Rational design of the benzothiazole-based fluorescent scaffold for tunable emission

Article abstract of DOI:10.1016/j.tetlet.2019.03.029

The 2-(2-hydroxyphenyl)-benzothiazole (HBT) fluorophore has attracted considerable attention due to its excited-state intramolecular proton transfer (ESIPT) based emission and its large Stokes shift. However, this fluorophore possesses several disadvantag

Full text of DOI:10.1016/j.tetlet.2019.03.029

Tetrahedron Letters xxx (xxxx) xxx
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Rational design of the benzothiazole-based fluorescent scaffold
for tunable emission
⇑
Yong Ren, Dong Fan, Huazhou Ying, Xin Li
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The 2-(2-hydroxyphenyl)-benzothiazole (HBT) fluorophore has attracted considerable attention due to
its excited-state intramolecular proton transfer (ESIPT) based emission and its large Stokes shift.
However, this fluorophore possesses several disadvantages including low quantum yield and short emis-
sion in the blue range. In this study, by coupling HBT at the ortho-, meta-, and para-positions to the hydro-
xyl group with different heterocycles to extend the conjugation system, we have successfully obtained
new fluorophores with tunable emissions both in solution and in the solid-state (409–652 nm).
Notably, all of the derivatives demonstrated improved quantum yields compared with the parent HBT
structure. Moreover, selected compounds have been shown to shine brightly in live cells, indicating
promising potential for bioimaging.
Received 28 January 2019
Revised 8 March 2019
Accepted 11 March 2019
Available online xxxx
Keywords:
Benzothiazole
Fluorescence
Quantum yield
Ó 2019 Elsevier Ltd. All rights reserved.
Introduction
[35]. However, the introduction of polyenes usually increases the
lipophilicity of the fluorophore, decreasing their water solubility.
Fluorescent probes have been widely used in the fields of bio-
logical and environmental analysis due to their good photophysical
properties, high sensitivity, good selectivity [1–12]. For the design
of fluorescent probes, the choice of a proper fluorophore is of pri-
mary importance. Many fluorophores have been widely used for
probe construction, such as BODIPY [4,13,14], coumarin, xanthene
[15–17], cyanine [18–20], HBT [21–24]. Among them, HBT has
attracted considerable attention because of the easy manipulation
of its brightness due to its excited-state intramolecular proton
transfer (ESIPT)-based emission [25–27]. HBT has been used in flu-
orescent molecular probes [7,8,23,28], molecular logic gates [7],
fluorescent bioimaging [29–32], and for fundamental photophysi-
cal studies [33,34]. However, there are still some disadvantages
that limit its application, such as low quantum yield and short
emission wavelength. Generally, the emission wavelength of this
fluorophore is in the range of 400–550 nm, which overlaps with
the autofluorescence from biomolecules. To decrease the back-
ground signal from autofluorescence when bioimaging experi-
ments are performed, it is preferable for the probe to emit in the
green, red, or ideally, in the near infrared region.
In 2016, You and co-workers reported that the introduction of
five-membered heterocyclic rings may also red shift the emission
of fluorophores [36]. Inspired by this strategy, we have developed
a series of HBT derivatives by introducing various substituted hete-
rocycles to the HBT skeleton. The photophysical properties of the
compounds were measured and the structure-photophysical prop-
erty relationships are discussed. Notably, tunable emission was
observed and all compounds show improved quantum yields com-
pared with HBT. We have also confirmed the compatibility of the
compounds with live cells and their brightness for live cell
imaging.
Result and discussion
In solution HBT may emit via its phenolate form (blue emission)
or an ESIPT-based mechanism (green emission, Fig. 1a). To red shift
its emission, our strategy was to extend the conjugation system
with its structure. Various five-membered heterocycles such as
thiophene, furan and their derivatives were therefore introduced
to the HBT skeleton (Fig. 1b), and the effect of the substitution
position on the emission wavelength was determined.
The target fluorophores were synthesized as shown in Scheme 1.
First, the starting compound 1 was synthesized according to a
reported literature procedures [37]. Next, coupling 1 with bis(pina-
colato)diboron in the presence of PdCl2(dppf) and potassium acet-
ate in 1,4-dioxane afforded the key intermediate 2. Suzuki coupling
It is well known that extending the
p system of a fluorophore
will red shift its emission, as illustrated by the gradual increase
of the emission wavelength of the cyanine series of fluorophores
⇑
Corresponding author.
E-mail address: lixin81@zju.edu.cn (X. Li).
https://doi.org/10.1016/j.tetlet.2019.03.029
0040-4039/Ó 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Y. Ren, D. Fan, H. Ying et al., Rational design of the benzothiazole-based fluorescent scaffold for tunable emission, Tetrahedron
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Y. Ren et al. / Tetrahedron Letters xxx (xxxx) xxx
Fig. 1. ESIPT-based emission of HBT (a) and design rationale (b) of the HBT derivatives.
(360 nm, 4 W) (Figs. 2 and S1). The emission wavelength was
dependent on both the length of the conjugated system, and the
position of the derivatization. To make clear the structure-photo-
physical property relationships, all data are shown in Tables 1, 2
and S1.
As shown by the data in Tables 1 and 2, extending the conju-
gated system works well to red shift its emission. Comparing with
HBT, incorporation of either the furanyl (3) or thienyl (6) moiety
significantly red-shifts the emission wavelengths by approxi-
mately 40–140 nm. Further extension of the conjugated system
with a dicyanovinyl moiety (5, 8) causes a more dramatic red-shift-
ing effect with a further 50–100 nm shift (Tables 1, 2 and S1). Tak-
ing the data collected in acetonitrile for example, HBT exhibits an
emission maximum at 369 nm, while its furanyl counterparts (3-o)
show much longer emission maxima at 575 nm in acetonitrile.
Notably, emission in the near-infrared region was observed for
compound 5-p and 8-p (Fig. 3a). To further understand the rela-
tionship between the fluorescence properties and the chemical
structure of these compounds, density functional theory (DFT)
and time dependent density functional theory (TDDFT) calcula-
tions were carried out with the B3LYP/6-31+G(d) method basis
set using the Gaussian 09 C.01 program. The optimized geometry,
the highest occupied molecular orbital (HOMO) and the lowest
unoccupied molecular orbital (LUMO) of selected compounds are
presented in Figs. 4 and S2,S3. The HOMO–LUMO energy gap for
4-o was determined as 4.223 eV which is reduced in 5-o to
3.514 eV. The emission at a longer wavelength for 5-o is well cor-
related with the reduced HOMO–LUMO energy gap.
After exploring the relationship between the substituent posi-
tions and emission wavelength, it was found that the incorporation
of a furanyl/thienyl substituent in the ortho-position to the hydroxyl
group induced the most dramatic red-shifting effect, followed by the
para-position and then the meta-position (Fig. 3b). We hypothesize
that extending the conjugated system of HBT makes it a more ICT
(intramolecular charge transfer)-based fluorophore. The substitu-
tion of electron withdrawing furanyl/thienyl moiety in the ortho-
or para-position is more favorable than the meta-position for the
ICT effect, and therefore compounds with substituents in these
two positions demonstrated the most dramatic red-shift effect [38].
Scheme 1. Reagents and conditions: (a) bis(pinacolato)diboron (1 eq.), PdCl2(dppf)
(0.03 eq.), AcOK (3.5 eq.), 1,4-dioxane, reflux, overnight, 77%; (b) Pd(OAc)2
(0.05 eq.), PPh3 (0.2 eq.), K2CO3 (3.0 eq.), PhMe, reflux, overnight, 48–94%; (c)
pyridine (0.06 eq.), malononitrile (3.0 eq.), EtOH, reflux, overnight, 23–72%.
reaction of 2 with 2-bromofuran, 2-bromothiophene or their ana-
logues in the presence of Pd(OAc)2, PPh3 and potassium carbonate
in anhydrous toluene gave the target compounds 3, 4, 6 and 7. Fur-
ther derivatization of compounds 4 and 7 with malononitrile
employing the Knoevenagel condensation reaction gave com-
pounds 5 and 8, respectively. The structures of the final fluo-
rophores were characterized by 1H NMR, 13C NMR spectroscopy
and LCMS. Detailed synthetic data are given in the Supporting
information.
With the HBT derivatives in hand (Scheme 2), their photophys-
ical properties including absorption, emission, and quantum yields
were measured in various solutions. Interestingly, these com-
pounds demonstrate a wide range of emission (kem: 409–637 nm
in CH3CN; 491–612 nm in toluene) with large Stokes shifts (up to
230 nm in toluene; 232 nm in CH3CN). Blue to red color emission
was observed when the solutions were placed under a UV light
Please cite this article as: Y. Ren, D. Fan, H. Ying et al., Rational design of the benzothiazole-based fluorescent scaffold for tunable emission, Tetrahedron
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Scheme 2. Structures of the HBT derivatives.
Fig. 2. (a) Fluorescence of selected compounds in CH3CN (excited at 365 nm under a UV lamp). (b) Fluorescence of selected compounds in toluene (excited at 365 nm under a
UV lamp).
Table 1
Optical properties of compounds 3-p-HBT in CH3CN.
Comp.
kabs (nm)
kem (nm)
Stokes shift (nm)
e
(MÀ1 cmÀ1
)
UF
UF/UHBT
3-p
4-p
5-p
6-p
7-p
8-p
3-m
4-m
5-m
6-m
7-m
8-m
3-o
4-o
5-o
6-o
7-o
8-o
HBT
363
336
426
352
363
419
357
368
419
360
375
419
356
361
419
354
365
414
333
553
553
623
409
556
637
516
528
524
514
541
529
575
563
622
586
566
618
369
190
217
197
57
3910
3530
4620
4230
3900
2980
2540
2840
3500
3500
2680
3520
4040
3680
4130
1960
3450
3790
4220
0.0140
0.0103
0.0333
0.0088
0.0149
0.0305
0.0223
0.0780
0.0070
0.0267
0.0554
0.0068
0.0063
0.0735
0.1825
0.0119
0.1334
0.1471
0.0014
9.96842631
7.3191883
23.742704
6.31737005
10.6180883
21.7810802
15.9247416
55.6561272
4.97869776
19.0248591
39.5317037
4.84030999
4.52995555
52.4665411
130.301992
8.52436485
95.2471313
105.01302
1
193
218
159
160
105
154
166
110
219
202
203
232
201
204
36
Furthermore, these compounds were also observed to demon-
strate solvent-dependent emission properties. In general, the emis-
sion wavelength of the compounds tends to red shift as the polarity
of the solvent increases. For instance, 5-o shows an emission max-
ima at 603 nm (toluene)/605 nm (CH2Cl2)/622 nm (CH3-
CN)/631 nm (EtOH)/635 nm (PBS), respectively (Fig. 3c). Other
compounds showed similar patterns (Tables 1, 2 and S1). We
hypothesize that this phenomenon should be due to the polar nat-
ure of the excited state which prefers a polar environment for sta-
bility. Consequently, a more polar solvent would decrease the
energy gap between the HOMO and LUMO, and therefore red-shift
its emission.
Interestingly, although all of the compounds showed low quan-
tum yields in the region of 400–650 nm, their quantum yields were
improved compared with the parent HBT in different organic sol-
vents (Fig. 5). Interestingly, we found that the incorporation of
Please cite this article as: Y. Ren, D. Fan, H. Ying et al., Rational design of the benzothiazole-based fluorescent scaffold for tunable emission, Tetrahedron
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Table 2
Optical properties of compounds 3-p-HBT in toluene.
Comp.
kabs (nm)
kem (nm)
Stokes shift (nm)
e
(MÀ1 cmÀ1
)
UF
UF/UHBT
3-p
4-p
5-p
6-p
7-p
8-p
3-m
4-m
5-m
6-m
7-m
8-m
3-o
4-o
5-o
6-o
7-o
8-o
HBT
370
339
436
365
350
426
363
374
425
366
380
425
364
364
425
359
371
421
336
550
553
600
555
562
606
520
532
496
518
535
491
581
568
603
589
578
612
518
180
214
164
190
212
180
157
158
71
152
155
66
217
204
178
230
207
191
182
3750
3820
4230
4380
4270
4600
5020
3780
4250
3460
4180
4130
2960
3440
4080
3410
3730
3900
3470
0.0228
0.0803
0.0230
0.0151
0.0910
0.0152
0.0707
0.0778
0.0151
0.0849
0.0696
0.0148
0.0195
0.0874
0.1342
0.0210
0.1528
0.1565
0.0103
2.21730818
7.81927843
2.24172205
1.46738319
8.86414898
1.4841833
6.88843995
7.5825541
1.47461093
8.27016456
6.77689507
1.44611956
1.89976061
8.50962898
13.0738351
2.05012388
14.887644
15.2478176
1
Fig. 3. (a) Fluorescence spectra of selected compounds in CH3CN; (b) Fluorescence spectra of selected compounds in toluene; (c) Emission spectra of 5-o in different solvents.
Fig. 4. Energy minimization structures of selected compounds and their HOMO-LUMO distribution.
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Fig. 5. All compounds show improved quantum yields compared with HBT in organic solvents (Y-axis: UF/UHBT, where UF is the quantum yield of the newly synthesized
compounds and UHBT is that of HBT.
Fig. 6. Fluorescence microscopic image of HeLa cells dosed with compound 3-p, 4-p, 5-p, 6-p, 7-p, 8-p (10)
lM for 30 min 3-p and 6-p: Ex: 360 nm-400 nm, Em: 410 nm-
480 nm; 4-p and 7-p: Ex: 360 nm-400 nm, Em: 530 nm-590 nm; 5-p and 8-p: Ex: 410 nm-430 nm, Em: 580 nm-650 nm.
thiophene or furan at the ortho-position to the hydroxyl group usu-
ally resulted in higher quantum yields than those at the meta- or
para-position. This may result from the fact that the introduction
of thiophene or furan at the ortho-position restricts the twist of
the molecule, reducing energy consumption. For instance, when
furan was introduced, 5-o shows much higher quantum yield
(UF = 0.1342) than 5-m (UF = 0.0151) and 5-p (UF = 0.0230) in
toluene (Table 2).
were washed with PBS and then imaged under microscopy. As
shown in Fig. 6, all of the tested compounds demonstrate good cell
membrane permeability and shine brightly in live cells. Interest-
ingly, their emission color agrees well with that observed in solu-
tions and red-shifts with the conjugated system becoming longer.
Notably, for selected compounds, for example 3-p, 4-p and 5-p,
their emission color separate well with each other, suggesting their
potential for the multi-color imaging of live cells.
Finally, these compounds were observed to emit in the solid
state (Fig. S4). Furthermore, the emission color of these compounds
in the solid state is also significantly dependent on the length of
the conjugated system, with red emission being observed for com-
pounds bearing the longest conjugated system. Also, substitution
at the ortho-position to the hydroxyl group resulted in the bright-
est emission. All these observations agree well with those obtained
in solution. We argue that the solid-state fluorescence of the com-
pounds is desirable as it will not cause aggregation-induced
quenching.
Conclusion
By coupling HBT with furanyl/thienyl rings, a series of novel
compounds with tunable emission were obtained. All of the com-
pounds demonstrate red-shift emissions with their conjugated sys-
tem getting longer, and some compounds were observed to emit in
the near-infrared region. It was determined that the position of
derivatization significantly effects the photophysical properties of
the compounds, and the ortho-position to the hydroxyl group is
the ideal derivatization position for red-shifting emission. The fea-
sibility of the compounds for live cell imaging was also confirmed
and representative compounds were found to shine brightly in live
To test the applicability of the compounds for live cell imaging
experiments, representative compounds 3-p, 4-p, 5-p, 6-p, 7-p, 8-p
were fed to HeLa cells. After an incubation time of 30 min, the cells
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[15] M. Sibrian-Vazquez, J.O. Escobedo, M. Lowry, F.R. Fronczek, R.M. Strongin, J.
Am. Chem. Soc. 134 (2012) 10502.
[16] Y. Yang, M. Lowry, C.M. Schowalter, S.O. Fakayode, J.O. Escobedo, X. Xu, H.
Zhang, T.J. Jensen, F.R. Fronczek, I.M. Warner, R.M. Strongin, J Am. Chem. Soc.
128 (2006) 14081.
[17] T. Miura, Y. Urano, K. Tanaka, T. Nagano, K. Ohkubo, S. Fukuzumi, J. Am. Chem.
Soc. 125 (2003) 8666.
[18] W. Cao, E.M. Sletten, J. Am. Chem. Soc. 140 (2018) 2727.
[19] G.T. Dempsey, M. Bates, W.E. Kowtoniuk, D.R. Liu, R.Y. Tsien, X. Zhuang, J. Am.
Chem. Soc. 131 (2009) 18192.
[20] H.-J. Chen, C.Y. Chew, E.-H. Chang, Y.-W. Tu, L.-Y. Wei, B.-H. Wu, C.-H. Chen, Y.-
T. Yang, S.-C. Huang, J.-K. Chen, I.C. Chen, K.-T. Tan, J. Am. Chem. Soc. 140
(2018) 5224.
[21] M.A. Rios, M.C. Rios, J. Phys. Chem. 102 (1998) 1560.
[22] C. Rodriguez-Rodriguez, N.S. de Groot, A. Rimola, A. Alvarez-Larena, V.
Lloveras, J. Vidal-Gancedo, S. Ventura, J. Vendrell, M. Sodupe, P. Gonzalez-
Duarte, J. Am. Chem. Soc. 131 (2009) 1436.
[23] X.F. Yang, Q. Huang, Y.G. Zhong, Z. Li, H. Li, M. Lowry, J.O. Escobedo, R.M.
Strongin, Chem. Sci. 5 (2014) 2177.
[24] T.I. Kim, H.J. Kang, G. Han, S.J. Chung, Y. Kim, Chem. Commun. (2009) 5895.
[25] P. Yang, J. Zhao, W. Wu, X. Yu, Y.T. Liu, J. Org. Chem. 77 (2012) 6166.
[26] S. Goswami, S. Das, K. Aich, B. Pakhira, S. Panja, S.K. Mukherjee, S. Sarkar, Org.
Lett. 15 (2013) 5412.
[27] D. Maity, V. Kumar, T. Govindaraju, Org. Lett. 14 (2012) 6008.
[28] C.-C. Hsieh, C.-M. Jiang, P.-T. Chou, Acc. Chem. Res. 43 (2010) 1364.
[29] X. Li, R.-R. Tao, L.-J. Hong, J. Cheng, Q. Jiang, Y.-M. Lu, M.-H. Liao, W.-F. Ye, N.-N.
Lu, F. Han, Y.-Z. Hu, Y.-H. Hu, J. Am. Chem. Soc. 137 (2015) 12296.
[30] K.C. Song, J.S. Kim, S.M. Park, K.-C. Chung, S. Ahn, S.-K. Chang, Org. Lett. 8
(2006) 3413.
[31] S.K. Patra, S.K. Sheet, B. Sen, K. Aguan, D.R. Roy, S. Khatua, J. Org. Chem. 82
(2017) 10234.
[32] Y. Zhang, L. Guan, H. Yu, Y. Yan, L. Du, Y. Liu, M. Sun, D. Huang, S. Wang, Anal.
Chem. 88 (2016) 4426.
[33] G.-J. Zhao, K.-L. Han, Acc. Chem. Res. 45 (2012) 404.
[34] M.A. Ríos, M.C. Ríos, J. Phys. Chem. 102 (1998) 1560.
[35] W. Sun, S. Guo, C. Hu, J. Fan, X. Peng, Chem. Rev. 116 (2016) 7768.
[36] Y. Cheng, G. Li, Y. Liu, Y. Shi, G. Gao, D. Wu, J. Lan, J. You, J. Am. Chem. Soc. 138
(2016) 4730.
cells. Given the tunable emission of the compounds and their
improved quantum yields compared with the parent HBT struc-
ture, we believe that they may work as alternatives for future flu-
orescent probe development.
Acknowledgments
We appreciate Prof. Jianzhong Chen for the instruction on DFT
and TDDFT calculations, Dr Ying Dong for the generous help in cell
imaging experiments, and Dr Jianyang Pan for collecting the NMR
data. This work was supported by the National Natural Science
Foundation of China (21778048, 21642007), and the Natural
Science Foundation of Zhejiang province (LR18H300001).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2019.03.029.
References
[1] B.N.G. Giepmans, S.R. Adams, M.H. Ellisman, R.Y. Tsien, Science 312 (2006) 217.
[2] I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Nat. Mater. 4 (2005) 435.
[3] S. Weiss, Science 283 (1999) 1676.
[4] N. Boens, V. Leen, W. Dehaen, Chem. Soc. Rev. 41 (2012) 1130.
[5] H.N. Kim, W.X. Ren, J.S. Kim, J. Yoon, Chem. Soc. Rev. 41 (2012) 3210.
[6] Y. Zhou, J.F. Zhang, J. Yoon, Chem. Rev. 114 (2014) 5511.
[7] J. Wu, W. Liu, J. Ge, H. Zhang, P. Wang, Chem. Soc. Rev. 40 (2011) 3483.
[8] J.F. Zhang, Y. Zhou, J. Yoon, J.S. Kim, Chem. Soc. Rev. 40 (2011) 3416.
[9] S. Luo, E. Zhang, Y. Su, T. Cheng, C. Shi, Biomaterials 32 (2011) 7127.
[10] H.M. Kim, B.R. Cho, Acc. Chem. Res. 42 (2009) 863.
[11] M. Vendrell, D.T. Zhai, J.C. Er, Y.T. Chang, Chem. Rev. 112 (2012) 4391.
[12] M.H. Lee, J.S. Kim, J.L. Sessler, Chem. Soc. Rev. 44 (2015) 4185.
[13] A. Loudet, K. Burgess, Chem. Rev. 107 (2007) 4891.
[37] S. Samanta, S. Das, P. Biswas, J. Org. Chem. 78 (2013) 11184.
[38] Z.R. Grabowski, K. Rotkiewicz, W. Rettig, Chem. Rev. 103 (2003) 3899.
[14] G. Ulrich, R. Ziessel, A. Harriman, Angew. Chem. Int. Ed. 47 (2008) 1184.
Please cite this article as: Y. Ren, D. Fan, H. Ying et al., Rational design of the benzothiazole-based fluorescent scaffold for tunable emission, Tetrahedron
Letters, https://doi.org/10.1016/j.tetlet.2019.03.029