Bright near-infrared chemiluminescent dyes: Phthalhydrazides conjugated with fluorescent BODIPYs

Article abstract of DOI:10.1016/j.dyepig.2020.108339

A series of phthalhydrazides conjugated with NIR fluorescent BODIPY dyes were synthesized (three steps, overall yields 68–83%) and found to show strong NIR chemiluminescence (CL) with maxima in the range of 670–736 nm. One of these showed a CL quantum yield reaching 8.4 times higher than that of luminol. Tuning of the fluorescence band of the BODIPY unit directly leads to a red-shifted CL, developing a strategy for designing NIR chemiluminogens.

Full text of DOI:10.1016/j.dyepig.2020.108339

Journal Pre-proof
Bright near-infrared chemiluminescent dyes: Phthalhydrazides conjugated with
fluorescent BODIPYs
Guanglei Li, Takashi Hirano, Koji Yamada
PII:
S0143-7208(20)30319-3
DOI:
https://doi.org/10.1016/j.dyepig.2020.108339
DYPI 108339
Reference:
To appear in: Dyes and Pigments
Received Date: 5 February 2020
Revised Date: 5 March 2020
Accepted Date: 6 March 2020
Please cite this article as: Li G, Hirano T, Yamada K, Bright near-infrared chemiluminescent dyes:
Phthalhydrazides conjugated with fluorescent BODIPYs, Dyes and Pigments (2020), doi: https://
doi.org/10.1016/j.dyepig.2020.108339.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Guanglei Li: Methodology, Formal analysis, Investigation, Writing - Original Draft.
Takashi Hirano: Methodology, Formal analysis, Investigation, Resources, Writing - Review & Editing,
Funding acquisition
Koji Yamada: Conceptualization, Methodology, Resources, Funding acquisition

1
2
3
4
5
6
7
8
9
Bright Near-Infrared Chemiluminescent Dyes: Phthalhydrazides Conjugated with
Fluorescent BODIPYs
Guanglei Li† Takashi Hirano,‡ and Koji Yamada*†
† Division of Materials Science, Graduate School of Environmental Science, Hokkaido University,
Sapporo, Hokkaido 0860-0810, Japan
‡ Department of Engineering Science, Graduate School of Informatics and Engineering, The University
of Electro-Communications, Chofu, Tokyo 182-8585, Japan
* Corresponding author, E-mail: yamada@ees.hokudai.ac.jp
10
23 Keywords ： Near-infrared, Chemiluminescent
11 ABSTRACT: A series of phthalhydrazides24 dyes, Phthalhydrazide, BODIPY
12 conjugated with NIR fluorescent BODIPY dyes
13 were synthesized (three steps, overall yields 68–
25
26 INTRODUCTION
14 83%) and found to show strong NIR27 Chemiluminescence (CL) is a phenomenon
15 chemiluminescence (CL) with maxima in the28 whereby photons are generated through
16 range of 670–736 nm. One of these showed a CL29 chemical reaction of chemiluminogen.
a
a
17 quantum yield reaching 8.4 times higher than that30 Compared with fluorescence analysis using an
18 of luminol. Tuning of the fluorescence band of31 excitation light source, CL analysis provides data
19 the BODIPY unit directly leads to a red-shifted32 with no background noise arising from
20 CL, developing a strategy for designing NIR33 autofluorescence in a biological sample or from
21 chemiluminogens.
34 scattering of excitation light. Because of the high
35 sensitivity and high signal-to-noise ratio of CL
22
1

36 detection, CL has been widely utilized in various58 has recently been used in in vivo imaging with
37 chemical and biological analyses.1
38 Luminol
59 intermolecular energy transfer.4 In this system,
60 luminol functioned as an energy donor to
39 (5-amino-2,3-dihydrophthalazine-1,4-dione) is a61 stimulate
a
NIR fluorescent dye through
40 chemiluminescent compound whose anion62 sequential dual Förster resonance energy transfer
41 generated by deprotonation can be easily63 (FRET). Because an efficiency of an
42 oxygenated by an appropriate biological,64 intermolecular energy transfer is determined by
43 chemical, or electronic reaction leading to blue65 the factors for FRET including the distance
44 light emission with emission maximum around66 between an energy donor and an energy acceptor,
45 420–450 nm. Besides its readily obtained67 emission intensities of the intermolecular CL
46 luminescence,
47 advantages
luminol
possesses
with
many68 systems will be affected by the reaction
other69 conditions. To overcome the weaknesses of this
compared
48 bio/chemiluminogens, such as good stability, low70 intermolecular
energy
transfer
system,
49 cost, and safety. The CL of luminol is widely71 development of bio/chemiluminogens that
50 used in biological analysis and in vitro imaging as72 produce NIR light is urgently required. Although
51 a well-established sensitive technology.1b, 2 The73 efforts
have
been
made
to
prepare
52 blue CL of luminol, however, hinders its74 phthalhydrazide-based CL systems,5 to the best of
53 application to in vivo systems due to significant75 our knowledge, no NIR systems with
54 light attenuation by tissues.3 Hence, increasing76 phthalhydrazide have been reported. To prepare
55 the emission wavelength to the near-infrared77 NIR phthalhydrazide-based CL systems with high
56 (NIR) region would facilitate the in vivo78 CL quantum yields, it is important to select an
57 application of luminol. For this purpose, luminol79 appropriate NIR fluorophore unit to combine
2

80 with the phthalhydrazide unit. As regards the NIR82 choose. Recently, we developed NIR fluorescent
81 fluorophores, there are many candidates to83 BODIPYs 2a–c6 (Figure 1).
84
a: R =
b: R =
c: R =
Me
85
O
O
O
S
S
S
1'
8'
5'
8
1
7'
7
NH 2'
NH 3'
NH
NH
2
OMe
6
4
86
6'
N
N
N
N
3
5
O
O4'
B
B
NH2
R
R
R
R
OtBu
87
F
F
F
F
N
H
luminol
1
2
88
89
Figure 1. Structures of luminol, phthalhydrazide NIR BODIPY conjugates 1a–c, and NIR BODIPYs
2a–c.
90 The bulky 2,6-dimethylphenyl group at the C8100 by intramolecular energy transfer, and the
91 position in 2a–c reduced aggregation and101 methodology of the direct connection will expand
92 imparted good solubility and high fluorescence102 the derivatization range of the NIR CL compound.
93 quantum yields. Phthalhydrazide NIR BODIPY103 We report here the synthesis, spectroscopic
94 conjugates 1a–c were then designed by directly104 properties, and CL properties of 1a–c.
95 connecting the phthalhydrazide unit at the C6’
105 RESULTS AND DISSCUSSION
96 position with the NIR BODIPY units at the C2106 Phthalhydrazide
97 position. The direct connection of the107 synthesized from BODIPYs 2a–c and
98 phthalhydrazide and NIR BODIPY units i1s08 commercially available 5-bromophthalic
conjugates
1a–c
were
99 preferable to modulate the emission wavelength109 anhydride 7 as shown in Scheme 1.
110
111
112
113
3

114
115
116
117
118
119
Scheme 1. Synthesis scheme of compounds 1a–c
120 To synthesize the phthalhydrazide unit1,28 readily reacted with excess hydrazine to afford
121 -methylphthalimide 6 was prepared from 7 with129 1a–c. As we expected, 1a–c showed good
122 methylamine, and subsequent Miyaura–130 solubility in many organic solvents.
123 Ishiyama–Hartwig borylation of yielded131 The spectroscopic properties of 1a–c were
N
6
124 boronate 5. BODIPYs 2a–c were treated with one132 investigated in acetone and DMSO (Figure 2),
125 equivalent of NBS to afford monobromides 3a–c1,33 and the data are summarized in Table 1 together
126 which were coupled with
127 phthalhydrazide precursors 4a–c. Precursors 4a–c135 for comparison.
136
5
to give134 with those of 2a–c6 in dichloromethane (DCM)
4

1.0
0.5
0.0
8
6
4
2
0
500
500
500
600
600
600
700
700
700
800
800
800
900
1.0
0.5
0.0
6
4
2
0
900
1.0
0.5
0.0
6
4
2
0
900
137
138
Figure 2. UV/vis absorption (Abs: dotted line) and fluorescence (FL: solid line) spectra of 1a (A), 1b
(B), and 1c (C) in acetone (blue) and DMSO (red).
139
140
141
Table 1. Spectroscopic data for 1a–c in acetone and DMSO together with those for 2a–c in DCM.
a
c
d
dye solvent λabs
εb
λfl
Φfl
Stokes
shift/cm−1
1220
1250
520
/nm
acetone 617
DMSO 626
/M−1 cm−1 /nm
81,000
72,000
66,000
63,000
56,000
78,000
66,000
60,000
82,000
667
679
662
733
754
725
740
761
740
0.85
0.39
0.81
0.55
0.27
0.68
0.31
0.09
0.53
1a
2ae DCM
640
acetone 657
1580
1660
720
1b
DMSO 670
2be DCM
689
acetone 662
1590
1610
960
1c
DMSO 678
2ce DCM
691
a
b
c
d
142
143
144
Absorption maximum. Molar absorption coefficient. Fluorescence emission maximum. Absolute
fluorescence quantum yield. e From reference 6.
5

145 The absorption and emission bands of 1a–c167 quantum yields (Φfl) of 1a–c in DMSO were
146 spanned a wide range of over the red and NIR168 about 1/3–1/2 lower than those in acetone. The
147 regions. The absorption maxima (λabs) of the169 polarization properties of the S1 states of 1a–c are
148 lowest energy bands of 1a–c were observed in the170 known to influence the rate of non-radiative
149 range of 617–678 nm with large mola1r71 decay from the S1 state in a polar solvent,
150 absorptivities (56,000–81,000 M−1 cm−1)1,72 resulting in lower Φfl values.8 This result was
151 indicating that these absorption bands could be173 consistent with previous reports on BODIPY
152 attributed to
π–π
* transitions. Fluorescence174 systems.9 It is noteworthy that the λabs values of
153 emission maxima (λfl) were in the 667–761 nm175 1a–c were blue-shifted compared with those of
154 range with moderate Stokes shifts. As the solven1t76 2a–c, but the λfl values of 2a–c were similar to
N
155 polarity increased from acetone (ET 0.335) to177 those of 1a–c in acetone. The result indicates that
N
156 DMSO (ET 0.444),7 both the λabs and λfl value1s78 the phthalhydrazide units in 1a–c showed
157 were red-shifted by 9–16 nm and 12–21 nm1,79
π-conjugation with the BODIPY unit in the
158 respectively. The Stokes shifts of 1a–c in DMSO180 ground states, while the
π-conjugation of the
159 are slightly larger than those in acetone1,81 BODIPY and phthalhydrazide units was
160 indicating that the dipole moments of the excited182 attenuated in the S1 states of 1a–c. Thus, the
161 singlet (S1) states of 1a–c were a little larger than183 fluorescence emission properties of 1a–c are
162 those of the ground states. As the result, the S184 mainly determined by the BODIPY unit. To
1
163 states of 1a–c were more stabilized by the more185 rationalize the spectroscopic properties of 1a–c
164 polar solvent (DMSO).7 Therefore, the electronic186 and 2a–c, DFT and TD-DFT calculations were
165 excitations of 1a–c have weak intramolecula1r87 performed for them using the B3LYP/6-31G(d)
166 charge-transfer character. The fluorescence188 method (Figure 3, Table 2).9a, 10
6

189
190
191
Figure 3. Frontier orbitals and optimized molecular structures of dyes 1a–c and 2a–c. The energy levels
of the molecular orbital are denoted by blue and red bars. ∆EH-L denotes the energy gap between HOMO
and LUMO.
192
193
194
195
Table 2. Calculation data for 1 and 2 with DFT and TD-DFT using B3LYP/6-31G(d).
a
c
dye HOMO LUMO ∆EH−L
Transition b λtr
Configuration d,e
/eV
/eV
/eV
/nm (f)
1a
−5.20
−2.82
2.37
S0 → S1
S0 → S1
S0 → S1
S0 → S1
S0 → S1
546
(0.76)
626
(0.92)
633
(0.97)
541
(0.69)
616
(1.03)
H → L (99.5%)
H ← L (2.2%)
H → L (98.7%)
1b −4.95
−2.80
−2.80
−2.65
−2.64
2.15
2.14
2.33
2.12
1c
2a
−4.94
−4.98
H → L (98.5%)
H → L (101.3%)
H ← L (2.9%)
H → L (100.2%)
H ← L (2.1%)
2b −4.76
7

2c
−4.76
−2.64
2.12
S0 → S1
624
H → L (99.8%)
(1.10)
a
b
196
197
198
Energy difference between HOMO and LUMO. The π,π* transition to the excited singlet state with
c
d
the lowest excitation energy. Wavelengths estimated from transition energies. Configuration of
excitation. e H and L denote the HOMO and LUMO, respectively.
199
200 The optimized geometries of 1a–c had dihedra2l18 and 4-BOC-aminophenyl groups to the BODIPY
201 angles between the phthalhydrazide and BODIPY219 units increased the HOMO levels more
202 units of 45.2° for 1a, 46.3° for 1b, and 45.1° fo2r20 effectively than the LUMO levels, leading to
203 1c. The relatively non-planar molecular structure2s21 smaller HOMO–LUMO gaps (∆EH−L) for 1b and
204 of 1a–c may hinder aggregation of the dyes and222 1c compared with that of 1a. The slightly
205 also maintain communication between the223 blue-shifted λabs values of 1a–c compared with
206 phthalhydrazide units and BODIPY frameworks2.24 those of 2a–c were supported by the ∆EH−L values.
207 The HOMO and LUMO levels of 1a–c were225 The ∆EH−L values of 1a–c were larger than those
208 lower than those of the corresponding BODIPY226 of 2a–c, while the calculated wavelengths (λtr)
209 2a–c
compounds,
suggesting
that
the227 estimated from the transition energies showed the
a2s28 opposite trend. Configuration interactions in the
210 phthalhydrazide
units behaved
211 electron-withdrawing groups in 1a–c. The229 calculations would decrease the transition
212 HOMO levels of 1b and 1c were higher than tha2t30 energies of 1a–c. Interestingly, the LUMOs of
213 of 1a as with the series of 2, indicating that the231 1a–c had electron distributions only at the
214 4-methoxyphenyl
and
4-BOC-aminopheny2l32 BODIPY frameworks, while their HOMOs had
215 groups in the BODIPY units effectively233 small electron distributions at the phthalhydrazide
216 functioned as electron-donating π-conjugated234 units.
217 substituents. Introducing the 4-methoxypheny2l35 The CL properties of 1a–c and luminol were
8

236 evaluated with horseradish peroxidase (HRP) and238 4 shows normalized CL spectra of 1a–c together
237 H2O2 in MeOH/K2CO3 aqueous solutions. Figure239 with that of luminol.
240
241
242
Figure 4. Normalized CL spectra of 1a (a), 1b (b), 1c (c), and luminol (d).
243
244 Phthalhydrazide conjugates 1a–c showed single254 states, and the excited state energies were used to
245 sharp emission bands in the NIR region with255 generate the S1 states of the BODIPY units.
246 maxima (λCL) in the 670–738 nm range, and no256 The time courses of the CL reactions of 1a–c
247 CL derived from the isolated phthalhydrazide257 traced by monitoring emission intensities showed
248 units was observed. The λCL values of 1a–c were258 that the emission intensities maximized
249 similar to the λfl values of the corresponding259 immediately after mixing the reagents and
250 BODIPY 2a–c compounds, indicating that the260 gradually decreased (Figure S1). The half-lives of
251 BODIPY moieties in 1a–c functioned effectively261 the emission intensities (τ1/2) are summarized in
252 as fluorophore units. The CL reactions of 1a–c262 Table 3.
253 gave the phthalate products oxy-1a–c in the S1
263
264
Table 3. Chemiluminescent parameters obtained for dyes 1a–c and luminol.
c
dye
λCL/nma
τ1/2/sb
rel. ΦCL
9

670
735
736
451
145
42
22
6
8.4
0.8
0.4
1
1a
1b
1c
luminol
265
266
a CL emission maximum. b Half-life of light emission. c Relative CL quantum yield.
267 The τ1/2 values follow the order 1a > 1b > 1c2,75 that of luminol), while 1b and 1c showed lower
268 suggesting that the 4-methoxyphenyl and276 efficiencies (0.8-fold for 1b and 0.4-fold for 1c).
269 4-BOC-aminophenyl groups in the BODIPY277 In the CL reactions of 1a–c, the S1 states of the
270 units accelerated the CL reactions a2s78 phthalate units in oxy-1a–c (Figure 5) were
271 electron-donating substituents. The relative CL279 initially generated by the reactions of the anions
272 quantum yields (rel. ΦCL) of 1a–c were estimated280 of the phthalhydrazide units with superoxide
273 using luminol as a standard.11 It is noteworthy281 anions.12
274 that 1a showed higher CL efficiency (8.4-fold
282
283
284
Figure 5. Phthalate products oxy-1a–c of CL reactions of 1a–c.
285
286
287
288
289
The phthalate and BODIPY units in oxy-1a–c are connected by the single bond, leading to make a
twisted but partially conjugated energy donor and acceptor system. Because the two units in oxy-1a–c
are close to each other, the energy transfer process between the phthalate and BODIPY units will not be
hindered by the need of spectral overlap for FRET as in the case of the through-bond energy transfer
10

290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
cassettes.13 Because of the partially π-electronic conjugation between the phthalate and BODIPY units, it
may be better to understand that the CL reactions of 1a–c directly generate the S1 states of the whole
molecules of oxy-1a–c. Characterization of the energy transfer processes in conjugated CL systems
remained challenging.5c
CONCLUSIONS
In summary, we have developed the phthalhydrazide derivatives 1a–c, which show NIR CL with
HRP-H2O2. The NIR chemiluminogens were obtained by directly introducing NIR fluorescent BODIPYs
into the phthalhydrazide skeleton at the C2 position. Their CL properties indicated that the direct
conjugation strategy was suitable for providing a superior CL performance. Particularly, the observed CL
maximum data indicated that various NIR chemiluminescent dyes could be developed by simply
exchanging the BODIPY unit for one with a desired emission wavelength. Unfortunately, however, 1a–c
are insoluble in a 100% aqueous solution. Thus, it is necessary for in vivo applications to give further
modification of the dye structure such as introducing a hydrophilic group. We believe that our present
study provides a strategy for designing and preparing BODIPY-based NIR CL dyes, which can
potentially be utilized for chemical and biological applications.
EXPERIMENTAL SECTION
General experimental. All commercially available solvents and reagents were used as received unless
otherwise noted. Reactions were monitored with a silica gel TLC. Purification was performed on a silica
gel chromatography. Melting points were determined with a Yamato MP-21 apparatus. 1H and 13C NMR
spectra were recorded on a JEOL 400 spectrometer (400 MHz for 1H NMR, 100 MHz for 13C NMR) at
11

312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
room temperature. Chemical shifts were expressed in parts per million (ppm) relative to TMS (0 ppm).
Coupling constants (J) were expressed in Hz. HRMS were obtained on a Thermo Scientific Exactive
under ESI or ACPI conditions. FT-IR spectra were recorded on a Thermo Nicolet iS10 spectrometer.
UV/vis absorption spectra were measured with an Agilent Technologies Cary 60 spectrophotometer.
Fluorescence spectra and fluorescence quantum yields were measured with Hamamatsu Photonics
Quantaurus-QY Absolute PL quantum yield spectrometer C11347. CL spectra were recorded using an
ATTO AB-1850 spectrophotometer or a PMA-12 Photonic multichannel analyzer C10027-02. CL
kinetics were measured with an ATTO Luminescencer-Octa AB-2270. DFT calculations using the
B3LYP functional with the 6-31G(d) basis set were performed on Gaussian 09 program, and the results
were visualized in GaussView 5.
Chemiluminescence measurements. The relative CL quantum yields of the 1a–1c were relatively
determined by using that of luminol. Emission spectra of the 1a–1c and luminol were measured with an
AB-1850 spectrophotometer for detecting light intensity from the reaction mixture until the intensity of
light decreased to the background level. For the measurements, a small portion of 500 µL stock solution
of 1a–1c (80 µM) or luminol (7 µM) in MeOH was added in a quartz cuvette placed in the
spectrophotometer and was mixed with H2O2 (0.12 M in H2O, 17 µL). The CL reaction was initiated by
injecting HRP solution (10 µM in 0.1 M K2CO3, 500 µL) into the reaction mixture. All the measurements
were conducted at room temperature (22 to 25 °C). Areas of the CL spectra obtained by integration per
number of molecules of the 1a–1c were calculated, and the ratios of the area values for 1a–1c to that for
luminol gave the relative CL quantum yields. As the CL bands of 1a–1c exceeded the limitation of the
AB-1850 spectrophotometer (400–800 nm), in order to show the whole spectra of 1a–1c, the CL spectra
were recorded with a PMA-12 photonic multichannel analyzer following the similar procedure.
12

334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
Synthesis
of
2-bromo-8-(2,6-dimethylphenyl)-3,5-bis(5-methylthien-2-yl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-ind
acene (3a). To the solution of the starting 2a (118 mg, 0.24 mmol) in DCM (5 mL) in an ice bath was
added NBS (43 mg, 0.24 mmol) slowly. The reaction mixture was stirred at room temperature for 3 h.
The reaction was quenched by adding an ice-cold saturated NaHCO3 aqueous solution, and the products
were extracted with DCM. The organic layers were combined, washed with H2O and brine successively,
and dried over Na2SO4. The solvent was evaporated under reduced pressure. The crude sample was
purified by silica gel column chromatography using eluent gradients with the eluent pair hexane/ ethyl
1
acetate (EA), to give 3a as a dark red solid (133 mg, 97%). mp 189−191 °C. H NMR (400 MHz,
CDCl3): δ (ppm) = 8.03 (d, J = 3.9 Hz, 1H), 7.65 (d, J = 4.0 Hz, 1H), 7.30−7.24 (m, 1H), 7.14 (d, J = 7.6
Hz, 2H), 6.87−6.84 (m, 2H), 6.74 (d, J = 4.3 Hz, 1H), 6.60 (d, J = 4.6 Hz, 1H), 6.52 (S, 1H), 2.58 (s, 3H),
2.53 (s, 3H), 2.19 (s, 6H). 13C NMR (100 MHz, CDCl3) δ: 167.74, 153.4, 147.1, 145.3, 144.3, 139.1,
137.5, 137.0, 133.6 (t, JC-F = 7.4 Hz), 132.7, 132.6 (t, JC-F = 4.8 Hz), 131.2, 130.7, 129.0, 128.4, 128.3,
127.4, 127.3, 125.8, 122.1, 20.2, 15.7, 15.5. FT-IR (ATR) ν/cm-1: 2915, 2847, 1558, 1538, 1486, 1462,
1438, 1403, 1375. HRMS (ESI, Orbitrap) m/z: [M+Na]+ Calcd for C27H22N210BBrF2NaS2 588.0401,
Found 588.0402.
Synthesis
2-bromo-8-(2,6-dimethylphenyl)-3,5-bis[5-(4-methoxyphenyl)thien-2-yl]-4,4'-difluoro-4-bora-3a,4a
-diaza-s-indacene (3b) A preparation according to the procedure similar to 3a by using 2b (150 mg,
of
.
0.22 mmol), NBS (40 mg, 0.22 mmol) and DCM (4 mL) afforded 3bas a black solid (157 mg, 95%). mp
230−231 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) = 8.24 (d, J = 4.1 Hz, 1H), 7.88 (d, J = 4.3 Hz, 1H),
7.64 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.33−7.27 (m, 3H), 7.15 (d, J = 7.6 Hz, 2H), 6.95-
13

356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
6.91 (m, 4H), 6.84 (d, J = 4.4 Hz, 1H), 6.62 (d, J = 4.5 Hz, 1H), 6.56 (s, 1H), 3.85 (s, 3H), 3.83 (s, 3H),
2.22 (s, 6H). 13C NMR (100 MHz, CDCl3) δ: 160.2, 159.6, 152.8, 150.2, 148.2, 145.0, 138.6, 137.8,
137.1, 134.4 (t, JC-F = 8.2 Hz), 134.0, 133.6 (t, JC-F = 5.6 Hz), 132.7, 131.6, 130.6, 129.2, 129.0, 127.5,
127.46, 127.42, 126.2, 124.8, 122.5, 122.3, 114.5, 114.3, 107.9, 55.41, 55.36, 20.2. FT-IR (ATR) ν/cm-1:
2988, 2901, 1603, 1559, 1536, 1510, 1479, 1449, 1430. HRMS (ESI, Orbitrap) m/z: [M+Na]+ Calcd for
C39H30O2N210BBrF2NaS2 772.0922, Found 772.0931.
Synthesis
of
8-(2,6-dimethylphenyl)-2-(2-methyl-1,3-dioxoisoindolin-5-yl)-3,5-bis(5-methylthien-2-yl)-4,4'-diflu
oro-4-bora-3a,4a-diaza-s-indacene (4a). Compounds 3a (96 mg, 0.17 mmol), 5 (70 mg, 0.26 mmol),
tris(dibenzylideneacetone)dipalladium (0) (Pd2dba3) (16 mg, 0.017 mmol), tri-tert-butylphosphonium
tetrafluoroborate ([(t-Bu)3PH]BF4) (10 mg, 0.034 mmol) and Cs2CO3 (222 mg, 0.68 mmol) were placed
in a two-necked round bottom flask. Prior to the addition of THF/H2O (3 mL/20 µL), the flask was
purged with N2 three times. The reaction mixture was stirred at 25 °C for 24 h, then the product was
extracted with DCM. The organic layers were combined, washed with H2O and brine successively, and
dried over Na2SO4. The solvent was evaporated under reduced pressure. The crude sample was purified
by silica gel column chromatography using eluent gradients with the eluent pair hexane/DCM, to give 4a
as a dark red solid (96 mg, 88%). mp 288−289 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) = 8.06 (d, J =
3.8 Hz, 1H), 7.65−7.63 (m, 2H), 7.39 (dd, J = 7.6 Hz, J = 1.6 Hz, 1H), 7.33−7.29 (m, 2H), 7.17 (d, J =
7.8 Hz, 2H), 6.87 (d, J = 3.9 Hz, 1H), 6.79 (d, J = 4.1 Hz, 2H), 6.65 (d, J = 4.6 Hz, 1H), 6.57 (s, 1H),
3.14 (s, 3H), 2.55 (s, 3H), 2.50 (s, 3H), 2.25 (s, 6H). 13C NMR (100 MHz, CDCl3): 168.4, 168.3, 153.6,
147.2, 144.9, 144.1, 141.1, 139.9, 137.9, 137.0, 134.1, 133.8 (t, JC-F = 7.4 Hz), 133.7, 132.9, 132.4,
131.9 (t, JC-F = 3.2 Hz), 131.6, 131.1, 130.8, 129.8, 129.2, 129.0, 128.3, 127.5, 126.0, 124.5, 123.0,
14

378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
122.9, 122.3, 23.9, 20.2, 15.7, 15.5. FT-IR (ATR) ν/cm-1: 2913, 1768, 1708, 1558, 1537, 1481, 1463,
1422, 1374. HRMS (ESI, Orbitrap) m/z: [M+Na]+ Calcd for C36H28O2N310BF2NaS2 669.1622, Found
669.1622.
Synthesis
8-(2,6-dimethylphenyl)-2-(2-methyl-1,3-dioxoisoindolin-5-yl)-3,5-bis[5-(4-methoxyphenyl)thien-2-y
l]-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (4b) A preparation according to the procedure similar
of
.
to 2a by using 3b (120 mg, 0.16 mmol), 5 (65 mg, 0.24 mmol), Pd2dba3 (15 mg, 0.016 mmol),
[(t-Bu)3PH]BF4 (9 mg, 0.03 mmol), Cs2CO3 (209 mg, 0.64 mmol) and THF/H2O (3 mL/20 µL) afforded
1
4b as a dark green solid (115 mg, 87%). mp 155−157 °C. H NMR (400 MHz, CDCl3): δ (ppm) = 8.26
(d, J = 4.0 Hz, 1H), 7.70 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.56-7.54 (m, 3H), 7.50 (d, J = 8.8 Hz, 2H),
7.45 (d, J = 8.6 Hz, 1H), 7.32−7.27 (m, 2H), 7.25−7.24 (m, 1H), 7.17 (d, J = 7.6 Hz, 2H), 6.90−6.86 (m,
5H), 6.67 (d, J = 4.6 Hz, 1H), 6.61 (s, 1H), 3.79 (s, 6H), 3.12 (s, 3H), 2.26 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ: 168.3, 168.2, 160.1, 159.5, 153.1, 150.4, 148,0, 144.4, 140.9, 139.5, 138.2, 137.0, 134.6 (t,
JC-F = 7.8 Hz), 134.4, 133.8, 132.9 (t, JC-F = 3.5 Hz), 132.8, 132.4, 131.9, 131.4, 130.8, 130.0, 129.8,
129.0, 127.5, 127.4, 127.2, 126.6, 126.0, 124.8, 124.7, 123.0, 122.9, 122.6, 122.5, 114.40, 114.2, 55.3,
23.9, 20.2. FT-IR (ATR) ν/cm-1: 2988, 2901, 1770, 1711, 1605, 1536, 1510, 1476, 1488, 1431. HRMS
(ESI, Orbitrap) m/z: [M+Na]+ Calcd for C48H36O4N310BF2NaS2 853.2137, Found 853.2155.
Synthesis
of
8-(2,6-dimethylphenyl)-2-(2-methyl-1,3-dioxoisoindolin-5-yl)-3,5-bis{5-[4-((N-tert-butoxycarbonyl)
amino)phenyl]thien-2-yl}-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (4c). The starting compound
3c was prepared according to the procedure similar to 3a by using 2c (60 mg, 0.07 mmol), NBS (13 mg,
0.07 mmol) and DCM (2 mL). The crude compound 3c was used for the next reaction without
15

400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
purification. A preparation according to the procedure similar to 2a by using the crude 3c, 5 (29 mg, 0.1
mmol), Pd2dba3 (6 mg, 0.007 mmol), [(t-Bu)3PH]BF4 (4 mg, 0.014 mmol), Cs2CO3 (91 mg, 0.28 mmol)
and THF/H2O (1.5 mL/10 µL) afforded 4c as a dark red solid (51 mg, two step overall yield 72%). mp
185−187 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) = 8.25 (d, J = 4.2 Hz, 1H), 7.70 (s, 1H), 7.65 (d, J =
7.8 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.53−7.49 (m, 3H), 7.45 (d, J = 7.8 Hz, 1H), 7.41 (d, J = 8.6 Hz,
2H), 7.37−7.30 (m, 4H), 7.28−7.26 (m, 1H), 7.19 (d, J = 7.7 Hz, 2H), 6.88 (d, J = 4.6 Hz, 1H), 6.69 (d, J
= 4.6 Hz, 1H), 6.60 (s, 1H), 6.59 (bs, 1H), 6.54 (bs, 1H), 3.14 (s, 3H), 2.27 (s, 6H), 1.53 (s, 9H) , 1.52 (s,
9H). 13C NMR (100 MHz, CDCl3) δ: 168.4, 168.3, 153.1, 152.5, 152.4, 150.2, 147.9, 144.5, 141.0, 139.7,
139.0, 138.3, 137.1, 134.6 (t, JC-F = 7.6 Hz), 134.5, 133.9, 133.0 (t, JC-F = 3.7 Hz), 132.8, 132.5, 131.8,
130.9, 130.2, 130.1, 129.1, 128.6, 128.1, 127.6, 126.9, 126.6, 125.1, 124.8, 123.1, 123.0, 122.6, 118.65,
118.59, 80.9, 80.7, 28.3, 24.0, 20.3. FT-IR (ATR) ν/cm-1: 2973, 2901, 1771, 1711, 1607, 1538, 1478,
1435, 1409, 1378. HRMS (ESI, Orbitrap) m/z: [M+Na]+ Calcd for C56H50O6N510BF2NaS2 1023.3192,
Found 1023.3212.
Synthesis
of
8-(2,6-dimethylphenyl)-2-(1,4-dioxo-1,2,3,4-tetrahydrophthalazin-6-yl)-3,5-bis(5-methylthien-2-yl)-
4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (1a). A solution of 4a (65 mg, 0.10 mmol) and hydrazine
monohydrate (NH2NH2·H2O, 50 µL, 1 mmol) in ethanol/DCM (3 mL/6 mL) was refluxed under N2 for
24 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue
was purified by silica gel column chromatography using eluent gradients with the eluent pair
1
hexane/acetone to give 1a as a dark purple solid (64 mg, 98%). mp 240−242 °C. H NMR (400 MHz,
CDCl3): δ (ppm) = 13.55 (bs, 1H), 12.34 (bs, 1H), 8.14 (s, 1H), 8.07−8.03 (m, 2H), 7.53 (d, J = 8.2 Hz,
1H), 7.33−7.30 (m, 2H), 7.19 (d, J = 7.6 Hz, 2H), 6.87 (d, J = 3.4 Hz, 1H), 6.79−6.78 (m, 2H), 6.69 (s,
16

422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
1H), 6.66 (d, J = 4.5Hz, 1H), 2.55 (s, 3H), 2.51 (s, 3H), 2.27 (s, 6H). 13C NMR (100 MHz, CDCl3) δ:
158.2, 157.5, 153.5, 147.1, 145.3, 144.2, 140.3, 140.1, 137.9, 137.0, 134.3, 133.74, 133.69 (t, JC-F = 7.8
Hz), 132.9, 132.1 (t, JC-F = 3.7 Hz), 131.8, 131.2, 130.8, 129.3, 129.0, 128.3, 127.6, 127.2, 126.1, 125.4,
125.1, 124.9, 122.2, 20.3, 15.7, 15.5. FT-IR (ATR) ν/cm-1: 2988, 2901, 1661, 1559, 1543, 1489, 1462,
1408. HRMS (ESI, Orbitrap) m/z: [M+Na]+ Calcd for C35H27O2N410BF2NaS2 670.1565, Found
670.1574.
Synthesis
of
8-(2,6-dimethylphenyl)-2-(1,4-dioxo-1,2,3,4-tetrahydrophthalazin-6-yl)-3,5-bis[5-(4-methoxyphenyl
)thien-2-yl]-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (1b). A preparation according to the
procedure similar to 1a by using 4b (42 mg, 0.05 mmol), NH2NH2·H2O (28 µL, 0.75 mmol), and
ethanol/DCM (3 mL/3 mL) afforded 1b as a dark purple solid (38 mg, 90%). mp 202−204 °C. 1H NMR
(400 MHz, CDCl3): δ (ppm) = 13.60 (bs, 1H), 12.27 (bs, 1H), 8.28 (d, J = 4.2 Hz, 1H), 8.20 (s, 1H), 8.06
(d, J = 8.3 Hz, 1H), 7.62−7.59 (m, 3H), 7.54−7.51 (m, 3H), 7.33−7.31 (m, 2H), 7.26−7.25 (m, 1H), 7.20
(d, J = 7.7 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H), 6.90−6.87 (m, 3H), 6.71−7.69 (m, 2H), 3.85 (s, 3H), 3.81 (s,
3H), 2.29 (s, 6H). 13C NMR (100 MHz, CDCl3) δ: 160.2, 159.5, 158.3, 157.4, 152.9, 150.2, 148.2, 144.8,
140.2, 139.8, 138.2, 137.1, 134.6, 134.5 (t, JC-F = 7.4 Hz), 133.9, 133.1 (t, JC-F = 4.5 Hz), 132.9, 132.1,
131.6, 131.0, 130.8, 130.0, 129.1, 127.6, 127.5, 127.3, 126.7, 126.2, 125.4, 125.3, 125.0, 124.9, 122.7,
122.4, 114.5, 114.3, 55.4, 55.3, 20.3. FT-IR (ATR) ν/cm-1: 2989, 2901, 1720, 1657, 1605, 1538, 1475,
1450, 1435, 1407. HRMS (ESI, Orbitrap) m/z: [M+Na]+ Calcd for C47H35O4N410BF2NaS2 854.2089,
Found 854.2105.
Synthesis
of
8-(2,6-dimethylphenyl)-2-(1,4-dioxo-1,2,3,4-tetrahydrophthalazin-6-yl)-3,5-bis{5-[4-((N-tert-butoxy
17

444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
carbonyl)amino)phenyl]thien-2-yl}-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (1c). A preparation
according to the procedure similar to 1a by using 4c (30 mg, 0.015 mmol), NH2NH2·H2O (45 µL, 0.45
mmol), and ethanol/DCM (3 mL/3 mL) afforded 1c as a dark purple solid (29 mg, 95%). mp > 300 °C.
1H NMR (400 MHz, CDCl3): δ (ppm) = 13.59 (bs, 1H), 12.28 (bs, 1H), 8.27 (d, J = 3.7 Hz, 1H), 8.19 (s,
1H), 8.06 (d, J = 8.2 Hz, 1H), 7.61−7.58 (m, 3H), 7.54 (d, J = 3.4 Hz, 1H), 7.50 (d, J = 7.9 Hz, 2H), 7.41
(d, J = 8.0 Hz, 2H), 7.35−7.32 (m, 4H), 7.28−7.26 (m,1H), 7.20 (d, J = 7.6 Hz, 2H), 6.88 (d, J = 4.1 Hz,
1H), 6.70−6.69 (m, 2H), 6.60 (s, 2H), 2.29 (s, 6H), 1.53 (s, 9H), 1.51 (s, 9H). 13C NMR (100 MHz,
CDCl3) δ: 158.2, 157.4, 152.9, 152.6, 152.5, 149.9, 147.9, 144.9, 140.1, 139.9, 139.0, 138.3, 137.0,
134.7, 134.4, 133.9, 133.1, 132.8, 132.2, 131.9, 131.0, 130.8, 130.2, 129.1, 128.6, 128.1, 127.6, 126.8,
126.7, 125.5, 125.4, 125.1, 123.0, 122.4, 118.7, 118.6, 80.9, 80.6, 28.3, 20.3. FT-IR (ATR) ν/cm-1: 2973,
2917, 1705, 1652, 1538, 1475, 1447, 1409, 1364. HRMS (ESI, Orbitrap) m/z: [M+Na]+ Calcd for
C55H49O6N610BF2NaS2 1024.3145, Found 1024.3160.
Synthesis of 5-bromo-2-methylisoindoline-1,3-dione (6). 5-Bromophthalic anhydride 7 (1.14 g, 5.0
mmol), methylamine (0.65 mL of 40 wt% in H2O, 7.5 mmol) and toluene (25 mL) were placed in a
round bottom flask and purged with N2 three times. The flask fitted with a Dean-Stark trap, and the
mixture was refluxed for 12 h. After cooling to room temperature, the solvent was evaporated under
reduced pressure. The residue was purified by silica gel column chromatography (EA: hexane 1: 9) to
yield 6 as white solid (1.52 g, 90%). mp 145−146 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.97 (d, J
=1.0 Hz, 1H), 7.84 (dd, J =7.8 Hz, J =1.5 Hz, 1H), 7.70 (d, J = 7.9 Hz, 1H), 3.17 (s, 3H). 13C NMR (100
MHz, CDCl3) δ: 167.6, 167.0, 136.9, 133.8, 130.7, 128.8, 126.6, 124.6, 24.1. FT-IR (ATR) ν/cm-1: 1762,
1707, 1601, 1432, 1415, 1380. HRMS (ESI, Orbitrap) m/z: [M+H]+ Calcd for C9H7O2NBr 239.9655,
Found 239.9660.
18

466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
Synthesis of 5-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-2-methylisoindole-1,3-dione (5). Compound 6
(1.51 g, 6.29 mmol), bis(neopentyl glycolato)diboron (1.42 g, 6.29 mmol), potassium acetate (KOAc)
(1.85 g, 18.9 mmol) and PdCl2(dppf)·CH2Cl2 (154 mg, 0.19 mmol, 3 mol%) were placed in a
two-necked round bottom flask. The flask was then purged with N2 three times before addition of 16 mL
of DMSO. The mixture was stirred at 80 °C for 22 h. After the reaction was complete, the mixture was
extracted with EA, washed with H2O and brine successively, and the organic layer was collected, dried
over Na2SO4 and evaporated to dryness under reduced pressure. The residue was purified by silica gel
column chromatography (EA: hexane 1: 9 to 1: 4) to yield 5 as white solid (1.04 g, 61%). mp
189−190 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) = 8.27 (s, 1H), 8.13 (d, J = 0.7 Hz, 1H), 7.80 (d, J =
0.8 Hz, 1H), 3.80 (s, 4H), 3.18 (s, 3H), 1.04 (s, 6H). 13C NMR (100 MHz, CDCl3) δ: 168.8, 168.7, 139.5,
133.9, 131.2, 128.4, 122.1, 72.5, 31.9, 23.9, 21.8. FT-IR (ATR) ν/cm-1: 2959, 2930, 1770, 1709, 1618,
1480, 1593, 1432, 1414, 1375. HRMS (ACPI, Orbitrap) m/z: [M+H]+ Calcd for C14H17O4N10B 273.1282,
Found 273.1282.
Supplementary information
The supplement information is available.
1
CL kinetic profiles of 1a–1c and luminol. H and 13C NMR spectra. DFT and TD-DFT calculation
results for 1a-1c, 2a-2c.
ACKNOWLEDGEMENT
This work was partially supported by JSPS KAKENHI Grants (JP17H06371 and JP18K05075 for TH).
We acknowledge Mr. Yasuhide Fukuyo for his help on synthesis experiments. We thank the Information
Technology Center of UEC for technical assistance in computing DFT calculations. We thank Ms. Miho
Yamada, the Instrument Analysis Division, Hokkaido University for measuring the mass spectra.
19

488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
References
1. (a) Roda, A.; Guardigli, M., Analytical chemiluminescence and bioluminescence: latest achievements
and new horizons. Anal. Bioanal. Chem. 2012, 402, 69-76; (b) Roda, A.; Pasini, P.; Guardigli, M.; Baraldini,
M.; Musiani, M.; Mirasoli, M., Bio-and chemiluminescence in bioanalysis. Fresenius J. Anal. Chem. 2000,
366, 752-759.
2. Alshetaiwi, H. S.; Balivada, S.; Shrestha, T. B.; Pyle, M.; Basel, M. T.; Bossmann, S. H.; Troyer, D. L.,
Luminol-based bioluminescence imaging of mouse mammary tumors. J. Photochem. Photobiol. B 2013, 127,
223-228.
3. (a) Weissleder, R., A clearer vision for in vivo imaging. Nat. Biotechnol. 2001, 19, 316-317; (b) Lee, E.
S.; Deepagan, V.; You, D. G.; Jeon, J.; Yi, G.-R.; Lee, J. Y.; Lee, D. S.; Suh, Y. D.; Park, J. H., Nanoparticles
based on quantum dots and a luminol derivative: implications for in vivo imaging of hydrogen peroxide by
chemiluminescence resonance energy transfer. Chem. Commun. 2016, 52, 4132-4135; (c) Tseng, J.-C.; Kung,
A. L., In vivo imaging of inflammatory phagocytes. Chem. Biol. 2012, 19, 1199-1209; (d) Gross, S.; Gammon,
S. T.; Moss, B. L.; Rauch, D.; Harding, J.; Heinecke, J. W.; Ratner, L.; Piwnica-Worms, D., Bioluminescence
imaging of myeloperoxidase activity in vivo. Nat. Med. 2009, 15, 455.
4. Liu, R.; Tang, J.; Xu, Y.; Dai, Z., Bioluminescence Imaging of Inflammation in Vivo Based on
Bioluminescence and Fluorescence Resonance Energy Transfer Using Nanobubble Ultrasound Contrast Agent.
ACS nano 2019, 13, 5124-5132.
5. (a) Degirmenci, A.; Algi, F., Synthesis, chemiluminescence and energy transfer efficiency of 2,
3-dihydrophthalazine-1, 4-dione and BODIPY dyad. Dyes Pigm. 2017, 140, 92-99; (b) Bag, S.; Tseng, J.-C.;
Rochford, J., A BODIPY-luminol chemiluminescent resonance energy-transfer (CRET) cassette for imaging
20

510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
of cellular superoxide. Org. Biomol. Chem. 2015, 13, 1763-1767; (c) Han, J.; Jose, J.; Mei, E.; Burgess, K.,
Chemiluminescent Energy‐Transfer Cassettes Based on Fluorescein and Nile Red. Angew. Chem. Int. Ed.
2007, 46, 1684-1687.
6. Li, G.; Otsuka, Y.; Matsumiya, T.; Suzuki, T.; Li, J.; Takahashi, M.; Yamada, K., A Straightforward
Substitution Strategy to Tune BODIPY Dyes Spanning the Near-Infrared Region via Suzuki–Miyaura
Cross-Coupling. Materials 2018, 11, 1297.
7. Reichardt, C., Solvatochromic dyes as solvent polarity indicators. Chem. Rev. 1994, 94, 2319-2358.
8. Umezawa, K.; Matsui, A.; Nakamura, Y.; Citterio, D.; Suzuki, K., Bright, color-tunable fluorescent dyes
in the Vis/NIR region: establishment of new "tailor-made" multicolor fluorophores based on
borondipyrromethene. Chem. Eur. J. 2009, 15, 1096-1106.
9. (a) Xuan, S.; Zhao, N.; Ke, X.; Zhou, Z.; Fronczek, F. R.; Kadish, K. M.; Smith, K. M.; Vicente, M. G.
H., Synthesis and spectroscopic investigation of a series of push–pull boron dipyrromethenes (BODIPYs). J.
Org. Chem. 2017, 82, 2545-2557; (b) Shi, W.-J.; Lo, P.-C.; Singh, A.; Ledoux-Rak, I.; Ng, D. K., Synthesis
and second-order nonlinear optical properties of push-pull BODIPY derivatives. Tetrahedron 2012, 68,
8712-8718; (c) Qin, W.; Baruah, M.; Van der Auweraer, M.; De Schryver, F. C.; Boens, N., Photophysical
properties of borondipyrromethene analogues in solution. J. Phys. Chem. A 2005, 109, 7371-7384.
10. (a) Nano, A.; Ziessel, R.; Stachelek, P.; Harriman, A., Charge‐Recombination Fluorescence from Push–
Pull Electronic Systems Constructed around Amino‐Substituted Styryl–BODIPY Dyes. Chem. Eur. J. 2013,
19, 13528-13537; (b) Jiao, C.; Huang, K.-W.; Wu, J., Perylene-fused BODIPY dye with near-IR
absorption/emission and high photostability. Org. Lett. 2011, 13, 632-635.
11. Ando, Y.; Niwa, K.; Yamada, N.; Irie, T.; Enomoto, T.; Kubota, H.; Ohmiya, Y.; Akiyama, H.,
Development of a Quantitative Bio/Chemiluminescence Spectrometer Determining Quantum Yields: Re‐
21

532
533
534
535
536
537
538
539
examination of the Aqueous Luminol Chemiluminescence Standard. Photochem. Photobiol. 2007, 83,
1205-1210.
12. Merényi, G.; Lind, J.; Eriksen, T. E., Luminol chemiluminescence: chemistry, excitation, emitter. J.
Biolumin. Chemilumin. 1990, 5, 53-56.
13. (a) Wan, C. W.; Burghart, A.; Chen, J.; Bergström, F.; Johansson, L. B. Å.; Wolford, M. F.; Kim, T. G.;
Topp, M. R.; Hochstrasser, R. M.; Burgess, K., Anthracene–BODIPY cassettes: syntheses and energy transfer.
Chem. Eur. J. 2003, 9, 4430-4441; (b) Fan, J.; Hu, M.; Zhan, P.; Peng, X., Energy transfer cassettes based on
organic fluorophores: construction and applications in ratiometric sensing. Chem. Soc. Rev. 2013, 42, 29-43.
22

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: