Red-emitting salicylaldehyde Schiff base with AIE behaviour and large Stokes shift

Article abstract of DOI:10.1016/j.cclet.2018.05.043

Three salicylaldehyde Schiff base (SSB), iso-PBP, PBP and EPB, were facilely synthesized and exhibited aggregation-induced emission. The introduction of C = N-N = C moiety in these SSB dyes largely extend the conjugation system and push their emission to yellow to red spectral region. These SSB dyes were negligibly fluorescent in dilute THF solution. In THF/water mixtures with high water fractions, they displayed strong yellow to red fluorescence (up to 617 nm) and large Stokes shifts (up to 152 nm). Single crystal analysis on EBP showed the longer emission of in aggregated state was attributed to the molecular packing effect as compared with that in dilute solution. The bio-imaging application indicated EBP could specifically accumulate in lipid droplets in living cells.

Full text of DOI:10.1016/j.cclet.2018.05.043

Accepted Manuscript
Title: Red-emitting salicylaldehyde Schiff base with AIE
behaviour and large Stokes shift
Authors: Junyi Gong, Peifa Wei, Yuanan Su, Ying Li, Xing
Feng, Jacky W.Y. Lam, Dongzhou Zhang, Xiangzhi Song, Ben
Zhong Tang
PII:
DOI:
Reference:
S1001-8417(18)30239-0
https://doi.org/10.1016/j.cclet.2018.05.043
CCLET 4575
To appear in:
Chinese Chemical Letters
Please cite this article as: Gong J, Wei P, Su Y, Li Y, Feng X, Lam JWY, Zhang D, Song X,
Tang BZ, Red-emitting salicylaldehyde Schiff base with AIE behaviour and large Stokes
shift, Chinese Chemical Letters (2018), https://doi.org/10.1016/j.cclet.2018.05.043
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Communication
Red-emitting salicylaldehyde Schiff base with AIE behaviour and large Stokes shift
Junyi Gonga, Peifa Weib, Yuanan Sua, Ying Lib, Xing Fengb, Jacky W. Y. Lamb, Dongzhou Zhangc, Xiangzhi Songa,*, Ben Zhong
Tangb,*
a College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China
b
Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Division of Life
Science and Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Hong Kong, China
c Partnership for Extreme Crystallography, Hawaii Institute of Geophysiscs and Planetology, University of Hawaii at Manoa, Honolulu HI 96822, United States
 Corresponding authors.
E-mail addresses: xzsong@csu.edu.cn (X. Song), tangbenz@ust.hk (B.Z. Tang).
ARTICLE INFO
Article history:
Received
Received in revised form
Accepted
Available online
Graphical abstract
Three salicylaldehyde Schiff base (SSB) with AIE behaviour were designed and facilely synthesized through a condensation reaction. In solid and aggregation
states, these SSB dyes exhibited yellow to red emission with large Stokes shift. One of SSB dyes could specifically stain lipid droplets in living cells.
ABSTRACT
Three salicylaldehyde Schiff base (SSB), iso-PBP, PBP and EPB, were facilely synthesized and exhibited aggregation-induced emission. The
introduction of C=N-N=C moiety in these SSB dyes largely extend the conjugation system and push their emission to yellow to red spectral region.
These SSB dyes were negligibly fluorescent in dilute THF solution. In THF/water mixtures with high water fractions, they displayed strong yellow to
red fluorescence (up to 617 nm) and large Stokes shifts (up to 152 nm). Single crystal analysis on EBP showed the longer emission of in aggregated state
was attributed to the molecular packing effect as compared with that in dilute solution. The bio-imaging application indicated EBP could specifically
accumulate in lipid droplets in living cells.
Keywords:
Aggregation-induced emission
Salicylaldehyde Schiff base
Red-emitting
ESIPT
Aldehyde-amine condensation
Red-emitting fluorophores such as rhodamines, BODIPY derivatives and some coumarins have been widely applied as bio-imaging
agents, photo-sensitizers and optical materials [1-5]. Usually, these dyes are strongly fluorescent in dilute solutions, however, are weakly
fluorescent in high concentration and/or in solid state due to the aggregation-caused quenching (ACQ) effect [6-8]. In 2001,
multiphenylsiloles had been discovered to display strong emission in high aggregation and solid state and are weakly fluorescent in dilute
solutions, which is called aggregation-induced emission (AIE) [9,10]. Later on, more materials have been found to exhibit AIE properties
with different features and functions [11-19]. The typical prototypical AIE luminogens (AIEgens) such as 1-methyl-1,2,3,4,5-
pentaphenylsilole and tetraphenylethylene (TPE) emit in a short-wavelength spectral region [20,21]. To date, not many AIEgens with
simple structures have exhibited emissions in red/NIR spectral region. It is well known that fluorophores with long-wavelength emissions
are more favorable because long-wavelength photos have deeper tissue penetration, less photodamage to the biological tissue, and less
interference from auto fluorescence in organism. Therefore, it is very meaningful to develop new red-emitting AIEgens.
Most of the reported red-emitting AIEgens are subjected to complicated preparation [22]. Salicylaldehyde Schiff base (SSB) dyes
could be easily obtained and were reported as AIEgens by Tong’s group in 2009, which displayed a large Stokes shift due to the excited

state intramolecular proton transfer (ESIPT) process [23]. Later on, more SSB dyes have been investigated [24-32]. However, so far SSB
dyes emitting in red/NIR spectral region (above 600 nm) are still rare.
SSB dyes belong to donor-acceptor dyes and undergo an intramolecular charge transfer (ICT) process upon excited. It is known that
long-wavelength emission can be obtained by enhancing ICT effect in SSB dyes through two approaches: 1) using strong electron donors
such as dialkylamino groups and/or strong electron acceptors such as nitro group [33-36]; 2) extending conjugation system to significantly
narrow the energy gap between ground state and first singlet excited-state [37].
Herein, we reported the design and facile synthesis of three new SSB dyes, iso-PBP, PBP and EPB, in which C=N-N=C segments
were readily introduced through the condensation reaction between hydrazine and aldehydes to extend their conjugation system and
thereby to shift their emissions to red spectral region (Scheme 1). It is noteworthy that the ESIPT process in these SSB dyes would
provide a large Stokes shift which is beneficial for practical applications.
The synthetic routes of iso-PBP, PBP and EBP were shown in Scheme 1. First, salicylaldehyde reacted with hydrazine in methanol at
room temperature to afford salicylaldehyde hydrazone with a 75 % yield. Next, the hydrazone reacted with dialdehydes: glyoxal,
terephthalaldehyde and isophthalaldehyde to yield iso-PBP, PBP and EBP, respectively, with 88 %, 77 % and 90 % yields. Pleasantly,
all the starting materials are commercially available and low-cost; the reaction conditions are mild; especially the purification of these
products can be realized by simple recrystallization. All the new compounds were fully characterized by NMR and high-resolution mass
spectral analysis.
Scheme 1. Synthetic route and chemical structures of iso-PBP, PBP and EBP.
We further investigated their optical behaviour in solid form. iso-PBP, PBP and EBP were light yellow powder and were strongly
fluorescent in solid-state. The photoluminescence (PL) spectra of these SSB dyes in solid state were shown in Fig. 1. iso-PBP, PBP and
EPB emitted yellow (maxEm = 551 nm), orange (maxEm = 584 nm) and red (maxEm = 617 nm) fluorescence, respectively. All the SSB dyes
exhibited large Stokes shifts (up to 152 nm for EBP) due to the ESIPT process. The absolute fluorescent quantum yields for iso-PBP,
PBP and EBP in solid-state (ss) are 7.21%, 4.60% and 2.00%, respectively.
EM spectra of EBP
EM spectra of PBP
1.0
EM spectra of iso-PBP
EX spectra of EBP
0.8
EX spectra of PBP
EX spectra of iso-PBP
0.6
0.4
0.2
0.0
400 500 600 700 800
Wavelength (nm)
Fig. 1. Normalized emission (solid) and excitation (dash) spectra of iso-PBP, PBP and EBP in solid state.
iso-PBP, PBP and EBP were soluble in common organic solvents such as hexane, toluene, dichloromethane, THF, ethyl acetate and
DMF. As expected, iso-PBP, PBP and EBP were weakly fluorescent in these organic solvents due to the rotation of N-N single bonds,
as shown in Fig. S1 (Supporting information). Their quantum yields of iso-PBP, PBP and EBP in dilute THF solutions were 0.31 %,
0.11 % and 0.03 %, respectively.
Next, their AIE properties were investigated by measuring their PL spectra in THF-water mixtures with different water fractions (fw)
(Fig. 2). For EPB, the fluorescence started to occur when the water fraction was increased to 60% and the fluorescence enhancement was
up to 70-fold when the fw reached 90%. The absolute fluorescent quantum yields (aggr) of iso-PBP, PBP and EBP in the mixture of THF-
water with fw = 90% were 21.00%, 13.12 % and 5.10 %, following the same order in solid state. Dynamic light scattering (DLS) analysis
was performed to determine the particle size of EBP, PBP and iso-PBP in the mixture of THF-water with fw = 90%, which were 171.59
nm, 393.81 nm and 300.35 nm, respectively (Fig. S2 in Supporting information). These results indicated that the fluorescence
enhancement of these SSB dyes was ascribed to the restriction of intramolecular rotation in aggregation state (Scheme S1 in Supporting
information). The AIE activity of them is 170.0, 119.3 and 67.7 (as shown in Table S2 in Supporting information). In the mixture of

THF/water with small fw, the dyes did not aggregate and the ESIPT process was inhibited to some extent due to the formation of
intermolecular hydrogen bonds between dye molecules and the solvent. In contrast, dyes aggregated in the mixture of THF/water with
high fw, which prevented the formation of intermolecular hydrogen bonds between dye molecules and recovered intramolecular hydrogen
bonds between dye molecules to favor the ESIPT process [13]. It was noted that a blue-shift in the absorption spectra was observed for
PBP when the solvent changed from pure THF to the mixture of THF/water with fw = 90%. In contrast, EBP displayed a red-shift in its
absorption spectra when making the same solvent change (Fig. S3 in Supporting information).
To provide further mechanistic insight, we performed density function theoretical (DFT) calculations on these SSB dyes (Fig. S4 in
Supporting information). It was found that they all had a highly co-planar geometry. The energy gaps between HOMO and LUMO of
iso-PBP, PBP and EPB were 3.54 eV, 3.20 eV and 3.21 eV, which well agreed with their absorption spectra in THF.
In order to gain profound information about the AIE behavior of these SSB dyes, we managed to obtain the single crystal structure of
EBP, as shown in Fig. S5 (Supporting information). The distance of O−H···N was 1.84 Å, indicating the formation of strong
intramolecular hydrogen bonding which facilitated the ESIPT process in EPB. EBP had a completely co-planar conformation and the
intermolecular distance C(1)···N(2) and C(2)···N(1) was 3.43 Å, which implied the strong intermolecular interaction. The strong dipole
coupling between two neighbouring molecules of EPB enhanced the ICT process in EPB to induce the bathochromic shift, which gave
a good explanation of red-shift in the emission of EPB in aggregated state as compared with that in THF solution [13]. Since the single
crystal of PBP was not obtained, we did DFT calculation on PBP and EPB to obtain their electrostatic potential surfaces. As shown in
Fig. S6 (Supporting information), the negative charge on nitrogen atoms in PBP were localized by the middle benzene ring, which result
in a loose packing in aggregation form or solid state and weak intermolecular dipole interactions between neighbouring molecules. As a
result, the less conjugated EBP showed a longer emission compared to PBP.
Moreover, since C=N bond is usually unstable in strong acidic condition, the stability of EBP and PBP should be concerned. It was
investigated at different pH values by monitoring their fluorescence behavior (Fig. S7 in Supporting information). In acidic media, the
emission spectra of EBP and PBP hardly changed. However, in basic media a large blue-shift in their absorption spectra and a serious
fluorescence quench was observed for EPB and PBP. This result was ascribed to the prohibition of ESIPT process and was consisted
1
with the previous report on SSB dye [30]. H NMR spectral analysis showed that the signal of the active proton of hydroxyl group in
PBP disappeared when NaOH was added (Fig. S8 in Supporting information). The optical spectral change of EPB in acidic and basic
media was reversible for many cycles, indicating the good stability of these SSB dyes (Fig. S9 in Supporting information).
fw
fw
5x103
4x103
3x103
2x103
1x103
0
A 800
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
fw
B
C
40%
30%
20%
10%
0%
90%
80%
70%
60%
50%
8.0x106
6.0x106
4.0x106
2.0x106
0.0
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
600
400
200
0
500
550
600
650
700
500
550
600
650
700
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
F
D
E
fw
fw
fw
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Water fraction (%)
Water fraction (%)
Water fraction (%)
Fig. 2. Fluorescence spectra of iso-PBP (A), PBP (B) and EBP (C) in THF/water mixtures. Fluorescence intensity of iso-PBP (D), PBP (E) and EBP (F) in
THF/water mixtures as a function of the water fraction. Inset: fluorescence images of iso-PBP, PBP and EBP in THF/water mixture under a 365 nm light irradiation.

Fig. 3. Fluorescence (A, D), bright-field (B, E) and merged (C, F) images of HeLa cells. Top row (A, B, C): cells without the treatment of EPB; bottom row (D,
E, F): cells incubated with EBP (20 μmol/L) at 37 oC for 1 h. Scale bar: 10 μm.
Finally, we used EPB to perform bio-imaging experiments in living HeLa cells (Fig. 3). When cells were incubated with 0.02 mmol/L
o
EBP at 37 C for 1 h, a strong red fluorescence was observed. It is noted that EBP selectively accumulated in lipid droplets (LDs) of
cells. LDs, as important subcellular organelles, are intimately involved in many diseases and are indicators for cell’s living status [38,39].
However, there were few red-emitting AIEgens that could specifically accumulate in LDs [30,40,41]. Thus, EBP was a promising AIEgen
for specific imaging LDs in living cells.
In conclusion, we developed a new type of SSB dyes exhibiting AIE behavior with long-wavelength emissions, large Stokes shifts
and LDs-targeting ability in living cells. Particularly, the facile preparation of these SSB dyes and the low cost of starting materials made
them have a good potential for the future application.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. U1608222), the State Key Laboratory of Fine
Chemicals (No. KF1606), the State Key Laboratory of Chemo/Biosensing and Chemometrics (No. 2016005), the Research Grants
Council of Hong Kong (Nos. 16308116 and 16305015) and the Innovation and Technology Commission (No. ITC-CNERC14SC01).

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