) the switching of the triplet
excited state is thoroughly studied with the nanosecond transient
and the fluorescence modules.
These results are useful for
1
0
future designing of activatable theranostic PDT reagents and for
12
real-time treatment/monitoring.
O
2
5
B-1-C; X = C
B-1-S; X = S
I
I
N
N
N
N
N
O
F
O
O
B
F
X
O
O
N
N
F
F
O
O
X
B
O
O
O
N
N
N
O
NO2
O N
2
O
3
0
S
O
O
I
F
B-2-C: X = C
B-2-S: X = S
N
N
B
F
F
N
N
B
O
N
F
O
3
4
4
5
5
5
0
5
0
5
O
N
N
O
X
I
O
N
N
X
O
O
O
O
N
O
O
I
I
N
B-3-C: X = C
B-3-S: X = S
N
N
N
N
F
O
O
O
B
F
X
O
O
N
N
N
X
F
F
O
O
B
O
O
O
N
N
N
O
The mechanism of thiol activated triplet photosensitizers
O2N
O
NO
2
R2
SR
R2
S
NO2
I
O
O
+
F
N
B
NO2
F
N
S
N
R1
F
F
I
I
N
N
S
N
N
R1
B
O
S
N
+
B
B
+
I
F
F
O
F
F
B-2-S
O
OH
O
OH
O
O
R =
2
O
N
N N
R =
1
N
N N
O
O
6
0
Scheme 1. The thiol-activatable Bodipy-derived dyad triplet photosensitizers used in the study.
2
|
Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 3 of 18
Journal of Materials Chemistry B
CHO
CH OH
O
2
O
View Article Online
DOI: 10.1039/C5TB01857A
a
b
X
O
O
X
OH
O
O
X
HO
X
I
I
O
N
N
O
O
O
B
3
-C, X = C
3-S, X = S
4-C, X = C
1
F
F
2
5
4-S, X = S
B-4
N3
N3
N3
N3
O
O
B
O
O
1
1
2
2
3
3
4
4
0
5
0
5
0
5
0
5
e
d
I
I
N
N
F
c
N
N
I
I
B
N
N
N
N
F
F
F
B
F
B
F
F
F
N
H3CO
OCH3
10
1
1
6
5
N3
N3
N3
N3
O
O
O
O
g
f
h
I
I
N
N
I
I
N
N
N
N
N
F
N
F
B
B
B
B
B
F
F
F
F
F
F
O2N
O
O S
N3
9
8
6
7
NO2
OH
O
B
OH
N
F
O
N
O
N
O
F
O
N
N
X
O
O
O
O
X
i
4
-C
-S
O
4
+
O
N
N
F
1
1
2-C, X = C
2-S, X = S
F
N3
I
O
N
F
O S O
O
N
N
B
F
O
NO2
O
N
N
X
O
O
O
O
X
i
O
O
NO2
O
I
O
4
4
-C
-S
I
I
+
15
N
N
F
13-C, X = C
13-S, X = S
B
F
N3
I
O
N
F
N
O
N
O
B
I
F
O
N
N
X
O
O
O
O
X
i
O
4
-C
-S
O
+
I
I
O
4
N
F
N
1
4-C, X = C
B
14-S, X = S
F
H3CO
OCH3
i
i
i
1
1
2-C
2-S
13-C
13-S
9
B-2-C
B-2-S
14-C
14-S
B-3-C
B-3-S
B-1-C
B-1-S
+
+
10
+
11
5
0
Scheme 2. Preparation of the Dyads. (a) NaBH
acetic acid, toluene. (e) 4-N,N-dimethylbenzaldehyde, piperidine, acetic, toluene. (f) 4-Hydroxybenzaldehyde, piperidine, acetic, toluene. (g) Phenyl
boronic acid, Pd(PPh , K CO , toluene/EtOH/H O = 2/2/1 (v/v). (h) 2,4-Dinitrosulfonylchloride, Et N, DCM. (i) CHCl /EtOH/H O = 12/1/1 (v/v),
CuSO ·5H O, sodium ascorbate.
4
, EtOH. (b) Triphosgene, DCM. (c) Iodine, iodic acid, EtOH. (d) 4-Methoxybenzaldehyde, piperidine,
5
5
3
)
4
2
3
2
3
3
2
4
2
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

Journal of Materials Chemistry B
Page 4 of 18
Experimental Section
129.7, 129.6, 129.4, 128.6, 128.3, 124.2, 121.4, 116.9, 155.5,
1
2
15.2, 115.0, 114.5, 82.9, 69.4, 68.1, 66.5, 62.1, 55.4, 49.9, 28.7,
Analytical Measurements
View Article Online
5.5, 17.9, 14.8. HRMS (MALDI): m/z calcd for
DOI: 10.1039/C5TB01857A
−
Luminescence lifetimes were measured on
a
OB920
60 [C H B F O N I ] : 1800.4482; found m/z = 1800.4457
8
6
84
2
4
12 10 2
fluorescence/phosphorescence
lifetime
instrument. The
Synthesis of complex B-2-S
5
nanosecond time-resolved transient difference absorption spectra
were detected by Edinburgh LP920 instruments (Edinburgh
Instruments, UK). The signal was digitized with a Tektronix TDS
The synthesis is similar to that of B-1-S. Black solid. Yield:
1
21.7 mg (54 %). H NMR (500 Hz, CDCl
3
) δ = 8.65 (d, 1H, J =
3012B oscilloscope and was analyzed by the LP900 software. All
5.0 Hz), 8.47−8.45 (m, 1H), 8.11−8.09 (m, 1H), 7.85 (d, 2H, J =
samples in flash photolysis experiments were deaerated with N2 65 10.0 Hz), 7.62 (d, 1H, J = 20.0 Hz), 7.42−7.36 (m, 5H), 7.34−
1
0
for ca. 15 min before measurement and the gas flow is kept
during the measurement.
7.29 (m, 5 H), 7.22−7.20 (m, 3 H), 7.17−7.14 (m, 3 H), 7.07 (d,
2H, J = 10.0 Hz), 6.99−6.94 (m, 8 H), 6.42 (d, 1 H, J = 15.0 Hz),
5
4
.24 (d, 4H, J = 15.0 Hz), 5.09 (d, 4H, J = 5.0 Hz), 4.83−4.80 (m,
H), 4.46−4.42 (m, 4H), 4.37−4.34 (m, 4H), 2.93−2.90 (m, 4H),
Synthesis of complex B-1-S
General methods. Under Ar atmosphere,
compound 12-S (48.5 mg, 0.04 mmol), 9 (44.7 mg, 0.05 mmol)
and CuSO ⋅5H O (0.08 mmol, 8.0 mg) were dissolved in
CHCl /EtOH/H O (12/1/1, v/v, 12 mL). Then sodium ascorbate
0.08 mmol, 7.5 mg) was added. The mixture was stirred at room
a mixture of 70 2.63 (s, 6H), 2.56 (s, 3 H), 1.69 (s, 3 H), 1.40 (s, 6 H), 1.36 (s, 3
1
3
H). C NMR (125 Hz, CDCl ) δ = 167.7, 158.6, 158.4, 156.8,
156.3, 154.9, 150.9, 149.0, 148.4, 148.1, 145.2, 141.0, 140.9,
140.0, 139.4, 137.2, 135.0, 134.8, 134.7, 134.1, 133.2, 132.8,
132.6, 132.3, 131.6, 130.9, 130.4, 130.1, 129.7, 129.4, 128.8,
3
1
2
2
3
3
5
0
5
0
5
4
2
3
2
(
temperature (RT) for 12 h. After completion of the reaction, 75 128.7, 128.6, 128.4, 128.1, 127.9, 127.6, 127.4, 126.4, 122.1,
water (10 mL) was added, the mixture was extracted with
dichloromethane (3×30 mL). The organic layer was dried over
Na SO . After removal of the solvent under reduced pressure, the
2 4
120.7, 120.3, 115.4, 115.3, 114.9, 114.8, 85.8, 69.5, 66.2, 65.6,
61.6, 60.4, 53.4, 50.2, 37.0, 31.9, 30.6, 29.7, 29.4, 19.2, 17.2,
16.0, 14.1, 13.7, 13.2, 12.6. Determination of mass spectra failed.
mixture was purified by column chromatography (silica gel,
Synthesis of complex B-2-C
DCM/EtOAc = 5/2, v/v) to give orange solid (41.9 mg). Yield:
1
5
1 %. H NMR (500 MHz, CDCl ) δ = 8.16 (d, 2H, J = 16.8 Hz),
80
The synthesis is similar to that of B-1-C. Black solid (30.6 mg).
3
1
7.84 (d, 2H, J = 8.4 Hz), 7.62−7.56 (m, 6H), 7.34−7.30 (m, 4H),
.18−7.15 (m, 4H), 7.00−6.93 (m, 12H), 5.96 (s, 2H), 5.24 (d, 4H,
J = 8.0 Hz), 5.09 (d, 4H, J = 4.0 Hz), 4.83−4.77 (m, 4H), 4.46−
Yield: 85 %. HNMR (500 MHz, CDCl
3
) δ = 8.64 (s, 1 H), 8.47−
7
8.45 (m, 1H), 8.11 (s, 1H, J = 10.0 Hz), 7.85 (d, 2H, J = 15.0 Hz),
7.62 (d, 1H, J = 20.0 Hz), 7.42−7.36 (m, 5H), 7.34−7.30 (m, 7H),
7.23−7.20 (m, 4H), 7.17−7.14 (m, 4H), 7.07−7.06 (d, 2H, J = 5.0
4
.39 (m, 4H), 4.37−4.33 (m, 4H), 3.85 (s, 6H), 2.93−2.89 (m, 4H),
13
2
.54 (s, 6H), 1.46 (s, 6H), 1.38 ppm (s, 6H). C NMR (125 Hz, 85 Hz), 7.00-6.95 (m, 8H), 6.42 (d, 1H, J = 15.0 Hz), 5.24 (d, 4H, J
CDCl ) δ = 160.7, 158.6, 158.5, 158.3, 155.4, 154.8, 150.5, 145.6,
= 15.0 Hz), 5.08 (d, 4H, J = 10.0 Hz), 4.84−4.79 (m, 4H), 4.46−
4.42 (m, 4H), 4.32−4.29 (m, 4H), 2.63 (s, 6H), 2,56 (s, 3H), 1.65-
1.63 (m, 4H), 1.40 (s, 6H), 1.39 (s, 6H), 1.36−1.34 ppm (m, 4H).
3
1
1
1
6
44.1, 144.0, 143.0, 141.2, 139.1, 137.9, 133.1, 131.7, 130.4,
29.9, 129.4, 129.3, 128.7, 128.4, 128.1, 127.8, 124.1, 121.1,
16,7, 115.3, 115.0, 114.8, 114.6, 114.3, 82.7, 69.6, 66.3, 66.2,
1
3
C NMR (125 MHz, CDCl ) δ = 158.9, 158.7, 158.6, 156.9,
3
5.6, 61.9, 55.4, 49.8, 17.8, 14.6.ppm. HRMS (MALDI): m/z 90 155.4, 151.1, 149.1, 148.5, 148.3, 145.4, 141.2, 141.1, 140.0,
+
calcd for [C H B F O N I S ] : m/z = 1836.3610; found m/z
139.6, 137.3, 135.1, 135.0, 134.8, 134.2, 133.6, 133.5, 133.4,
132.9, 132.8, 132.5, 131.8, 131.1, 130.5, 130.4, 130.2, 129.8,
8
4
80
2
4
12 10 2 2
=
1836.3622.
Synthesis of complex B-1-C
General methods. Under Ar atmosphere,
1
1
29.5, 129.0, 128.9, 128.7, 128.6, 128.3, 128.0, 127.8, 127.5,
26.5, 124.2, 122.2, 120.9, 120.4, 115.5, 115.4, 115.0, 114.9,
a
mixture of 95 85.8, 68.1, 66.5, 65.7, 62.1, 50.0, 32.1, 30.7, 29.9, 29.5, 28.7,
compound 12-C (23.1 mg, 0.02 mmol), 9 (26.8 mg, 0.03 mmol)
25.5, 22.9, 19.4, 17.4, 16.2, 14.3, 13.9, 13.3, 12.8 ppm.
Determination of mass spectra failed.
4
4
5
5
0
5
0
5
and CuSO ⋅5H O (0.05 mmol, 5.0 mg) were dissolved in
4
2
CHCl
3
2
/EtOH/H O (12/1/1, v/v, 12 mL). Then sodium ascorbate
Synthesis of complex B-3-S
(0.05 mmol, 4.9 mg) was added. The mixture was stirred at RT
for 12 h. After completion of the reaction, water (10 mL) was
added. The mixture was extracted with dichloromethane (3×20 100 mg). Yield: 65 %. H NMR (500 MHz, CDCl
mL), the organic layers were dried over Na SO . After removal
of the solvent under reduced pressure, the mixture was purified
by column chromatography (silica gel, DCM/EtOAc = 5/2, v/v)
to give orange solid (18.5 mg). Yield: 45 %. H NMR (500 MHz,
CDCl ) δ = 8.15 (d, 2H, J = 15.0 Hz), 7.84 (d, 2H, J = 10.0 Hz), 105 4.39−4.33 (m, 4H), 3.86 (s, 6H), 3.01 (s, 6H), 2.92−2.90 (m, 4H),
7.62−7.57 (m, 6H), 7.34−7.31 (m, 4H), 7.19−7.16 (m, 4H), 7.00−
The synthesis is similar to that of B-1-S. Orange solid (25.5
1
3
) δ = 8.15 (d, 2H, J
= 15.0 Hz) 7.82 (s, 2H), 7.62−7.57 (m, 6H), 7.50−7.47 (m, 3H),
7.33−7.31 (m, 4H), 7.22−7.14 (m, 5H), 7.00−6.92 (m, 12H), 6.68
(d, 2 H, J = 10.0 Hz), 6.58 (s, 1H), 5.95 (s, 1H), 5.23 (d, 4H, J =
5.0 Hz), 5.09 (s, 4H), 4.80−4.76 (m, 4H), 4.44−4.40 (m, 4H),
2
4
1
3
1
3
2.57 (s, 3H), 1.45 (s, 6H), 1.42 (s, 3H), 1.38 ppm (s, 3H).
C
6.94 (m, 12H), 5.97 (s, 2H), 5.25 (d, 4H, J = 10.0 Hz), 5.08 (d,
4H, J = 10.0 Hz), 4.84−4.78 (m, 4H), 4.47−4.41 (m, 4H), 4.13−
4.09 (m, 4H), 3.86 (s, 6H), 2.54 (s, 6H), 1.65−1.62 (m, 4H), 1.47
NMR (125 MHz, CDCl ) δ = 160.9, 158.8, 158.7, 158.4, 155.0,
3
150.6, 145.8, 144.2, 139.3, 138.1, 133.3, 130.5, 130.3, 130.0,
129.9, 129.4, 128.5, 128.0, 124.2, 117.8, 116.8, 115.5, 115.1,
1
3
(
s, 6H), 1.39 (s, 6H), 1.38−1.36 (m, 4H). C NMR (125 Hz, 110 115.0, 82.9, 69.7, 66.5, 66.4, 65.8, 62.1, 55.6, 49.9, 37.2, 29.9,
CDCl ) 160.9, 158.8, 158.6, 158.5, 155.6, 155.4, 150.6, 145.7,
17.9, 17.4, 15.1, 14.8, 14.7. ppm. HRMS (MALDI): m/z calcd for
3
+
1
44.3, 143.2, 141.4, 139.3, 138.1, 133.3, 131.9, 130.5, 130.1,
[C93
H
89
B
2
F
4
O
12
N
11
S
2
I
2
] : 1967.4345; found m/z = 1967.4329.
4
|
Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 5 of 18
Journal of Materials Chemistry B
Synthesis of complex B-3-C
55 capability, with different chromophores in a dyad containing
1
9b
cleavable linker. To this end, we use the disulfide bond (−S−
The synthesis is similar to that of B-1-C. Orange solid (26.1
View Article Online
1
S−) as a linker between the PDT and the fluorescence modules
mg). Yield: 71 %. H NMR (500 MHz, CDCl ) δ = 8.15 (d, 2H, J
DOI: 10.1039/C5TB01857A
3
(
B-1-S, B-2-S and B-3-S, Scheme 1). It is known that disulfide
=
15.0 Hz), 7.82 (s, 2H), 7.62−7.57 (m, 6H), 7.50−7.47 (m, 3H),
7.34−7.32 (m, 4H), 7.22−7.15 (m, 5H), 7.00−6.93 (m, 12H), 6.68
d, 2H, J = 10.0 Hz), 6.58 (s, 1H), 5.95 (s, 1H), 5.24 (d, 4H, J =
bond can be selectively cleaved by thiols such as Cys and
5
0
5
2
1,22,24
6
6
7
7
8
8
0
5
0
5
0
5
GSH.
These reagents may demonstrate targeted PDT effect
(
toward cancer cells or tissues because it is known that the thiol
compound concentration is higher than the normal thiols or
tissues. At the same time the PDT treatment can be imaged with
the activated fluorescence.
In order to achieve efficient PDT and strong fluorescence upon
the activation (cleavage by thiol), the PDT reagents were
designed as FRET dyads in which the PDT and fluorescence
5
4
1
.0 Hz), 5.07 (s, 4H), 4.81−4.77 (m, 4H), 4.45−4.39 (m, 4H),
.11−4.09 (m, 4H), 3.86 (s, 6H), 3.01 (s, 6H), 2.57 (s, 3 H), 1.64−
.62 (m, 4H), 1.46 (s, 6H), 1.43 (s, 3H), 1.38 (s, 3H), 1.37−1.35
2
4
1
3
1
(m, 4H). C NMR (125 MHz, CDCl ) δ = 160.9, 158.8, 158.6,
3
1
1
1
6
58.5, 155.3, 150.6, 145.7, 144.3, 142.5, 139.3, 138.1, 133.3,
30.5, 130.1, 129.9, 129.7, 129.4, 129.2, 128.5, 128.4, 128.3,
24.2, 117.8, 116.8, 115.5, 115.2, 115.0, 114.5, 82.9, 69.4, 68.1,
1
8,19
modules were based on separated chromophores (Scheme 1).
6.5, 66.4, 62.1, 55.6, 49.9, 28.7, 25.5, 17.8, 15.0, 14.8 ppm.
+
The singlet energy donor and the acceptor were selected in such a
way that the PDT module is singlet energy donor, thus the ISC of
PDT module can be substantially inhibited by the FRET process
B-2-S and B-2-C, Scheme 1). The fluorescence of the singlet
energy acceptor (the fluorophore) was caged by 2,4-
1
HRMS (MALDI): m/z calcd for [C H B F O N I ] :
9
5
93
2
4
12 11 2
1
931.5217; found m/z = 1931.5236.
Cell Culture and Luminescence Imaging.
(
Human endometrial carcinoma cell line (Hela) was obtained
from School of Life Science and Biotechnology, Dalian
University of Technology. HeLa cell line was cultured in
Dulbecco's modified Eagle's medium (DMEM) containing 10%
19a,28−33
dinitrobenzenesulfonate (DNBS) group.
Thus the singlet
energy acceptor is a caged fluorophore so that the fluorescence
can be switched ON by the same stimuli that activate the PDT
module, i.e. biological thiols, such as Cys or GSH (Scheme 1). B-
2-S was designed following these rationales. Bodipy was selected
as the fluorophore due to the strong absorption of visible light,
high fluorescence quantum yield, good photostability and feasible
2
0
−
1
fetal bovine serum (FBS), 100 µg mL streptomycin and 100
−
1
U mL penicillin at 37°C in a humidified incubator containing
% CO2 and 95% air. After HeLa cells were cultured for 24
5
2
5
hours, the dyads (25 µM) was added and the cells were cultured
another 4 hours. Before HeLa cells were observed with confocal
laser fluorescence microscopic images, the cells were rinsing
three times with PBS buffer solution in order to
remove excess dyads.
3
4−38
derivatization.
The expected PDT properties are as
followings, in the absence of thiols, the PDT effect is in OFF
state because the ISC of the PDT module is inhibited by the
FRET to the styrylBodipy moiety. On the other hand, the
fluorophore is a caged by 2,4-dinitrobenzenesulfonyl (DNBS)
thus no fluorescence can be observed.
3
9
3
0
MTT Assay.
19a,29
In the presence of
thiols, the disulfide bond is cleaved, FRET ceased thus the ISC of
MTT assay was carried out to investigate the dark
toxicity and phototoxicity of dyads for cell growth. Hela cells
1
PDT module is activated, cytotoxic O will be produced upon
2
photoirradiation. At the same time, the fluorophore will be
were first seeded to two 24-well plates at a seeding density of
4
90 simultaneously un-caged by cleavage of the DNBS moiety by the
thiols, thus the red fluorescence will be switched on
1
×10 cells per well in 400 µL complete medium, which was
3
4
4
5
5
0
5
0
incubated at 37 °C for 24 h. After rinsing with PBS, Hela cells
were incubated with 400 µL culture media containing serial
concentrations of Dyad for 4 h. One plate was kept in the dark
for studying dark toxicity, and another plate was irradiated using
29
concomittantly. Thus B-2-S is a thiol-activatable PDT reagents
which show activatable fluorescence upon activation to
1
19
selectively illuminate the O producing region.
2
2
9
5
We also designed reference dyads with different FRET/cage
strategies to demonstrate B-2-S is the optimal (Scheme 1). In B-
a 520-530 nm LED at a power of 5W/ m for 15 min. Afterward,
-1
the cells were grown for another 24 h. Then, 40 µL of 5 mg mL
1
-S, the fluorophore was caged by FRET. However, the PDT
MTT solution in pH 7.4 PBS was added to each well. After 4 h
incubation, the medium containing un-reacted MTT was removed
carefully, and 400 µL DMSO was added to each well to dissolve
the produced blue formazan. After 5 min, the optical density (OD)
at a wavelength of 490 nm was measured with Bio-Rad
microplate reader. The percentage of growth inhibition was
calculated with Eq 7.
1
9b
module is not caged.
In the presence of thiols and thus
cleavage of the disulfide bond, the fluorescence will be switched
1
12
1
00 ON but the O producing capability is not altered. Thus the
2
switching capability of B-1-S is only half to that of B-2-S. On the
other hand, we used a fluorophore which shows longer absorption
wavelength in B-3-S than that used in B-1-S and B-2-S. Similar
to B-1-S, the PDT module is not caged. However, we found that
⎛
⎝
OD value of test
⎞
⎟
105 the fluorescence is very weak either in the absence or presence of
thiols, which is due to the intramolecular charge transfer
(
Eq. 7)
Growth Inhibition= 1-
×100
⎜
OD value of control ⎠
4
0
feature.
In order to prove the activation effect with thiols on the
photophysical properties, we prepared the references compounds
10 without the disulfide bonds, i.e. the −S−S− linker was replaced
with the un-cleavable −C−C− linker (B-1-C, B-2-C and B-3-C.
Scheme 1).
Results and discussion
Molecules Design and Synthesis.
1
The main goal of the new PDT reagents is to attain the
simultaneously activated PDT effect and the fluorescent imaging
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

Journal of Materials Chemistry B
Page 6 of 18
It should be pointed out that the PDT reagent should show
some extent of solubility in aqueous solution. The dyads studied
herein are soluble in aqueous solvent (the concentration is up to 35 studied (Fig. 1d-1f). For B-1-S alone, weak fluorescence at 513
The fluorescence of the dyads in the absence of thiols were
View Article Online
DOI: 10.1039/C5TB01857A
–
5
1
.0 × 10 M). However, we did not take much effort to add
nm and 699 nm were observed. The weak emission band at 513
nm is due to the quenched emission of the fluorescence module
by the FRET effect, whereas the weak fluorescence band at 699
5
water-soluble segments to the molecular structure.
The preparation of the six dyads were present in Scheme 2.
Firstly the thiol-cleavable and the un-cleavable linkers were
4
2
nm is due to the PDT module (efficient ISC). Upon addition of
prepared with the alkynyl intermediates. Then different 40 Cys thus the cleavage of the disulfide bond, the FRET ceased in
fluorescence modules or the PDT modules were consecutively
attached to the linkers. All the compounds were obtained in
moderate to satisfactory yields.
B-1-S and the fluorescence of the fluorophore module recovered
(enhanced by 9.4-fold). Conversely, the emission at 699 nm
decreased, which can be attributed to cease of FRET (λex = 475
nm, excitation into the fluorescence module, i.e. the energy
donor). For B-2-S, the emission of the caged fluorophore
enhanced by 28.5-fold in the presence of Cys (Fig. 1e), due to the
cleavage of the DNS moiety, an electron trap to quench the
1
0
UV−vis Absorption and Fluorescence Spectra.
4
5
The UV−vis absorption spectra of the compounds were studied
(
Fig. 1). The common feature of the dyads is the dual absorption
2
8,29,31,32,43
1
2
2
5
0
5
bands. For example, B-1-S shows absorption at 501 nm and 662
nm, respectively (Fig. 1a), which are attributed to the
fluorophore.
Thus ratiometric response was observed
for B-1-S in the presence of Cys, with the I513nm/I699nm varied
fluorescence module (Bodipy) and the PDT module (iodo- 50 from 1.8 from 38.3. The separation of the two emission band is as
3
6
StyrylBodipy part), respectively. These absorption bands are
highly identical to those of the reference components of the dyad,
thus we propose that there is no significant electronic interaction
larger as ca. 186 nm. Previously a ratiometric caspase-specific
PDT/luminescence imaging reagent was studied with porphyrin
as the energy acceptor and rhodamine as energy donor, separation
3
6,41
12
between the chromophores in the dyad at ground state.
For B-
of the two emission band is 90 nm.
2
-S, the absorption at 535 nm and 580 nm is due to the
55
We noted the emission at 622 nm of B-2-S is slightly red-
diiodoBodipy and the caged styrylBodipy fluorophore,
respectively (Fig. 1b). For B-3-S, the absorption of the
dimethylaminostyrylBodipy is at 608 nm, the PDT module shows
absorption at 656 nm (Fig. 1c).
shifted upon cleavage of the DNS moiety, which is caused by
2
9
intramolecular charge transfer (ICT) effect. The emission at 554
nm increased slightly upon addition of Cys, which is due to the
cease of the FRET effect. The emission band at 554 nm is still
The variation of the UV−vis absorption spectra of the 60 very weak upon addition of Cys, which is due to the efficient ISC
compounds in presence of thiols were studied. Only minor
variation were observed (Fig. 1a−1c). The UV−vis absorption
spectra of B-1-C, B-2-C and B-3-C also was studied in the
presence of Cys, the change of spectra is no significant (See ESI
of this PDT module. For B-3-S, the emission band at 695 nm was
slightly enhanced upon addition of Cys. This increase upon
cleavage of the disulfide bond is due to the ceasing of the
3
6
0
5
4
2
intramolecular electron transfer (see later section).
†
, Fig. S48).
1
0
0
.2
.8
.4
1.5
B-1-S
B-1-S + Cys
B-2-S
B-2-S + Cys
B-3-S
B-3-S+Cys
1
0
0
0
.2
.8
.4
.0
a
c
b
1.0
0.5
7
0
0.0
0.0
3
00 400 500 600 700 800
Wavelength / nm
300 400 500 600 700 800
Wavelength / nm
3
00 400 500 600 700 800
Wavelength / nm
7
5
2
2
1
1
50
00
50
00
50
0
1
60
20
4
5
B-2-S
B-2-S+Cys
B-1-S
B-1-S + Cys
d
e
f
1
B-3-S
B-3-S+Cys
30
80
1
5
8
0
40
0
0
500
600
700
800
7
00
800
900
6
00
700
800
Wavelength / nm
Wavelength / nm
Wavelength / nm
8
5
Fig. 1 UV−vis absorption spectra of (a) B-1-S, (b) B-2-S and (c) B-3-S in the presence and in the absence of Cys. The corresponding fluorescence
–5
emission spectral changes are presented in (d) B-1-S (λex = 475 nm), (e) B-2-S (λex = 520 nm) and (f) B-3-S (λex = 650 nm). c[Dyads] = 1.0 × 10 M,
–
3
2
c[Cys] = 3.0 × 10 M. In DMF/H O = 4/1 (v/v. pH = 7.4), 25 °C.
6
|
Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 7 of 18
Journal of Materials Chemistry B
a
Table3. Photophysical Parameters of Dyads and the Reference Compounds
5
-1
-1 b
c
d
e
f
g
λ
abs (nm)
ε / 10 M cm
λ
em (nm)
Φ
L
(%)
τ
F
(ns)
τ
T
(μs)
Φ
Δ
(%)View Article Online
DOI: 10.1039/C5TB01857A
1
1
0
1
661
608
535
0.98
0.78
0.83
694
713
556
7.0
2.80
2.4
2.10
1.20
0.13
1.40
−
110.2
61.2
−
B-4
78.1
In the absence of Cys
501/662
i
B-1-S
B-1-C
B-2-S
B-2-C
B-3-S
B-3-C
9
0.97/1.02
1.09/1.05
0.96/0.92
0.96/0.92
1.24/1.00
1.13/0.92
0.93
513/699
513/699
610
610
694
5.8
5.5
1.3
1.1
5.4
5.4
1.1
1.58/1.94
1.61/1.97
2.14
2.27
1.55
1.4
1.4
66.8h/69.3
i
501/662
535/580
535/580
608/656
608/655
580
62.4h /69.4
k
179.9
152.6
0.91
0.85
−
16.7j/6.3
k
16.1 j/5.9
16.5l
15.3l
−
695
610
1.59
2.07
In the presence of Cys
501/663
i
B-1-S
B-1-C
B-2-S
B-2-C
B-3-S
B-3-C
9
0.96/0.90
1.08/0.99
0.88/0.85
0.88/0.86
1.21/0.92
1,10/0.90
513/699
513/699
622
622
694
45.2
6.6
47.6
42.9
6.8
4.27/1.96
1.61/1.98
3.93
3.91
2.08
1.4
1.4
45.4
172.2
1.38
0.92
22.8 h /61.1
59.3h /68.9k
i
501/664
535/591
535/592
608/654
608/655
591
71.5 j /6.5
19.1j /6.9
59.5l
12.8l
−
k
695
622
5.2
50.1
1.60
3.95
0.86
−
a
-5
b
5
-1
-1
c
d
c = 1.0 × 10 M in DMF/H
2
O (4/1). 25 °C. Molar absorption coefficient at the absorption maxima.ε: 10 M cm . The emission maxima.
e
f
g
Fluorescence quantum yields determined with Rhodamine B as standard (Φ
F
i
= 65 % in EtOH). Fluorescence lifetime. Triplet excited state lifetime.
1
h
j
k
Singlet oxygen ( O
2
) quantum yield. Recorded upon excitation at 500 nm. Recorded upon excitation at 665 nm. Recorded upon excitation at 535 nm.
l
5
Recorded upon excitation at 580 nm. Recorded upon excitation at 665 nm.
Thus the function of the thiol-activation fluorescence was
achieved with B-1-S.
The reference dyads with the un-cleavable C−C linker were
also studied. For B-1-C and B-3-C, no fluorescence enhancement
was observed. For B-2-C, fluorescence enhancement was
observed due to the cleavage of the DNS from the caged
fluorophore (Fig. S49).
45
Similar studies were carried out for B-2-S (Fig. 3). Upon
addition of Cys, thus cleavage of the DNS moiety from the
styrylBodipy, the uncaged fluorophore give strong fluorescence
1
1
2
2
0
5
0
5
2
9
emission in red spectral region (Fig. 3a), which is different from
that of B-1-S that gives green fluorescence upon activation. Fast
kinetics was observed with k = 1.85 ± 0.03 s for B-2-S (Fig. 3b),
2
2
1
1
50
00
50
00
−1
5
5
6
0
5
0
a
10
8
No
Cys
+Cys
b
which is much faster than that of B-1-S. This result can be
rationalized by the different caging mechanism for the
fluorescence, in B-1-S, it is the cleavage of the disulfide bond,
but in B-2-S it is the cleavage of the DNS moiety that switch on
the fluorescence (Scheme 1).
Minor fluorescence enhancement (1.21-fold) was observed for
B-3-S in the presence of Cys (See ESI †, Fig. S52). For B-3-C,
however, no such enhancement was observed.
3
2
00 s
00 s
6
.
..
4
0
s
5
0
0
2
0
5
00
600
700
800
0
100
200
300
400
Wavelength / nm
Reaction time / s
2
50
00
50
00
1.00
c
2
1
1
No +Cys
Cys
d
In order to study the FRET effect in the absence and in the
presence of Cys, the fluorescence excitation spectra of the dyads
0
0
.75
.50
4
5,46
were investigated (Fig. 4).
For B-1-S, intense excitation band
3
2
00 s
00 s
at 513 nm was observed (Figure 4a), where the fluorescence
module gives strong absorption. Thus, FRET exists in B-1-S. In
the presence of Cys, the disulfide linker was cleaved, the intense
65 band at 498 nm in the excitation spectrum disappeared (Fig. 4a),
indicated that the FRET ceased. For the dyad with un-cleavable
C−C linker, however, no such change was observed (Fig. 4b),
indicate that the FRET is persistent in the presence of Cys.
0.25
0.00
.
..
5
0
0
0
s
0
100
200
300
400
5
00 600 700 800 900
Wavelength / nm
Reaction time / s
3
3
0
5
Fig. 2 (a) The change of fluorescence of B-1-S in presence of Cys, (b)
The kinetics of B-1-S in presence of Cys. (c) and (d) The change of
fluorescence and the kinetics in different time in presence of Cys for B-1-
C, λex = 475 nm. c[Dyads] = 1.0 × 10 M, c[Cys] = 3.0 × 10 M in
O/DMF (1/4, v/v), pH = 7.4, 25 °C. Photograph was taken under hand-
Similarly, two excitation bands were observed for B-2-S (Fig. 4c),
–
5
–3
7
0
indicates the FRET from the diiodoBodipy to the caged
styrylBodipy fluorophore. In the presence of Cys, thus the
cleavage of the disulfide bond, the FRET ceased and the
excitation band at 535 nm disappeared (Fig. 4c). For B-2-C, i.e.
the dyad with the un-cleavable linker, no such change was
observed (Fig. 4d). Similar study was applied to the excitation
spectra of B-3-S. Two excitation bands at 608 nm and 665 nm
were observed for B-3-S (Fig. 4e), which are due to the
fluorophore module and the PDT module, respectively. Thus
H
2
hold UV lamp irradiation (λex = 365 nm).
The kinetic of the response of dyads to Cys, i.e. the cleavage
kinetics, was monitored by following the fluorescence variation.
For B-1-S, the emission intensity at 513 nm increased with elapse
of the time after addition of Cys (Fig. 2a). The response is fast
(Fig. 2b). Fast kinetics was observed k = 0.13±0.04 s . For B-
-C, however, no fluorescence enhancement was observed in the
7
5
4
4
−1
4
0
1
presence of Cys because the linker is unable to be cleaved by Cys.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

Journal of Materials Chemistry B
Page 8 of 18
FRET from the fluorophore to the PDT module exists. In the
presence of Cys, the FRET ceased and the excitation band at 608
nm disappeared. No such change was observed for B-3-C (Fig.
60
The selectivity of the dyads toward different thiols was
studied (Fig. 5). The dyads give strong response to the analytes
4
7
View Article Online
containing -SH group. Thus the dyads B-1-S and B-2-S can be
DOI: 10.1039/C5TB01857A
4
f).
used as probe for biological thiols. For B-3-S, however, only
5
0
5
30
weak fluorescence enhancement was observed in the presence of
65 thiols.
1
000
4
3
20 s No +Cys
a
00 s
8
6
4
2
00
00
00
00
0
Cys
.
..
250
800
2
1
0
0
0
3
0
0 s
s
a
b
200
150
100
6
00
00
00
4
1
b
7
0
2
50
6
00
700
800
0
20
40 60
80 100
0
0
Wavelength / nm
Reaction time / s
1
000
c
30
20
1
No +Cys
20 s Cys
300 s
..
8
6
4
2
00
00
00
00
0
4
7
8
5
0
Fig. 5 (a) Response of B-1-S to different analytes. Relative fluorescence
intensity of 10µM B-1-S at 511nm (λex = 475 nm) before and after
incubation in presence of 3 mM analytes. (b) Response of B-2-S to
different analytes. Relative fluorescence intensity of 10 µM B-2-S at 622
nm (λex = 560 nm) before and after incubation in the presence of 3 mM
analytes. 25 °C.
.
0
s
1
0
0
d
2
0
6
00 650 700 750 800
Wavelength / nm
0
20
40
60
80 100
Nanosecond transient absorption spectroscopy: switching of
the triplet excited state by thiols
Reaction time / s
Fig. 3 (a) The change of fluorescence of B-2-S in the presence of Cys, (b)
The kinetics of B-2-©S in presence of Cys. (c) and (d) The change of
fluorescence and the kinetics in different time in presence of Cys for B-2-
C, Photograph was taken under hand UV lamp irradiation (λex = 365 nm).
c[Dyads] = 1.0 × 10 M, c[Cys] = 3.0 × 10 M, λex = 560 nm, in
O/DMF (v/v, 1/4), pH = 7.4, 25 °C.
Previously pH- or enzyme-activatable PDT reagents were
reported, but the variation of the triplet excited states of the
2
5
–
5
–3
85 compounds were not studied in detail with nanosecond time-
7
,19b,48
H
2
resolved transient difference absorption spectroscopy.
1
0
0
0
.2
.8
.4
Herein the change of the triplet excited state property of the
activatable PDT dyads was investigated with the nanosecond
transient difference absorption spectroscopy.
For B-1-S, bleaching band at 665 nm was observed upon 640
nm nanosecond laser excitation (Fig. 6c). The lifetime of this
transient was determined as 1.8 μs (in toluene), which was
substantially reduced to 1.4 ns in aerated solution, indicating that
the transient is due to triplet excited state. Since the PDT module
1
0
0
0
.2
.9
.6
.3
B-1-C
B-1-C + Cys
a
b
B-1-S
3
3
4
4
5
5
0
5
0
5
0
5
9
0
B-1-S + Cys
.0
3
0.0
300 400 500 600 700
Wavelength / nm
00 400 500 600 700
Wavelength / nm
95 in B-1-S shows strong steady absorption at 640 nm, the triplet
4
1,49
state is localized on the PDT module,
1
due to its lower T state
1
.0
.8
1.00
0.75
c
d
energy level than the fluorescence module in B-1-S. Selective
excitation into the fluorescence module in B-1-S (λex = 504 nm)
gives a very similar transient absorption spectra (Fig. 6a).
0
B-2-S
B-3-C
0.6
B-2-S+Cys
0
0
0
.50
.25
.00
1.2
0
0
0
.4
.2
.0
100
Notably the optical density of the spectra upon 504 nm
excitation is larger than that upon 640 nm excitation (Fig. 6e).
4
1,50
B-3-C+Cys
This result is due to the FRET effect in B-1-S,
i.e. the triplet
4
00
500
600
400
500
600
state of the singlet energy acceptor can be produced upon
selective excitation into the energy donor. In order to
Wavelength / nm
Wavelength / nm
1
0
0
0
.2
.8
.4
*
e
f
105 unambiguously prove the FRET, the TA spectra of B-1-S upon
excitation at 504 nm and 640 nm was compared with that of
compound 10 (Fig. 6e and 6f. The reference structure of the PDT
module in B-1-S). The results show that the spectrum of 10 upon
B-3-C+Cys
B-3-C
B-3-S
0.8
0.4
0.0
6
40 nm excitation is with larger OD values than that upon 504
1
10 nm excitation. This result is reasonable since 10 shows stronger
absorption at 640 nm than at 504 nm. For dyad B-1-S, however,
the optical density (OD) values of the spectrum recorded upon
.0
3
B-3-S + Cys
00 400 500 600 700
300 400 500 600 700
Wavelength / nm
Wavelength / nm
6
40 nm is weaker than that with 504 nm excitation (Fig. 6e).
Fig. 4 The change of the fluorescence excitation spectra of B-1-S, B-1-C
and B-2-S in the presence of Cys. (a) B-1-S (λem = 750 nm). (b) B-1-C
Thus FRET exists for B-1-S, i.e. the FRET following the
15 selective excitation into the fluorescence module of B-1-S will
convey the energy to the PDT module, and the ISC of the PDT
1
(λem = 750 nm). (c) B-2-S (λem = 640 nm). (d) B-2-C (λem = 640 nm). (e)
–5
B-3-S (λem = 720 nm). (f) B-3-C (λem = 720 nm). c [Dyads] = 1.0 × 10
–
3
49
M and c[Cys] = 3.0 × 10 M in H
2
O/DMF (v/v =1/4), 25 °C.
module will produce the triplet state.
8
|
Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 9 of 18
Journal of Materials Chemistry B
0
.06
0.03
.00
6
6
7
0
5
0
0.000
0.00
View Article Online
DOI: 10.1039/C5TB01857A
0
.07
.00
-
0.015
0
τT = 1.4 μs
-
0.04
0
τT = 1.8 μs
5
0
5
0
5
7.0 μs
...
0.42 μs
0 μs
-0.030
-0.045
1
...
9.0 μs
-0.03
-0.06
-
-
0.07
0.14
-0.08
a
b
0
0
.73 μs
μs
a
b
400 500 600 700 800
Wavelength / nm
0
10
20
t / μs
30
40
-0.12
4
00 500 600 700 800
Wavelength / nm
0
10
20
Time / μs
30
0
.02
0.01
.00
0.005
0.000
0.005
0.010
1
1
2
2
0.02
0.00
-
-
0
-
0.01
0.02
0.03
0.04
7.0 μs
0.00
τT = 1.36 μs
τT = 1.8 μs
...
0.75 μs
0 μs
17.0 μs
-0.01
-0.02
-
.
..
-0.015
-0.020
c
-
0.02
0.04
d
0.73 μs
75
-
-
c
0 μs
400 500 600 700
Wavelength / nm
d
0
10
20
30
40
-
t / μs
4
00 500 600 700 800
Wavelength / nm
0.06
0
10
20
Time / μs
30
0
.04
0.02
.00
0
.00
5
6
04 nm
40 nm
0.03
0.07
80
-0.02
0
B-1-S + Cys
B-1-S
0.00
0.00
-0.02
-0.04
-0.06
B-1-S
B-1-S + Cys
e
-
-
0.04
0.06
6
40 nm
504 nm
f
-
0.07
0.14
-0.03
e
400 500 600 700
Wavelength / nm
f
0
10
20
30
40
8
5
-
Wavelength / nm
-
0.06
4
00
600
Wavelength / nm
800
400 500 600 700 800
Wavelength / nm
Fig. 7 Nanosecond time-resolved transient difference absorption spectra
of B-1-S in the presence of Cys. (a) B-1-S alone and (b) the decay trace at
60 nm. (c) Transient absorption spectra of B-1-S with Cys added and (d)
Fig. 6 Nanosecond time-resolved transient difference absorption spectra
of B-1-S upon excitation at different wavelength. (a) Transient difference
absorption spectra and (b) the corresponding decay trace at 660 nm, upon
excitation at λex = 504 nm; (c) transient difference absorption spectra and
6
3
3
0
5
9
0
the decay trace at 660 nm. (e) Comparison of the transient absorption
spectra of B-1-S and B-1-S + Cys with delay time of 0 μs and (f) the
corresponding decay traces at 660 nm. λex = 504 nm. In deaerated
(
d) the corresponding decay trace at 660 nm. upon excitation at λex = 640
–
5
–3
2
DMF/H O = 4/1 (v/v). c[Dyad] = 1.0 × 10 M, c(Cys) = 3.0 × 10 M, 25
nm. (e) Comparison of the TA spectra of B-1-S recorded at different
excitation wavelengths. The delay time is 0 μs, (f) Comparison of the TA
spectra of compound 10 recorded at different excitation wavelengths. The
°
C
–
5
95
Upon addition of Cys, interesting TA spectral profile was
delay time is 0 μs. c[Dyad] = 1.0 × 10 M in deaerated toluene, 25 °C.
observed (Fig. 8c). For the mixture of B-2-S with Cys, i.e. the
cleaved dyad, two bleaching band at 535 nm and 590 nm were
observed upon nanosecond pulsed laser excitation (Fig. 8c).
Furthermore, the transient at 535 nm decrease on the
Upon addition of Cys, thus cleavage of the dyad, the TA
spectra do not show any substantial change, except the OD value
decreased (Fig. 7a and 7b), due to the cease of FRET. The triplet
4
4
5
5
0
5
0
5
state lifetime remains the same (1.4 μs. In DMF/H
It is the same with triplet state lifetime 10 in DMF/H
2
O = 4/1, v/v).
O (Fig.
1
1
1
1
00 microsecond time scale, and the bleaching band at 591 nm
increase concomitantly. Thus we postulate the exist of the
2
5
1,52
S59). The variation of the OD values upon 504 nm excitation in
the presence of Cys was studied (Fig. 7e). It was found the OD
value decreased upon cleavage, which supports the FRET
postulation.
intermolecular triplet state energy transfer.
Moreover, the
transient bleaching at 591 nm was intensified firstly, which
indicated the accumulation of the triplet state of the un-caged
05 fluorophore of B-2-S. Then after ca. 30 μs, the transient
bleaching decreased, which is due to the decay of the triplet
excited state of the un-caged fluorophore. The rate of
The change of the triplet excited state manifold of B-2-S was
also studied with the nanosecond time-resolved transient
absorption spectra (Fig. 8). Upon selective excitation into the
PDT module (the singlet energy donor in the dyad), triplet
excited state localized on the caged fluorescence module was
observed, with bleaching band at 584 nm (Fig. 8a). The triplet
state lifetime was determined as 179.9 μs (Fig. 8b). No triplet
state can be observed with the caged fluorophore (compound 9)
alone with excitation at 585 nm (See ESI †, Fig. S60). Thus the
triplet state energy transfer in B-2-S was confirmed. It should be
4
-1
intermolecular energy transfer was calculated as 2.84 ×10 s
with Eq. 1 and energy transfer efficiency was calculated as 82.4%
5
1
10 (Eq. 2). It should be pointed out that investigation on the
5
2
intermolecular triplet excited energy transfer is rare. The
efficient intermolecular EnT, facilitated by the long-lived triplet
state of the energy donor, will he helpful for the study of
intermolecular energy transfer and artificial photosynthetic
15 studies.
pointed out that the triplet state yield of B-2-S is low
1
(
approximated with the O
2
quantum yield, Φ
Δ
= 16.7%), due to
1
1
(Eq. 1)
1
8
kEnT
=
−
2 1
the competition of FRET with the ISC of the PDT module.
τ
τ
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 9

Journal of Materials Chemistry B
Page 10 of 18
0
.01
τ
τ
2
1
60 0.01
ΦEnT =1−
(
Eq. 2)
0.00
0.01
0.02
View Article Online
0
.00
0.01
65 -0.02
-0.03
-
-
DOI: 10.1039/C5TB01857A
τ = 152.6 μs
The response of B-2-C to the presence of Cys was studied with
TA spectra. The triplet state lifetime of B-2-C is 152.6 μs (Fig.
a and 9b). In the presence of Cys, the TA spectra do not change,
the lifetime of the triplet excited state was slightly extended to
72.2 μs (Fig. 9c and 9d). Interestingly, no bleaching band of the
PDT module was observed, which indicated that the
intramolecular EnT is persistent in the presence of Cys, which is
consistent with the fact that the linker between the fluorophore
and the PDT module was not cleaved. Thus, no intermolecular
triplet energy transfer was observed. As a further proof, the TA
spectra of B-2-C and B-2-C+Cys show the same OD value upon
selective excitation into the EnT donor (Fig. 9e).
T
-
760 μs
...
5
0
5
0
5
0
5
0
5
56 μs
-0.03
-0.04
9
a
0 μs
b
1
400 500 600 700
Wavelength / nm
0
500 1000 1500 2000
t / μs
0
0
0
.02
.01
.00
1
1
2
2
3
3
4
4
c
70
75
80
85
0
.000
τT = 172.2 μs
860 μs
-
-0.02
0.03
0.01
-0.015
...
2
0
8 μs
μs
-
0.030 d
0
The transient absorption of B-3-S in the absence and in the
presence of Cys were also studied. The bleaching band indicate
that the triplet state is localized on the PDT module (Fig. 10).
-
4
00
500
600
700
500 1000 1500 2000
Wavelength / nm
5
3−55
t / μs
0
.01
The lifetime was determined as 0.94 μs. Upon addition of Cys,
the TA spectral feature did not change, the lifetime was slightly
extended to 1.39 μs. The extension of the triplet state lifetime
may be due to the elimination of the intramolecular electron
transfer in B-3-S.
In order to prove the electron transfer (ET) in B-3-S, we
compared the TA spectra of B-3-S and compound 10 in solvents
with different polarity, because it is known that ET is dependent
0
0
.01
.00
e
0.00
0.01
B-2-C + Cys
-
f
B-2-C
τT = 152.6 μs
-
-
-
0.01
0.02
0.03
-0.02
-0.03
B-2-C
B-2-C + Cys
τT = 172.2 μs
-
0.04
400
500
600
700
0
500 1000 1500 2000
5
6−60
Wavelength / nm
on the solvent polarity.
In DMF/H O = 4/1 (v/v), the OD
t / μs
2
values of the B-3-S spectra is smaller than that of compound 10
upon selective excitation into the singlet energy donor module
(Fig. S59), indicating intramolecular electron transfer. In
nonpolar solvent such as toluene, the OD value of the compound
B-3-S and compound 10 are the same. Very close triplet state
lifetimes of 1.78 μs and 1.74 μs were observed. These results
show that electron transfer is repressed in nonpolar solvent.
Fig. 9 Nanosecond time-resolved transient difference absorption spectra
of B-2-C in the presence of Cys. (a) Transient difference absorption
spectra of B-2-C alone and (b) the decay trace at 580 nm. (c) Transient
difference absorption spectra of B-2-C + Cys and (d) the decay trace at
9
9
0
5
5
90 nm. (e) Comparison of the transient difference absorption spectra of
B-2-C and B-2-C + Cys. The decay time is 0 µs and (f) the corresponding
decay trace at 590 nm of the two samples. λex = 535 nm (nanosecond
–
3
–5
pulsed laser). C(Cys) = 3.0 × 10 M and c[Dyads]= 1.0 × 10 M in
deaerated DMF/H O = 4/1 (v/v), pH = 7.4, 25 °C.
2
0
0
.03
.00
0.015
.000
a
0
In order to study intramolecular energy transfer for Bodipy
dyads, the rate constants of energy transfer (kEnT) and electron
transfer (kET) were estimated using fluorescence quantum yields
-
-
-
0.015
0.030
0.045
5
70.0 μs
.
..
τ = 179.9 μs
T
-0.03
100 and fluorescence lifetimes of the dyads and the energy
3
0
0.0 μs
μs
5
7
b
d
donor/acceptors (Table 2 and Eq3 and Eq 4). For B-1-S and B-
-S , kEnT is faster than kET, so the intramolecular electron transfer
can not quench triplet state. But for B-3-S, It is not neglect to
-
0.06
2
4
00
500
600
700
0
500 1000 1500 2000
Wavelength / nm
t / μs
0.03
57
0
.00
0.03
-0.06
0.09
triplet excited state quenching because of kET is larger than kEnT.
c
0.00
105 The result is consistent with nanosecond time resolved the
transient absorption.
-
590 nm
2
.
2
0
48.6 μs
-
0.03
0.06
5
45 nm
..
τT = 45.4 μs
2.6 μs
μs
-
⎡ΦF(electron accptor part)
⎤
-
k =
/τ (electron accptor part)
(Eq. 3)
ET
⎢
⎥
⎦
5
0
-0.09
1
10
⎣
Φ
F(target molecule)
4
00 500 600 700
Wavelength / nm
0
500
1000
t / μs
1500
Fig. 8 Nanosecond time-resolved transient difference absorption spectra
of B-2-S in the presence of Cys. (a) Transient difference absorption
spectra of B-2-S alone and (b) the decay trace at 580 nm. (c) Transient
difference absorption spectra of B-2-S with Cys added and (d) the decay
⎡
⎣
Φ
F(energy donor part)
⎤
kEnT
=
−1 /τ (energy donor part) (Eq. 4)
5
5
⎢
⎥
⎦
Φ
F(target molecule)
1
15
trace at 545 nm and 590 nm. In deaerated DMF/H
with nanosecond pulsed laser at λex = 535 nm, c[Cys] = 3.0 × 10 M, c
2
O = 4/1 (v/v). Excited
–3
where kET is the rate of intramolecular electron transfer, Φ
electron acceptor part) and (target molecule) are the
F
–
5
[
Dyad] = 1.0 × 10 M, 25 °C
(
Φ
F
1
0
|
Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 11 of 18
Journal of Materials Chemistry B
0
fluorescence quantum yields of electron acceptor part and the
target molecule and τ (electron acceptor part) is the electron
accept part fluorescence lifetime. kEnT is the rate of intramolecular
as −0.44 eV (in DCM), and the ΔG CS value was calculated as
55 −0.69 eV in DMF/H O (4:1, v/v). It is known that intramolecular
2
View Article Online
electron transfer is favourable in polar solvent. With the triplet
excited state of diiodoBodipy part as E0,0, ΔG CS was calculated
as +0.22 eV (in DCM), and ΔG CS was calculated as −0.032 eV in
DOI: 10.1039/C5TB01857A
0
energy transfer constants, Φ
F
(energy donor part) and Φ
F
(target
0
5
0
5
0
molecule) are the fluorescence quantum yields of energy donor
part and the target molecule and τ (energy donor part) is the
energy donor part fluorescence lifetime.
DMF/H
60 state also was calculated. ECTS is 1.77 eV in DCM, and ECTS is
1.52 eV in DMF/H O. However, the triplet state energy level of
O (4:1, v/v). The energy level of the charge transfer
2
0
0
0
.008
.004
.000
0
.003
2
a
the caged fluorophore module (1.24 eV, by DFT calculation) is
lower than charge transfer state. Therefore the effect of
intramolecular electron transfer on the triplet state is not
0
.000
1
1
2
b
-0.003
-0.006
-0.009
56−60
6
5
significant.
1
7.0 μs
-
-
0.004
0.008
τΤ = 0.94 μs
...
For B-3-S, the electron donor moiety is 4-N,N-
dimetylstylBodipy part because of the high oxidation potential
(+0.35 V). Large driving force (ΔG CS = −0.76 eV in DMF/H
and lower charge transfer state (CTS) energy level were found
(1.09 eV). Thus the triplet state can be quenched by the electron
transfer.
0.57 μs
0.0 μs
0
2
O)
4
00 500 600 700 800
Wavelength / nm
0
5
10 15 20 25
t / μs
7
0
0
0
.015
.000
c
0
.000
d
-0.007
0.0 μs
.57 μs
...
7.0 μs
0
τΤ = 1.39 μs
c
-
-
0.014
0.021
a
b
-
0.015
1
75
-0.030
3
00 400 500 600 700 800
Wavelength / nm
0
5
10 15 20 25
t / μs
2
5
Fig. 10 Nanosecond time-resolved transient difference absorption spectra
of (a) B-3-S and (b) the corresponding decay trace at 665 nm (c)
Transient difference absorption spectra of B-3-S + Cys and (d) the
corresponding decay trace at 665 nm. λex = 655 nm, nanosecond pulsed
1
0
-1
-2
1
0
-1
-2
1
0
-1
-2
Potential (V)
8
0
Potential (V)
Potential (V)
Fig. 12 Cyclic voltammogram of (a) 9, (b) B-4, (c) B-2-S. Ferrocene (Fc)
was used as internal reference (E1/2 = +0.64 V (Fc /Fc) vs. standard
hydrogen electrode). In deaerated CH
–5
+
3
0
laser. In deaerated DMF/H
2
O = 4/1 (v/v). c[Dyads] = 1.0 × 10 M, c[Cys]
–
3
=
3.0 × 10 M, pH = 7.4, 25 °C.
2
Cl
2 4 6
solutions with 0.10 M Bu NPF
as supporting electrode, Ag/AgNO
V/s.
3
reference electrode, Scan rates: 0.1
Table 2. Parameters of Fluorescence Quantum Yield and Fluorescence
Lifetimes
8
5
Φ
F
Φ
F
τ
F
τ
F
Φ
F
k
(s )
ET
k
(s )
EnT
-1
-
1
d
e
(
electron (energy (electron(energy (target
Table 3. Redox Potentials of Thiol-Activatable Dyads for Study. Anodic
accepter) donor) accepter) donor) molecule)
and Cathodic Peak Potential Were Presented. The Potential Values of the
Compounds Are Vs. Saturated Calomel Electrode
a
8
9
a
B-1-S 7.0% 72.0% 1.91 ns 3.86 ns 5.8% 6.3 × 10 3.2 × 109
b
8
B-2-S 1.4% 2.7% 1.86 ns 0.13 ns 1.3% 5.8 × 10 8.3 × 10
8
c
8
E(Ox) (V)
+0.79
E(Red) (V)
−0.83, −1.18, −1.30, −1.57
−1.09
B-3-S 7.0% 7.0% 1.91 ns 1.91 ns 5.4% 6.8 × 10 1.6 × 10
9
a
the electron acceptor part is 2I-distylBodipy part, energy donor part is
1
1
0
1
+0.67, +0.99
+0.34, +0.66
+0.94
b
3
5
Bodipy part. the electron accepter part is StyrylBodipy-DNS part,
−1.44
c
energy donor part is diiodoBodipy part. the electron accepter part is 2I-
Bodipy
−1.50
−0.84, −1.28, −1.52
−1.27
d
distylBodipy part, energy donor part is 2I-distylBodipy part . kET is the
b
1
5
−
e
rate of intramolecular electron transfer constants. the rate of
B-4
+1.08
+0.70, +1.00
+0.82, +1.04
intramolecular energy transfer constants.
B-1-S
B-2-S
B-3-S
−1.07, −1.51
−0.82, −1.15, −1.28, −1.53
−1.08, −1.43
4
0
+0.35, +0.68, +0.92, +1.00
Electrochemical Studies: the Cyclic Voltammetry and the
Free Energy Changes of the Intramolecular Electron
Transfer.
a
9
9
0
5
Cyclic voltammetry in N
Bu NPF supporting electrolyte; Counter electrode is Pt electrode;
working electrode is glassy carbon electrode; Ag/AgNO couple was used
as the reference electrode. c [Ag ] = 0.1 M. 20 °C. Conditions: 0.5 mM
photosensitizers in CH Cl
2 2 2
saturated CH Cl containing a 0.10 M
4
6
3
+
The cyclic voltammetry of B-1-S, B-2-S and B-3-S were studied
(Fig. 12). For B-2-S, two irreversible oxidation waves at +0.82 V
and +1.04 V were observed. From the cathodic scan, four
irreversible reduction waves were also observed at −0.82 V,
2
2
, 20 °C. Calculate relative to SCE (saturated
4
5
b
calomel electrode). Not observed.
The photophysical process of B-1-S was summarized in
Scheme 3. In the absence of thiols, fluorescence FRET from the
Bodipy to bis(4-methoxy distyryl)-2,6-diiodo-Bodipy leads to the
−
1.15 V, −1.28 V and −1.53V. The control compounds 9, 15 and
0
B-4 were also studied (Fig. 12). The Gibbs free energy (ΔG CS)
of the intramolecular electron transfer were calculated by Weller
equation. For example, in B-2-S, the caged PDT module is
electron donor and the DNS as electron acceptor. With the singlet
excited state of diiodoBodipy part as E0,0, ΔG was calculated
fluorescence quenching of Bodipy part. Moreover, the T state
5
0
1
4
1
00 energy level of the Bodipy part level (1.52 eV), is higher than
that of the PDT module (1.18 eV by DFT calculation), therefore
the triplet excited state is located on the PDT module. The charge
0
CS
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 11

Journal of Materials Chemistry B
Page 12 of 18
transfer state (CTS) is with energy level of 1.73 eV, thus the 50 that in the presence of Cys (λex = 500 nm, Fig. 13a). It was found
1
fluorescence of the PDT module in B-1-S was quenched as
compared with that of reference 10 (Table 1). The energy
diagram also shows that the triplet state of B-1-S should be close
to that of compound 10. The TA spectroscopy demonstrated that
that upon addition of Cys, the
2
O photosensitizers ability
1
View Article Online
decreased. The O quantum yield decreased from 66.8 % to 22.8
%. This is due to the ceased FRET upon cleavage of the S−S
linker by thiols, thus the termination of FRET, as a result, the
2
DOI: 10.1039/C5TB01857A
5
0
5
this is exact the case (triplet state lifetime of 1.30 μs and 1.40 μs 55 excitation energy harvested by the Bodipy moiety can not be
1
were observed for compound 10 and B-1-S, respectively). In the
presence of thiols, disulfide bond was cleaved, and FRET ceased,
as a result the strong fluorescence of the fluorescence module
was observed (Scheme 3b).
conveyed to the PDT module. No variation of the
O
2
photosensitizing was observed for B-1-S upon selective
excitation into the PDT module (664 nm. Fig. 13d). This result is
reasonable since the ISC of the PDT module in B-1-S will not be
1
The photophysical processes of B-2-S were summarized in 60 altered by the cleavage of the linker between the PDT module
Scheme 4. In the absence of thiols, upon photoexcitation into the
PDT module (2,6-diiodo-Bodipy moiety), the FRET and the ISC
will compete with each other. It is known that the ISC of the
diiodoBodipy takes about 100 ps, thus the FRET process (takes a
few ps) is much faster than the ISC. As a result, the triplet state
yield of B-2-S will be low, which was proved by the singlet
and the fluorescence module. B-1-C gives no response to the
presence of Cys (λex = 500 nm. See ESI †, Fig. S78).
2.0
0
s
1
1.2
a
b
10 s
..
60 s
1.5
1.0
.
65
B-1-S + Cys
0.9
1
oxygen ( O ) photosensitizing (Φ = 16.7%). The fluorescence of
2
Δ
4
-hydroxyl styryl 2,6-phenyl-Bodipy part was quenched by
0
.5
B-1-S
0
.6
2
0
Table 4. The Thermodynamic Driving Force for Photoinduced Electron
Transfer Was Calculated From the Weller Equation
a
0.0
1
0
20
40
60
300
350
400
450
7
0
Irradiation time (s)
0
b
0
c
2.0
Wavelength / nm
b
c
ΔG CS (eV)
ΔG CS (eV)
E
CTS (eV)
E
CTS (eV)
0 s
10 s
...
c
.2
1
1
0
0
.5
.0
.5
.0
d
d
d
B-1-S
B-2-S
B-3-S
−0.43
−0.68e
1.98
1.77
1.34
1.73
1.52
1.09
60 s
B-1-S + Cys
e
+
0.46
+0.21
d
e
d
e
d
0.9
−0.44
0.22
−0.51
0.10
−0.69
7
5
e
B-1-S
+
−0.032
d
−0.76 e
0.6
+
−0.15
300
350
Wavelength / nm
400
450
0
20
40
60
a
Photoinduced electron transfer from energy donor to energy acceptor,
Irradiation time (s)
o
ΔG CS was calculated by Weller approach and ECTS = EOX − ERED + ΔG
and ΔG
(v/v). The S
s
2
.0
2.0
1.5
1.0
0.5
b
c
80
S
is he static Coulombic energy. in DCM. in DMF/H
2
O = 4/1
state of energy donor
0
s
0 s
10 s
...
d
e
e
f
2
5
1
state of energy donor as E0,0
.
The T
1
10 s
...
1.5
1.0
0.5
0.0
as E0,0
60 s
60 s
2
F
,4- dinitrosulfonyl group (Φ = 1.3%). TTET from 2,6-diiodo-
Bodipy to 4-hydroxyl styryl 2,6-phenyl-Bodipy part was
observed because the triplet excited state level of 4-hydroxyl
styryl 2,6-phenyl-Bodipy part (1.24 eV, by DFT calculation) is
8
9
9
5
0
5
3
3
4
0
5
0
0.0
4
300
350
400
450
lower than the PDT module (1.50 eV).
300
350
400
450
Wavelength / nm
Wavelength / nm
In the presence of thiols (Fig. 4b), both the 2,4-
dinitrobenzenesulfonyl group and disulfide bond were cleaved.
As a result, strong red fluorescence of the fluorescence module
was observed (Φ = 47.6%). At the same time, the ISC of the
PDT module was recovered due to the cease of the FRET effect
1
Fig. 13 Switching of the singlet oxygen ( O
2
) photosensitizing ability of
1
the dyads by Cysteine. The decreasing of the absorption of O
2
scavenger
1
,3-diphenylisobenzofuran (DPBF) at 414 nm was monitored upon the
F
monochromic light irradiation, with B-1-S as photosensitizer. (a)
Absorbance of DPBF at 414 nm changes in the absence and in the
presence of Cys (λex = 500 nm). The UV−vis absorption spectra in the
absence (b) and (c) in the presence of Cys. (d), (e) and (f) the
corresponding change with B-1-S as photosensitizer (λex = 664 nm). c
(
ΦΔ = 71.5%). Moreover, due to the long triplet excited state
lifetime of PDT module (i.e. the 2,6-diiodo-Bodipy moiety),
intermolecular triplet excited state energy transfer (TTET) from
the (cleaved) PDT module to the uncaged fluorescence module is
not negligible. Our results will shed light on new approaches to
modulate triplet excited states of organic chromophores.
1
–
5
–3
[
photosensitizers] = 1.0 × 10 M, c[Cys] = 3.0 × 10 M, in H
1/4, v/v), pH = 7.4, 25 °C.
2
O/DMF
(
Switching of the Singlet Oxygen ( O ) Production by Thiols
1
2
1
00
The switching of the O2 photosensitizing ability of B-2-S
upon addition of Cys is different from that of B-1-S (Fig. 14a). It
2
photosensitizing ability of B-2-S increased
in the presence of Cys (Φ = 71.5%) as compared with that in the
1
The switching of the singlet oxygen ( O ) photosensitizing ability
of the dyads was studied. O scavenger 1,3-diphenylbenzofuran
(
2
1
1
4
5
2
was found that the O
1
DPBF) was used to follow the O
2
photosensitizing kinetics.
Δ
1
absence of Cys (Φ
Δ
= 16.7%). This is due to the recovered ISC of
decreased. Firstly, the O2 photosensitizing of B-1-S upon 105 the PDT module after removal of the FRET by cleavage of the
2
Upon reaction with O , the absorption of DPBF at 414 nm
1
39
singlet energy acceptor, and it is similar to that observed in the
selective excitation into the Bodipy moiety was compared with
1
4-16
photodynamic molecular beacon.
If the excitation is into the
12 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 13 of 18
Journal of Materials Chemistry B
View Article Online
1[
BDP*-Distyryl BDP] 2.40 eV
FRET
1[BDP*-DDOisI:ty10ry.1l0B3D9/PC]5TB01857A
a
b
FRET
×
1[
BDP-Distyryl BDP*]
1
[BDP-Distyryl BDP*]
8
-1
k
ET =6.3×10 s
9 -1
k
EnT = 3.2×10 s
CTS
1
.78 eV
ISC
1.73 eV
3[BDP-Distyryl BDP*]
501 nm
3
[
BDP*-DiStyryl BDP]
Cysteine
1
.52 eV
BDP-DiStyryl BDP*]
.18 eV
3[
1
662 nm
501 nm
S
0
S
0
S
0
5
Scheme 3. Simplified Jablonski Diagram Illustrating the Photophysical Processes Involved in B-1-S (a) in the Absence of Cysteine and (b) in the Presence
of Cysteine. [BDP-Distyryl BDP] stands for B-1-S. The component at the excited state was designated with red colour and a asterisk. The number of the
superscript designated either the spin manifold. The fluorescence and the triplet state of B-1-S were switched by the presence and the absence of Cysteine.
Note in the absence of Cysteine, the fluorescence of B-1-S were efficiently quenched by FRET. In the presence of Cysteine, disulfide bond is cleaved and
FRET disappear, as a result the strong fluorescence of Bodipy were observed.
1
0
1[
2l-BDP*-styryl BDP-DNS]
FRET
1[
FRET
2l-BDP*-styryl BDP-DNS]
b
×
2.32 eV
ISC
[2l-BDP*-styryl BDP-DNS]
Intermolecular TTET
ISC
3
3[
2l-BDP*-styryl BDP-DNS]
2
.14 eV
80 nm
CTS
.52 eV
Cysteine
1
1.50 eV
Intramolecular
TTET
3[
2l-BDP-styryl BDP-DNS*]
.24 eV
3[
2l-BDP-styryl BDP-DNS*]
1
1
.24 eV
5
535 nm
6
20 nm
S
0
S
0
S
0
S
0
Scheme 4. Simplified Jablonski Diagram Illustrating the Photophysical Processes Involved in (a) B-2-S in the Absence of Cysteine and (b) B-2-S in the
Presence of Cysteine. [I-BDP-styryl BDP-DNS] stands for B-2-S. The component at the excited state was designated with red colour and a asterisk. The
number of the superscript designated the spin manifold. The fluorescence and the triplet state of B-2-S were switched by the presence and the absence of
Cysteine. Note in the absence of Cysteine, the fluorescence of B-2-S were efficiently quenched by 2,4-nitrobenzenesulfonyl group. In the presence of
1
5
Cysteine, both the disulfide bond and 2,4-nitrobenzenesulfonyl group are cleaved and RET disappears, as a result the strong fluorescence of Bodipy and
1
high O
2
quantum yield were observed.
1
caged fluorescence module, negligible
O
2
photosensitizing
presence of Cys, as compared to that of B-3-S alone (Fig. S80).
This result indicated in B-3-S, the ISC of the PDT module is
slightly quenched by the intramolecular charge transfer, which is
supported by the calculation of the free energy changes of
2
2
3
0
5
0
was observed due to the weak ISC of this module. It is noted that
the sum of the fluorescence quantum yield and the O
larger than 100%. Mono-chromophore based PDT/fluorescence
1
2
yield is
bioimaging materials can only give the sum smaller than 35 intramolecular electron transfer, and the low-lying CTS (at 1.0
19a
1
00%.
For B-2-C, the O photosensitizing ability is weak and was
eV). For B-3-C, however, the singlet oxygen photosensitizing is
not affected by Cys. Recently Bodipy based dyad was studied
with the fluorescence be switched ON in the presence of thiols,
1
2
not affected by the addition of Cys (Fig. 14e). This result
indicated that the ISC module in B-2-C was efficiently quenched
by FRET, the charge recombination (CR) is inefficient to produce
the triplet state, and the S−S linker is unable to be cleaved by Cys.
1
19b
but the O photosensitizing ability was not switched.
2
1
40
Switching ON of fluorescence and O2 photosensitizing
ability was observed with DNBS caged zinc(II) phthalocyanine
1
1
Enhanced O2 photosensitizing was observed for B-3-S in the
photosensitizer, but the fluorescence and the O
photosensitizing
2
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 13

Journal of Materials Chemistry B
Page 14 of 18
were accomplished with the same chromophore, thus the 60 Incubation of B-2-S in HeLa cells for 3 h, red fluorescence was
1
fluorescence and the O
as B-1-S does.
2
photosensitizing cannot be maximized
observed (Fig. 16f). In order to further illustrate the red
19a
View Article Online
fluorescence come from the result in thiols and B-2-S reacted in
DOI: 10.1039/C5TB01857A
the cell, N-methylmaleimide was used as the thiol scavenger. A
control experiment was performed where the cellular thiols were
removed using N-methylmaleimide before incubation of the cells
with B-2-S. As a result, the red fluorescence was not observed
1
.5
0 s
0 s
..
5
0
5
0
5
0
1.2
B-2-S
1
.
6
5
0.9
1.0
0.5
60 s
(
Fig. 16j). It shows that B-2-S can specifically detect thiols
B-2-S + Cys
0.6
within the cell.
1
1
2
2
3
a
b
0.3
70
0.0
0
20
40
60
a
e
b
f
c
d
h
300
350
400
450
Irradiation time / s
Wavelength / nm
1.2
0.8
0.4
0.0
0
1
s
0 s
B-2-S + Cys
B-2-S
1
.2
...
1.0
g
60 s
7
5
0
0
0
.8
.6
.4
d
c
00
0
20
40
60
3
350
400
450
Irradiation time / s
8
0
i
j
k
l
Wavelength / nm
1
.2
.1
.0
.9
.8
B-2-C
1
1
1
.2
.1
.0
B-2-C
Fig. 15 Confocal laser scanning fluorescence microscopic images of
HeLa cells. Luminescence images of HeLa cells without B-1-S at
1
1
0
0
B-2-C + Cys
different excitation and imaging wavelength of (a) λex = 488 nm, λem
=
B-2-C + Cys
85 490–550 nm, and (b) λex = 635 nm, λem = 650–750 nm. Luminescence
images of HeLa cells incubated with B-1-S (25 μM) for 3 h, at different
excitation and imaging wavelength of (e) λex = 488 nm, λem = 490–550
nm, and (f) λex = 635 nm, λem = 650–750 nm. Luminescence images of
HeLa cells pretreated with N-methylmaleimide (0.5 mM) for 12 h and
0.9
0.8
f
e
0
20
40
60
0
20
40
60
Irradiation time / s
Irradiation time / s
9
0
incubated with B-1-S (25 μM) for 3 h at different excitation and imaging
wavelength of (i) λex = 488 nm, λem = 490–550 nm and (j) λex = 635 nm,
λ
em = 650–750 nm. The corresponding bright field images of (d, h, l) are
the overlay of respective luminescence (a, e, j) and bright images (c, g, k).
1
Fig. 14 Switching of the singlet oxygen ( O
the dyad B-2-S by Cys. The decreasing of the absorption of O
,3-diphenylisobenzofuran (DPBF) at 414 nm was monitored upon
2
) photosensitizing ability of
1
2
scavenger
25 °C.
1
3
4
5
0
monochromic light irradiation. (a) Absorbance of DPBF at 414 nm
95
00
05
changes in the absence and in the presence of Cys (λex =535 nm) with B-
2
-S as photosensitizer. (b) UV−vis absorption spectral changes in the
a
e
b
f
c
d
absence of Cys and (c) in the presence of Cys. (d) Absorption changes at
14 nm upon excitation at λex = 586 nm. Change of the absorbance of
4
DPBF at 414 nm in the absence and presence of Cys with B-2-C as triplet
photosensitizer upon excitation at (e) 535 nm and (f) 586 nm. c
1
–
5
–3
[
(
2
photosensitizers] = 1.0 × 10 M, c[Cys] = 3.0 × 10 M, in H O/DMF
v/v =1/4), pH = 7.4, 25 °C.
g
h
4
5
Intracellular luminescence Imaging and PDT Studies
The thiol-activatable fluorescence and PDT effect with the dyads
1
i
j
k
l
2
3,61,62
were studied with HeLa cells.
cells with B-1-S for 3 h, intense green fluorescence was observed
Fig. 15e). With removal of the intracellular thiols by N-
methylmaleimide, no such green fluorescence can be observed
Fig. 15e). These results demonstrated that the dyad B-1-S is
After incubation of the HeLa
(
5
0
110
(
Fig. 16 Confocal laser scanning fluorescence microscopic images of
selective to the intracellular thiols. Similar studies were carried
out for B-1-C. No green fluorescence was observed after
incubation with HeLa cells (See See ESI †, Fig. S81). This result
indicates that the fluorescence was completely caged by the
HeLa cells. Luminescence images of HeLa cells upon excitation at
different wavelength of (a) λex = 488 nm, λem = 530–550 nm and (b) λex
=
5
59 nm, λem = 600 – 650 nm. Luminescence images of HeLa cells
1
1
15 incubated with B-2-S (25 μM) for 3 h and then imaged at different
excitation wavelength of (e) λex = 488 nm, λem = 530–550 nm and (f) λex
= 559 nm, λem = 600–650 nm. Luminescence images of HeLa cells
pretreated with N-methylmaleimide (0.5 mM) for 12 h and incubated with
B-2-S (25 μM) for 3 h, and then imaged at different excitation
20 wavelength of (i) λex = 488 nm λem = 530–550 nm and (j) λex = 559 nm,
5
5
42,63−65
FRET effect.
We noted the abnormal morphology of the
cell after incubation with B-1-S, but the reason for this change is
unclear.
B-2-S was used for in situ detection of thiols in HeLa cell.
λ
em = 600–650 nm. 25 °C.
1
4
|
Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 15 of 18
Journal of Materials Chemistry B
3
4
4
5
0
5
fluorescence can be observed (Fig. 17j). However, our later study
a
e
b
c
show that although the fluorescence can be un-caged by the
d
View Article Online
intracellular thiols, the PDT ability of B-2-C cannot be activated
DOI: 10.1039/C5TB01857A
by intracellular thiols
5
The cellular PDT effect with B-2-S and B-2-C as triplet
1
photosensitizers was studied with an intracellular O2 probe,
f
j
g
h
48
DCFH-DA (Fig. 18). Green fluorescence was observed for B-2-
S with photoirradiation (Fig. 18g), but no such green emission
was observed with B-2-C as the photosensitizer (Fig. 18e). Thus
1
1
1
1
2
2
3
6
7
7
0
5
0
5
0
5
0
5
we conclude that no O
2 2
can be produced with B-2-C, but O can
i
k
l
be produced with B-2-S upon photoexcitation. Study in solution
1
also show that the production of O
Fig. 14f).
2
with B-2-C is negligible
(
Fig. 17 Confocal laser scanning fluorescence microscopic images of
a
Table 5. IC50 values of B-2-S and B-2-C (µM)
HeLa cells. Luminescence images of HeLa cells upon excitation at
different wavelength of (a) λex = 488 nm, λem = 530–550 nm and (b) λex
59 nm, λem = 600 – 650 nm. Luminescence images of HeLa cells
=
B-2-S
B-2-C
In dark
With irradiation
In dark
With irradiation
5
3
0.1 ± 1.8 4.9 ± 0.27
29.6 ± 1.3 9.7 ± 0.45
incubated with B-2-C (25 μM) for 3 h, upon different excitation
wavelength at (e) λex = 488 nm, λem = 530 – 550 nm and (f) λex = 559 nm,
em = 600 – 650 nm. Luminescence images of cells pretreated with N-
methylmaleimide (0.5 mM) for 12 h and incubated with B-2-C (25µM)
for 3 h, upon different excitation wavelength at (i) λex = 488 nm, λem
30 – 550 nm and (j) λex = 559 nm, λem = 600 – 650 nm. (c, g, k) are the
a
50 IC50, the concentration of the compound that inhibits the proliferation
rate by 50% compared with untreated cells, HeLa cell lines. Irradiated by
λ
2
a 515 − 525 nm LED, power intensity is 5 W/m .
=
5
IC50 values of B-2-S and B-2-C was studied in the absence
55 and in the presence of photoirradiation by MTT assay (Table 5).
IC50 values of B-2-S and B-2-C were found to be 37.50 μM and
corresponding bright field images of (a, e, i); (d, h, l) are the overlay of
respective luminescence and bright images. 25 °C.
3
1.25 μM, respectively, for HeLa cell lines. Moreover, In the
Later study shows that the PDT effect is significant, indicate
that the linker between the PDT module and the fluorescence
presence of photoirradiation, much higher toxicity was observed,
with IC50 values of 4.78 μM and 11.40 μM for B-2-S and B-2-
module was cleaved. After removal of the intracellular thiols by 60 C,respectively. IC50 values for B-2-S is lower than B-2-C because
N-methylmaleimide, no red fluorescence can be observed.
Similar studies were carried out for B-2-C. Red fluorescence
was observed after incubation with HeLa cells (Fig. 17f). After
pre-treatment of the cells with N-methylmaleimide, no red
of disulfide bond was activation by thiols, but the caged PDT for
B-2-C was not fully activated. The PDT effect of other
compounds in Scheme 1 were not studied.
a
c
g
h
e
f
i
j
b
d
8
0
Fig. 18 Confocal laser scanning fluorescence microscopic images of the PDT effect on HeLa cells with B-2-S and B-2-C as the photosensitizers. DCFH-
1
2
DA as intracellular singlet oxygen ( O ) probe. Imaging of cells without B-2-S and irradiation: (a) Dark field and (b) bright field images; Imaging of cells
8
5
with B-2-S but without photoirradiation: (c) dark field and (d) bright field images; Imaging of cells with B-2-C and photoirradiation: (e) dark field and (f)
bright field images; Imaging of cells with B-2-S and photoirradiation: (g) luminescence and (h) bright field images; Imaging of cells with B-2-S and
photoirradiation, but pretreated with N-methylmaleicimide: (i) luminescence and (j) bright field images. c [photosensitizer] = 2.5 μM, c[DCFH-DA] = 2.0
μM, λex = 488 nm, λem = 500 – 550 nm. For PDT study, the cells were incubated with the photosensitizers and the cells were irradiated by a 515 − 525 nm
2
LED, power intensity is 5 W/m . 25 °C.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 15

Journal of Materials Chemistry B
Page 16 of 18
b
School of Life Science and Biotechnology, Dalian University of
Conclusions
Technology, Dalian 116024, P. R. China.
E-mail: wuhj@dlut.edu.cn
View Article Online
DOI: 10.1039/C5TB01857A
In conclusion, thiol-activatable bifunctional treatment/monitoring
materials, i.e. photodynamic/fluorescence bioimaging dyads were
prepared. The unique molecular structural profile of these dyads
is that the activatable PDT and fluorescence were accomplished
with two different chromophores (modules) in the dyad.
†
Electronic Supplementary Information (ESI) available: molecular
6
0
structure characterization data, UV-vis absorption spectra, fluorescence
spectra and nanosecond transient absorption spectra. See
DOI: 10.1039/b000000x
5
0
5
0
5
0
5
0
Therefore, both the PDT and the fluorescence bioimaging effect
1
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2
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3
3
4
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) can be up to
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8
8
9
9
0
5
0
5
DNS moiety, thus the PDT module and the fluorescence module
1
were activated simultaneously. As a result,
2
O was produced
2
865–2874.
upon photoexcitation and fluorescence was greatly enhanced (ΦΔ:
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Acknowledgement
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We thank the NSFC (21073028, 21273028, 21473020 and
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(SRFDP-20120041130005), the Fundamental Research Funds for
the Central Universities (DUT14ZD226), Program for
Changjiang Scholars and Innovative Research Team in
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State Key Laboratory of Fine Chemicals, School of Chemical
Engineering, Dalian University of Technology, E-208 West Campus, 2
Ling Gong Rd., Dalian 116024, P. R. China.
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Journal Name, [year], [vol], 00–00 | 17

Journal of Materials Chemistry B
Page 18 of 18
View Article Online
DOI: 10.1039/C5TB01857A
Graphical Abstract:
Maximizing the Thiol-Activated Photodynamic and Fluorescence Imaging Functionalities of
Theranostic Reagents by Modularization: Preparation and Study of the Switching of Triplet
Excited State
a,
a
a
b
b,
By Jianzhang Zhao, * Ling Huang, Xiaoneng Cui, Shujing Li, and Huijian Wu *
The fluorescence imaging and the singlet oxygen production ability of the thiol-cleavable triplet
photosensitizers was maxized by disintergration the functionalities.
1