Synthesis of 13-substituted derivatives of berberine: Aggregation-induced emission enhancement and pH sensitive property

Article abstract of DOI:10.1016/j.jphotochem.2017.01.017

Five kinds of berberine derivatives with hydroxy, methyl, ethyl, benzyl and (4-methyl)benzyl group at 13-position have been synthesized and characterized. 13-hydroxyberberine chloride exhibits aggregation-induced emission enhancement (AIEE) property because of hydroxy group, which is beneficial to increase the rigidity of molecule by enlarging conjugated system. Optical properties in pure solution, CH₃OH/H₂O mixed solution, amorphous and crystalline state were comparatively investigated. Polymeric morphology and particle size of 13-hydroxyberberine chloride with different water fractions (0–90 vol%) were obtained by scanning electron microscope (SEM) and dynamic light scattering (DLS) method respectively, which provided reasonable explanation that the formation of small globular nanoparticles in mixed solution is conducive to the fluorescence emission. The single crystal structure of 13-hydroxyberberine chloride was determined by single-crystal X-ray diffraction. Crystallographic data indicated that the main mechanism of the AIEE phenomenon is the existences of J-aggregation (head to tail dipole stacking) combined with molecular planarization. The calculation done by DFT showed that the HOMO–LUMO bandgap is in accordance with experimental data. To further explore the biomedical application of 13-hydroxyberberine, its cell viability and cell imaging performance were examined, which demonstrate that 13-hydroxyberberine shows definite fluorescent intensity. In all, 13-hydroxyberberine should be a promising candidate for different biomedical application such as pH fluorescence probe because of its response to the stimuli of pH value.

Full text of DOI:10.1016/j.jphotochem.2017.01.017

Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Invited feature article
Synthesis of 13-substituted derivatives of berberine:
Aggregation-induced emission enhancement and pH sensitive
property
a,
Xuemei Tanga, Jianhui Zhangb, Liyan Liua, Depo Yanga, Huaqiao Wangc, , Feng He
*
*
a
School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou 510006, China
School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
b
c
Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong 510080, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Five kinds of berberine derivatives with hydroxy, methyl, ethyl, benzyl and (4-methyl)benzyl group at 13-
position have been synthesized and characterized. 13-hydroxyberberine chloride exhibits aggregation-
induced emission enhancement (AIEE) property because of hydroxy group, which is beneficial to increase
the rigidity of molecule by enlarging conjugated system. Optical properties in pure solution, CH3OH/H2O
mixed solution, amorphous and crystalline state were comparatively investigated. Polymeric
morphology and particle size of 13-hydroxyberberine chloride with different water fractions (0–
90 vol%) were obtained by scanning electron microscope (SEM) and dynamic light scattering (DLS)
method respectively, which provided reasonable explanation that the formation of small globular
nanoparticles in mixed solution is conducive to the fluorescence emission. The single crystal structure of
13-hydroxyberberine chloride was determined by single-crystal X-ray diffraction. Crystallographic data
indicated that the main mechanism of the AIEE phenomenon is the existences of J-aggregation (head to
tail dipole stacking) combined with molecular planarization. The calculation done by DFT showed that
the HOMO–LUMO bandgap is in accordance with experimental data. To further explore the biomedical
application of 13-hydroxyberberine, its cell viability and cell imaging performance were examined,
which demonstrate that 13-hydroxyberberine shows definite fluorescent intensity. In all, 13-
hydroxyberberine should be a promising candidate for different biomedical application such as pH
fluorescence probe because of its response to the stimuli of pH value.
Received 1 November 2016
Received in revised form 11 January 2017
Accepted 13 January 2017
Available online 19 January 2017
Keywords:
Berberine derivatives
Aggregation-induced emission
enhancement
Crystal structure
Cell viability
pH sensitivity
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
small organic molecules attract tremendous attention due to their
strong emission in the poor solvents [2–4]. For example, siloles
As we all know, an increasing number of scientists pay more
attention to the development of nano- and microsized fluorescent
materials [1]. Fluorescent materials possess highly fluorescence
efficiency in the state of nanoaggregation, solid and crystalline,
namely aggregation-induced emission enhancement (AIEE) prop-
erty, which is opposite to the “aggregation-caused quenching”
(ACQ). Nevertheless, a majority of fluorescent materials are
subjected to aggregation-caused quenching (ACQ) effect because
compound is considered as the most representative of an
aggregation-induced emission (AIE) compound in earlier study
of Tang’s group [5]. Following closely behind are tetraphenyl-
ethylene (TPE) compound [6], intramolecular charge transfer (ICT)
compound [7], and hydrogen bonding compound [8], which are
also exploited as the new AIE molecules. Tang’s group has also
reported the luminescence mechanisms of AIE(E) which involve
inter- and intramolecular phenomena, such as molecular confor-
of intensive
p
-
p
stacking and dipole–dipole interactions, which
mation distortion, J-aggregation, CꢀꢀHꢁ ꢁ ꢁ
p interaction and so forth
bring about low fluorescence emission, limiting the application of
fluorescent materials. As a kind of AIE(E) fluorescent material,
[9–12].
Recently, this kind of organic molecule is considered to have
great potential application in the field of photocatalysts [13],
organic light-emitting diodes (OLEDs) [14,15] and optical devices
[16], especially as chemical or biological probe [17]. Many
important physiological processes of the body are related to pH
* Corresponding authors.
E-mail addresses: wanghq@mail.sysu.edu.cn (H. Wang),
hefeng@mail.sysu.edu.cn (F. He).
http://dx.doi.org/10.1016/j.jphotochem.2017.01.017
1010-6030/© 2017 Elsevier B.V. All rights reserved.

72
X. Tang et al. / Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
value, and the fluorescence property of some compounds can
indicate the change of acid-base property of medium. Therefore,
the study of fabricating novel chemical or biological fluorescence
probe with AIE(E) molecules is valuable. In comparison to
traditional pH fluorescence probe with the problem of aggrega-
tion-caused quenching (ACQ), new pH fluorescence probe has
higher sensitivity and faster response speed [18,19].
Based on our previous researches [20–22], our group synthe-
sized a series of 13-substituted derivatives of berberine (Scheme 1)
and their spectroscopic properties in solution, nanosuspensions,
amorphous and crystalline were investigated [23,24]. With further
exploration of 13-hydroxyberberine chloride assembly feature, we
researched its polymeric morphology and particle size by scanning
electron microscope (SEM) and dynamic light scattering (DLS)
method respectively. Crystal structure elucidates the mechanism
of enhanced emission in the aggregation state. The geometrical
and electronic structures were obtained to evidence the AIEE study
of compound 1. Moreover, the relationships between photo-
luminescence (PL) intensity and pH values were explored [25]. To
further examine the biological potential of compound 1 as pH
fluorescence probe, the cell uptake behavior as well as cell viability
of it was further evaluated.
Germany) emission scanning electron microscope. Particle size
was got by dynamic light scattering (DLS) method on a Brookhaven
BI-90 plus particle size analyzer. Single crystal X-ray diffraction
(XRD) patterns were taken on a Xcalibur Nova (Agilent Technolo-
gies (China) Co., Ltd) diffractometer. The geometrical and
electronic structures were obtained at the B3LYP/6-31G** level.
Cell viability was analyzed with a Flex Station 3 microplate reader
(Moleculardevices, America). Cell imaging performance was
examined on a laser scanning confocal microscopy 710 (Zeiss,
Germany).
2.2. Synthetic procedures
The molecular structures and synthesis routes of 13-substituted
berberine are exhibited in Scheme 1. All the derivatives had a high-
quality synthesis and marked as compounds 1-5 (Table 1).
Additionally, an oxidation reaction using N-bromosuccinimide
(NBS) gave the reaction products 2-5 in good yield [26,27]. 13-
hydroxyberberine chloride was synthesized by a sequence of
nucleophilic substitution reactions with acetone and KMnO4.
2.2.1. Compound 1
Berberine chloride (3.71 g, 10 mmol) was dissolved in deionized
water (4 mL) and acetone (4 mL). 50 wt% NaOH (5.6 mL) was added
in the mixture with vigorous stirring at the room temperature for
30 min. The mixture was filtered and the residue was washed with
deionized water (15 mL) and methanol (15 mL) to obtain brown
intermediate 6a (3.85 g, yield: 53%).
2. Experimental
2.1. Materials and instruments
All the reagents were obtained commercially, analytical grade
and used without further purification. Berberine chloride, Sodium
borohydride (NaBH4), formaldehyde solution, benzyl bromide and
acetaldehyde solution were obtained from Sigma-Aldrich, and 4-
Methylbenzyl bromide was got from Macklin. NMR spectra were
record on a Bruker Avance III 400 MHz spectrometer in DMSO-d6
as the internal standard. Mass spectra were measured on Shimazu
LCMS-IT-TOF mass spectrometers. Ultraviolet (UV) absorption was
recorded on a Shimadzu UV-2600 spectrometer. Photolumines-
cence (PL) spectra were taken using an (Edinburgh Instruments)
FLS920 spectrophotometer. Absolute PL quantum yields and
fluorescence lifetime were collected on an (Edinburge Instru-
ments, England) FLS 980 spectrometer. Scanning electron micro-
scope (SEM) images were obtained with a Zeiss Merlin (Zeiss Co.,
The suspension of compound 6a (2.32 g, 5.9 mmol) was
dissolved in acetone (100 mL) and cooled to minus ten degrees
Table 1
Structures of compounds 1-5.
Compound
R1
X
1
2
3
4
5
OH
CH3
C2H5
CH2Ph
CH2PhCH3
Cl
Cl
Cl
Br
Br
Scheme 1. Synthetic routes of 13-substituted berberine derivatives. (a) acetone, 50 wt% NaOH, room temperature; (b) KMnO4(aq), acetone, ꢀ10 ꢂC; (c) HCl (concd), methanol,
reflux; (d) NaBH4, K2CO3, methanol, room temperature; (e) RCHO, HOAc, 80% ethanol, 85–95 ꢂC; (f) RPhCH2Br, NaI, CH3CN, room temperature, reflux, NBS.

X. Tang et al. / Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
73
in the ice-salt bath. KMnO4 (1.1 g, 7 mmol) was added in deionized
water (100 mL). KMnO4 solution was dropped dropwise into the
reaction system, stirring and maintaining the temperature at
minus ten degrees for 2 h. Next, the mixture was filtered and the
filter liquor was rotary evaporated to dry. The solid was redissolved
in methanol (40 mL), adding concentrated hydrochloric acid
(2.5 mL) afterwards. The suspension was heated to reflux for
1.5 h. The solvent was removed by evaporation and recrystallized
with ethanol, affording compound 1 (1.2 g, yield: 52%). Yellow
powders. 1H and 13C NMR spectra and HRMS-EI analysis are
supplied in Fig. S1 for compound 1. 1H NMR (400 MHz, CH3OD):
J = 9.6 Hz,1H), 7.79 (d, J = 9.6 Hz,1H), 7.27–7.39 (m, 3H), 7.17–7.18 (m,
3H), 6.98 (s,1H), 6.08 (s, 2H), 4.89 (t, J = 4.8 Hz, 2H), 4.76 (s, 2H), 4.13
(s, 3H), 4.03 (s, 3H), 3.16 (t, J = 5.2 Hz, 2H).13C NMR(100 MHz, DMSO-
d6):d= 150.7, 149.7, 146.9, 145.9, 144.8, 139.6, 137.7, 134.5, 133.3,
130.5,129.5,128.5,127.2,126.7,122.1,121.7,120.5,108.9,108.6,102.5,
62.6, 57.5, 36.0, 27.8. HMRS (EI-MS); m/z calcd for C27H24NO4 [M-
Cl]+: 426.1705; found: 426.1679.
2.2.5. Compound 5
Compound 5 was obtained from intermediate 6b using the
similar procedure as compound 4. Yield: 69%. Yellow powders. 1H
and 13C NMR spectra and HRMS-EI analysis are supplied in Fig. S5
d
= 9.42 (s, 1H), 8.23 (d, J = 9.3 Hz, 1H), 8.07 (d, J = 9.4 Hz, 1H), 7.99 (s,
1H), 6.95 (s,1H), 6.07 (s, 2H), 4.84 (t, J = 8.0 Hz, 2H), 4.18 (s, 3H), 4.11
(s, 3H), 3.17 (t, J = 8.0 Hz, 2H). 13C NMR (100 MHz, CH3OD):
= 152.4,
for compound 5.1H NMR(400 MHz, DMSO-d6):
d=10.04 (s,1H), 8.09
d
(d, J = 9.6 Hz, 1H), 7.76 (d, J = 9.2 Hz, 1H), 7.05–7.18 (m, 6H), 6.99 (s,
1H), 6.08 (s, 2H), 4.89 (t, J = 4.8 Hz, 2H), 4.69 (s, 2H), 4.13 (s, 3H), 4.03
(s, 3H), 3.16 (t, J = 5.6 Hz, 2H), 2.29 (s, 3H). 13C NMR (100 MHz,
150.6, 150.6, 148.4, 145.8, 138.2, 132.7, 127.6, 127.3, 125.8, 123.7,
120.8, 119.4, 110.4, 109.0, 103.3, 62.5, 58.6, 57.5, 29.0. HMRS (EI–
MS); m/z calcd for C20H18NO5 [M-Cl]+: 352.1185; found: 352.1166.
DMSO-d6):
d= 150.7, 149.7, 146.9, 145.9, 144.8, 137.6, 136.5, 136.4,
134.5, 133.3, 130.7, 130.1, 128.4, 126.7, 122.1, 121.7, 120.5, 108.9,
2.2.2. Compound 2
108.6, 102.5, 102.5, 62.6, 35.7, 27.76, 2. HMRS (EI-MS); m/z calcd for
Berberine chloride (5.57 g,15 mmol) and K2CO3 (5.4 g, 45 mmol)
were mixed with methanol (188 mL), then 5 wt% NaOH (7.5 mL)
solution containing NaBH4 (0.45 g, 11.3 mmol) was added in small
portions and stirred at room temperature. After 1.5 h, precipitation
was filtered, washed and recrystallized with ethanol to obtain a
dark green intermediate 6b (3.75 g, yield: 68%).
C
28H26NO4 [M-Cl]+: 440.1862; found: 440.1841.
2.3. Nanoaggregates preparation for UV–vis spectra, PL spectra, SEM
and DLS measurements
To obtain the nanosuspensions, a series of stock methanol
solutions of these derivatives were prepared with a concentration
of 10ꢀ4 M. Aliquots of the stock solutions were transferred to the
10 mL volumetric flasks. Cautiously adding water dropwise under
vigorous stirring, and then the appropriate volume of methanol
was added. Not only did we keep the constant of final
concentration at 10ꢀ5 M, but also the change of different water
fractions from 0 to 90 vol% was unified [28]. Once completed this
preparation, UV–vis absorption and PL spectra were measured
immediately with the excitation wavelengths of 307, 336, 336, 339,
and 339 nm for compounds 1-5 respectively in CH3OH/H2O
mixture. Through adjusting the pH values from 1 to 12 in
CH3OH/H2O (10/90 v:v) mixture of compound 1, the PL intensity
spectra with different pH values were obtained. The absolute PL
quantum yields (FF) of CH3OH, CH3OH/H2O (2:8 v:v) mixture and
CH3OH/H2O (1:9 v:v) mixture were recorded on an integrating
sphere and fluorescence lifetime of CH3OH, CH3OH/H2O (2:8 v:v)
mixture and CH3OH/H2O (1:9 v:v) mixture were measured by FLS
980 spectrometer. After standing for 24 h, drops of different
proportion of CH3OH/H2O (5:5, 2:8, 1:9 v:v) mixture were put onto
a silicon wafer by slow evaporation over 12 h at room temperature,
metal spraying after completely dry for SEM [29]. Size distribution
of compound 1 in CH3OH/H2O (5:5, 2:8, 1:9 v:v) mixture after 24 h
storage was evaluated with DLS method.
Compound 6b (255 mg, 10 mmol) was dissolved in 80% ethanol
(12 mL) and acetic acid (3 mL), formaldehyde solution (3 mL) was
added. The mixture was heated to 90–95 ꢂC for 4 h. The solvent was
evaporated to crude and acidified by 2 M hydrochloric acid (7.5 mL)
with stirring at room temperature for 1 h. Precipitation by filtration
purified by silica gel chromatography, affording the compound 2
(110 mg, yield: 41%). Yellow powders. 1H and 13C NMR spectra and
HRMS-EI analysis are supplied in Fig. S2 for compound 2. 1H NMR
(400 MHz, DMSO-d6):
(s, 1H), 6.18 (s, 2H), 4.82 (t, J = 5.6 Hz, 2H), 4.10 (s, 6H), 3.11 (s,
J = 5.6 Hz, 2H), 2.93 (s, 3H). 13C NMR (100 MHz, DMSO-d6):
= 150.8,
d= 9.88 (s, 1H), 8.19 (m, 2H), 7.47 (s, 1H), 7.15
d
149.4, 146.9, 144.6, 144.4, 136.4, 134.2, 133.5, 130.5, 126.5, 121.8,
121.2, 120.8, 111.1, 108.6, 102.5, 62.5, 57.5, 57.2, 27.8, 18.1. HMRS (EI–
MS); m/z calcd for C21H20NO4 [M-Cl]+: 350.1392; found: 350.1370.
2.2.3. Compound 3
Compound 3 was obtained from intermediate 6b using the
similar procedure as compound 2. Yield: 42%. Yellow powders. 1H
and 13C NMR spectra and HRMS-EI analysis are supplied in Fig. S3
for compound 3.1H NMR (400 MHz, DMSO-d6):
d= 9.93 (s,1H), 8.24
(d, J = 9.6 Hz,1H), 8.20 (d, J = 9.6 Hz,1H), 7.30 (s,1H), 7.17 (s,1H), 6.19
(s, 2H), 4.82 (t, J = 5.2 Hz, 2H), 4.10 (s, 3H), 4.10 (s, 3H), 3.37 (q,
J = 7.2 Hz, 2H), 3.09 (t, J = 5.6 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6):
d= 150.1, 149.0, 146.5, 144.4, 144.3, 135.5,
135.2,134.0,132.0,126.0,121.2,121.2,120.1,108.8,108.3,102.0, 62.0,
57.0, 56.9, 27.3, 22.4, 15.4. HMRS (EI–MS); m/z calcd for C22H22NO4
[M-Cl]+: 364.1549; found: 364.1531.
2.4. X-ray crystallography
Faint yellow single crystal suited for X-ray structural analysis was
obtained by slow evaporation the mixture of CH3OH/H2O (7:1v:v) in
room temperature. All data were measured at low temperature
T = 100 (2) K on a Xcalibur Nova (Agilent Technologies (China) Co.,
Ltd)diffractometer.Thestructurewasgotbydirectmethods(SHELXS
97) and non-hydrogen atoms were refined anisotropically using the
least-squares method on F2. Crystallographic data of 13-hydrox-
yberberine are in CIF files and deposited into the Cambridge
CrystallographicDataCenterassupplementarypublicationnumber
CCDC 1511256, via http://www.ccdc.cam.ac.uk/
2.2.4. Compound 4
A suspension of NaI (465 mg, 3.09mmol) in acetonitrile (24 mL),
benzyl bromide (525 mg, 3.09mmol) was added dropwise with
stirring at room temperature for 1.5 h. Then, the mixture was heated
torefluxafteraddingtheintermediate6b. Threehourslater,reaction
system was filtered and the solid was obtained from filtrate by
rotary evaporation. The solid was redissolved in dichloromethane
(40 mL) which contains NBS (550.5 mg, 3.09 mmol) and then the
mixture was stirred at room temperature for 1 h. Evaporating the
solvent and purifying the residue by silica gel chromatography,
affording the compound 4 (910 mg, yield: 70%). Yellow powders.1H
and13CNMRspectraandHRMS-EIanalysisaresuppliedinFig. S4for
2.5. Theoretical calculation
The orbital distribution of highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbital (LUMO) energy
compound 4.1H NMR(400 MHz, DMSO-d6):
d= 10.05 (s,1H), 8.10 (d,

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X. Tang et al. / Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
of compound 1 was calculated using density functional theory
(DFT) at the B3LYP/6-31G** level implemented with Gaussian 03.
enlarging conjugated system of compound 1 in CH3OH. Increasing
the rigidity of molecule can enhance the fluorescence emission. As
shown in Fig.1b, the excitationwavelengths for compounds 2-5 are
nearly 336 nm and 307 nm for compound 1, the emission trend
roughly remains unchanged due to the same luminescent ability of
nanoaggregates. In Fig. 1b, the emission maximums are 524 nm,
511 nm, 512 nm, 513 nm and 514 nm for compounds 1-5 respec-
tively, and the PL intensity spectra reveal two distinct emissive
bands, approximately at ꢄ373 nm (for compound 1), ꢄ426 nm (for
compounds 2-5) and ꢄ520 nm (for all compounds). The high-
energy band (ꢄ520 nm) is ascribed to a TICT emission, while the
low-energy bands (ꢄ373 nm and 426 nm) are assigned to the
emission of the local state [30]. Comparative absorption and
emission spectra of compounds 1-5 are represented in Fig. 1.
2.6. Cell viability evaluation
HepG2 cells were seeded in 96-well microplates at a density of
2.5 ꢃ104 cells per mL in 200
m
L of the respective media containing
g/ml of streptomycin and 10 g/
10% fetal bovine serum (FBS), 10
m
m
ml penicillin. After 24 h of cell attachment, HepG2 cells were
incubated with 10, 20, 40, 80 and 100 M of compound 1 for 8 and
24 h. Then the cells were washed with phosphate buffered saline
(PBS) buffer for three times. Thereafter, 0.5% MTT and 200 L of
m
m
Dulbecco’s Modified Eagle’s Medium (DMEM) cell culture media
was added to each well and incubated for 2 h at 37 ꢂC. Plates were
then analyzed with
a microplate reader (Moleculardevices,
America). Measurements of absorbance were carried out at
450 nm.
3.2. Aggregation-induced emission enhancement
In Fig. 2(1a), absorbance of compound 1 slowly decreases with
the increasement of water fractions due to Mie effect, which
reduces the light transmission in the nanosuspensions. The refined
structures of absorption spectrum gradually disappear at 370 nm
and a red shift with 9 nm appears at 445 nm that indicates the
formation of nanoaggregates of compound 1 [31,32]. There is a
slight red shift with 6 nm in Fig. 2(1b) with water fractions
changing from 0% to 80%. It is presumable that the number of single
light-emitting molecules in nanoaggregates has increased. The
fluorescence emission reaches its maximum value at 80% water
fraction, which is 1.7-fold higher than pure methanol. The
photograph of compound 1 in CH3OH/H2O mixtures with different
water fractions (0–90 vol%) under UV illumination at 365 nm is
shown in Fig. 3. However, the PL intensity of compound 1 decreases
when the water fraction reaches 90% with main emission peak at
524 nm in Fig. 2(1b). There are possible guesses for this
phenomenon. On the one hand, the molecules change the mode
of packing [33]. Nanoaggregates form in an orderly fashion when
water fraction ꢅ80%, like crystal particles. It assembles in a casual
way when water fraction ꢅ90%, like amorphous particles. On the
other hand, nanoaggregates tend to form greater particles even
precipitation, which decrease the concentration of light-emitting
nanoaggregates and weakens its fluorescence emission [34]. The
absorption spectra (Fig. 2(2a–5a)) of compounds 2-5 in CH3OH/
H2O mixture show that there are no significantly changes on
absorbance with the change of water fractions. Therefore, it is
difficult to clearly distinguish these curves and can’t use arrows to
2.7. Cells culture
Cell imaging of compound 1 in HepG2 cells were obtained on
LSM710 laser scanning confocal microscopy (Zeiss, Germany). The
excitation wavelength was set as 458 nm. The cells were cultured
in DMEM supplemented with 10% fetal bovine serum (FBS). Cell
culture was maintained in the medium of 95% air humidity and 5%
CO2 at 37 ꢂC. To maintain the exponential growth of the cells,
culture medium had to change every three days. Before treatment,
cells were seeded in a glass bottom dish with a density of 1 ꢃ105
cells per dish. The cells were incubated with compound 1 (1000 uL,
100 m
M) for 3 h at 37 ꢂC, and the excess was removed by washing
three times with phosphate buffered saline (PBS) buffer. Com-
pound 1 was dispersed in the completely cell culture medium.
3. Results and discussion
3.1. Photophysical properties
In Fig. 1a, compounds 2-5 exhibited the approximately
absorption spectra profile, which manifest that the length and
type of alkyl chain have no significant effect on conjugated
backbones. However, the absorption maximum of compound 1 is
red-shifted by 32 nm at 445 nm in comparison to others. Reason of
the consequence can be attributed to the 13-hydroxy group, which
is rich in lone pair electrons increasing the rigidity of molecule by
200000
1
2
3
4
5
1
2
3
4
5
(b)
(a)
0.60
160000
0.45
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0.15
0.00
80000
40000
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600
700
Wavelength (nm)
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Fig. 1. Absorption (a) and PL (b) spectra with the excitation wavelength of 307, 336, 336, 339, and 339 nm for compounds 1-5 with a concentration of 2.07 ꢃ10ꢀ5 M in
methanol, respectively.

X. Tang et al. / Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
75
300000
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(vol%)
0%
10%
10%
20%
30%
40%
50%
60%
70%
80%
90%
20%
30%
40%
50%
60%
70%
80%
90%
350
400
450
500
400 450 500 550 600 650 700 750
Wavelength (nm)
Wavelength (nm)
Water fraction
(vol%)
0%
(5a)
Water fraction
(vol%)
(5b)
40000
0%
0.51
10%
10%
20%
30%
40%
50%
60%
70%
80%
90%
20%
30000
20000
10000
0
30%
40%
0.34
0.17
0.00
50%
60%
70%
80%
90%
400
450
500
550
600
650
700
750
350
400
450
500
Wavelength (nm)
Wavelength (nm)
Fig. 2. Absorption (1a–5a) and PL spectra (1b–5b) of compounds 1-5 (c = 2.07 ꢃ10ꢀ5 M) in CH3OH/H2O mixtures with different water fractions (0–90 vol%). Arrows illustrate
the peak changes.

76
X. Tang et al. / Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
Water fraction
(vol%)
0%
80%
90%
103
102
0
10
20
30
40
Time (ns)
Fig. 3. Photograph of compound 1 in CH3OH/H2O mixtures with different water
fractions (0–90 vol%) under UV illumination at 365 nm.
Fig. 4. Time-resolved fluorescence curves in CH3OH, CH3OH/H2O (2:8 v:v) mixture
and CH3OH/H2O (1:9 v:v) mixture. The fluorescence lifetime is 1.67 ns, 3.16 ns and
3.10 ns, respectively.
illustrate the changes of absorbance. In Fig. 2(2b–5b), PL intensity
decreases with the increasement of water fractions, which is
characterized for ACQ behavior, and the photographs of compound
2-5 in CH3OH/H2O mixtures with different water fractions (0–90
vol%) under UV illumination at 365 nm are supplied in Fig. S6.
Results reveal that 13-hydroxyberberine chloride is an AIEE
active compound, while compounds 2-5 suffer from aggregation-
caused quenching (ACQ). To have a quantitative comparison, the
fluorescence quantum yields (FF) of compounds 1-5 in the CH3OH
were measured by using an integrating sphere, which are 3%, 2%,
4% and 6% respectively in Table 2. Besides, the fluorescence
quantum yields (FF) of compound 1 in CH3OH/H2O (2:8 v:v)
mixture and CH3OH/H2O (1:9 v:v) mixture are 8, 27, and 17%
respectively, which is accord with the behavior of PL intensity
spectrum. Time resolved fluorescence spectra of compound 1 were
measured in CH3OH, CH3OH/H2O (2:8 v:v) mixture and CH3OH/
H2O (1:9 v:v) mixture by a time-resolved technique. As shown in
Fig. 4, the emission enhancement of compound 1 in CH3OH and
CH3OH/H2O mixtures is in accordance with a corresponding
change in lifetime. The fluorescence lifetime in CH3OH/H2O (2:8 v:
v) mixture is 3.16 ns, which is longer than that in the CH3OH
(1.67 ns) and CH3OH/H2O (1:9 v:v) mixture (3.10 ns). The results are
similar to the emission fluorescence behavior of compound 1 in
different water fractions. The transition time in CH3OH is very fast,
leading to low fluorescence quantum yield and emission fluores-
cence intensity.
CH3OH/H2O (5:5 v:v), CH3OH/H2O (2:8 v:v) and CH3OH/H2O
(1:9 v:v). As shown in Fig. 5d, particle size of nanoaggregates
mainly distributes at 140 nm in CH3OH/H2O (5:5 v:v) mixture.
Molecules aggregate into smaller globular nanoparticles with
particle size round 100 nm in CH3OH/H2O (2:8 v:v) mixture
(Fig. 5e) compared with CH3OH/H2O (5:5 v:v) mixed solution.
The reason why the diameter of nanoaggregates decrease with the
increasement of water fraction is that more compact aggregation
formed in high water fraction. In Fig. 5f, most of nanoparticles
distribute at 170 nm even adhesion to each other in the mixture of
CH3OH/H2O (1:9 v:v). Therefore, we can conclude that the
formation of smaller dimensions nanoaggregates results in strong
emission with increasing the water fractions until 80%, which is
identical to the behavior of PL intensity spectrum [35–37].
In order to further explore the AIEE property of compound 1 in
different morphology, a series of PL spectra were investigated in
the pure solution, mixed solution with fw = 80%, amorphous and
single crystal state. In Fig. 6, emission wavelengths at 540 nm and
554 nm are observed for the amorphous and the crystal
respectively, which are obvious red shift with 16 nm and 30 nm
compared with the lem = 524 nm in the pure solution and mixed
solution. In comparison to the solution, the red shift emission of
the amorphous and the crystal are possibly caused by the strong J-
aggregation. For a clearly comparison, the fluorescence quantum
yields (FF) of compound 1 in the pure solution, mixed solution
with fw = 80%, amorphous and single crystal state were measured
by using an integrating sphere, which are 8%, 27%, 30% and 41%
respectively (Table 3). Furthermore, single crystal was prepared
and analyzed to elucidate this phenomenon.
Nanoaggregates of compound 1 with different water fractions
were observed by scanning electron microscope (SEM) and their
particle sizes distribution were measured by dynamic light
scattering (DLS) method. In Fig. 5a–c, many small uniform globular
nanoaggregates with different sizes form in all mixtures of
Table 2
Photophysical data of compound 1.
Absorptiona
lmax(abs) (nm)
Emissiona
lmax(em) (nm)
Stokes shiftb
Quantum yield KF
a
Compound
Solvent
(cmꢀ1
)
1
CH3OH
445
451
520
524
3241
3089
0.08
0.27
CH3OH/H2O
(2:8 v:v)
CH3OH/H2O
(1:9 v:v)
CH3OH
CH3OH
CH3OH
CH3OH
454
522
2869
0.17
2
3
4
5
420
421
424
425
511
513
513
514
4240
4221
4092
4071
0.03
0.02
0.04
0.06
a
Recorded in CH3OH, CH3OH/H2O (2:8 v:v), CH3OH/H2O (1:9 v:v) (c = 2.07 ꢃ10ꢀ5 M) at 25 ꢂC.
b
Stokes shift = lmax(abs)
ꢀ
lmax(em) (cmꢀ1).

X. Tang et al. / Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
77
Fig. 5. (a) SEM image of compound 1 in CH3OH/H2O (5:5 v:v) mixture; (b) SEM image of compound 1 in CH3OH/H2O (2:8 v:v) mixture; (c) SEM image of compound 1 in
CH3OH/H2O (1:9 v:v) mixture; (d) DLS image of compound 1 in CH3OH/H2O (5:5 v:v) mixture; (e) DLS image of compound 1 in CH3OH/H2O (5:5 v:v) mixture; (f) DLS image of
compound 1 in CH3OH/H2O (5:5 v:v) mixture.
3.3. Crystal structure
the pure solution
250000
200000
150000
100000
50000
0
To have a further understanding of the mechanism, all the
compound were prepared for single crystal growth by vaporizing
mixed solvents of methanol and pure water (7:1 v:v) at room
temperature. However, only compound 1 produced crystal. The
molecular stacking mode of the crystal was explored by X-ray
diffraction on a Xcalibur Nova (Agilent Technologies (China) Co.,
Ltd). Empirical absorption corrections were applied automatically.
The structures were solved with direct methods and refined with a
full-matrix least-squares technique. Crystallographic data are
summarized in Table 4 and the supplementary publication number
CCDC is 1511256. The unit cell of compound 1 is triclinic, space
group P-1, containing two discrete molecules. The ORTEP diagrams
the mixed solution
amorphous
crystal
with the
N and O atom numbering schemes and packing
interactions in the crystal of compound
1 are depicted in
350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
Table 4
Crystallographic data and refinement parameters for compound 1.
Fig. 6. PL spectra of compound 1 in the pure solvent, the mixed solvent with
fw = 80%, the amorphous and the crystal.
Compound 1
chemical formula C21H21NO6
volume (Å3)
Z
880.93(8)
2
1.445
formula wt
383.39
100(2)
temperature (K)
density (g/cm3)
crystal size (mm3) 0.50 ꢃ 0.1 ꢃ0.08
2u
range
4.7790ꢀ71.6280
crystal system
space group
a (Å)
triclinic
P-1
F (000)
404
no. of reflns
no. of unique reflns
no. of params
6321
2911
257
0.0415, 0.0370
0.1002, 0.0957
9.0561(5)
9.2618(5)
11.3096(7)
87.223(4)
68.861(5)
84.744(4)
Table 3
b (Å)
c (Å)
Photophysical data of compound 1 with different morphology.
a
Rall, Robs
b
a
b
g
(deg)
(deg)
(deg)
wR2,all, wR2,obs
Morphology
Emission lmax(em) (nm)
Quantum yield KF
Goodness-of-fit on F2 1.050
Solution
522
524
540
554
0.08
0.27
0.30
0.41
Aggregation
Amorphous
Crystal
w = 1/[
s
2(Fo)2 + (aP)2 + bP], where P = [(Fo2) + 2Fc2]/3.
a
R1
=
S
kFo| ꢀ |Fck/
S|Fo|.
b
2
wR2 = [
S
[w(Fo ꢀ Fc2)2]/
S .
w(Fo2)2]1/2

78
X. Tang et al. / Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
Fig. 8. Energy level and electron density distribution of frontier molecular orbital of
compound 1.
respectively, which leads to two adjacent molecules stacking in
“head-to-tail” mode that is J-aggregation consisting with the
crystal structure. The theoretical study also explains the AIEE
property of compound 1.
3.5. pH fluorescence probe
On the basis of above exploration, 13-hydroxyberberine
chloride has been proved possessing the effect of aggregation-
induced emission enhancement, which has an advantage over
traditional probes with the problem of aggregation-caused
quenching (ACQ). So its pH sensitivity was studied to find if it
has the potential value as pH fluorescence probe. A series of mixed
solution CH3OH/H2O (1:9 v:v) of compound 1 with different pH
values was prepared and pH values were adjusted by a spot of
dilute hydrochloric acid and dilute sodium hydroxide from 1 to 12.
As shown in Fig. 9a, the PL intensity increases slowly when pH
values < 5.52, while it increases remarkably when pH values > 5.52.
PL intensity maximum appears at pH = 6.84 because the molecules
of compound 1 forms a number of nanoparticles, which is 5-fold
higher than pH = 1.57. Nonetheless, PL intensity gradually
decreases when pH > 6.84. It is reasonable to infer that 13-hydroxy
group of compound 1 is transformed into sodium alkoxide salt,
which results in a portion of compound 1 dissolves in the solvent,
reducing the formation of nanoaggregates [41–43]. Experimental
results show that compound 1 has sensitive response to the
changes of pH values, especially under the low-acid environment.
pKa value is a main parameter of pH fluorescence probe, which
determines the appropriate pH detection range. Additionally, a bit
of pH variation can cause the obvious changes of PL intensity
around the pKa value. In Fig. 8b, pKa value can be confirmed as 5.12
when I/Imax = 0.5 (in Fig. 9b).
Fig. 7. (a) ORTEP diagram of compound 1, (b) Dipolar orientation of compound 1, (c)
Slip angle of compound 1, (d) Packing arrangement and CꢀꢀHꢁ ꢁ ꢁ
p interaction in the
adjacent molecules marked by dashed line (purple), (hydrogen atoms except H8 and
CH3OH solvent are omitted for charity).
Fig. 7a. As shown in Fig. 7b, the arrow on the molecule shows the
dipolar orientation. Due to the planar heterocycles, the molecule
tends to form the parallel conformation with the neighboring one,
which gives rise to molecular planarization to some extent. It is
also the reason why absorption band doesn’t become narrowed of
compound 1. The molecular dimer is stacked in “head-to-tail”
mode that is J-aggregation with slip angle
u
of 40ꢂ (Fig. 7c), which
creates an advantage on the enhancement of fluorescence
emission in aggregation state. In Fig. 7c, the distance between
adjacent molecules is d3 = 3.80 Å, which is too large to form strong
p
-
p
interaction. A kind of CꢀꢀHꢁ ꢁ ꢁ
p hydrogen bond forms between
two molecules with the angle and interaction distance (d1) are
3.33 Å and 132ꢂ, where the dimethoxy groups of one molecule acts
as H donor and the [1,3]dioxolane acts as H acceptor. Other
CꢀꢀHꢁ ꢁ ꢁ
p hydrogen bond isn’t formed because of the large distance
d2 = 3.62 Å. There is a weak interdipole interaction for compound 1,
which will decrease the aggregation-caused quenching. In a word,
the AIEE mechanism of compound 1 is the result of J-aggregation
combined with molecular planarization [38–40].
Cell viability is an important parameter, which decides the
biological application potential for biological probe. As shown in
Fig. 10, the cytotoxicity of compound 1 in HepG2 cells was
examined by MTT assay [44,45]. It can be found that the cell
viability values have not changed apparently after cells were
incubated with different concentrations (10, 20, 40, 80 and
3.4. Electronic structure
To understand the relationship between the optical property
and electronic structure, the highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbital (LUMO) of
compound 1 was optimized using density functional theory (DFT)
at the B3LYP/6-31G** level. The HOMO energy level for compound
1 was calculated as ꢀ8.75 eV. Meanwhile, the LUMO energy level
was calculated to be ꢀ5.65 eV, and the calculated band gap is
3.10 eV, As shown in Fig. 8, HOMO and LUMO of compound 1 are
mainly localized on dimethoxy unit and isoquinoline part,
100 mM) of compound 1 for 8 and 24 h. These values are greater
than 95% and some even greater than 100%. Result suggests that
compound 1 has a high potential for biological probe. Considered
the strong fluorescence and high cell viability, the cell uptake
behavior of compound 1 was evaluated using laser scanning
confocal microscopy (LSM) to explore their potential biological

X. Tang et al. / Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
79
150000
120000
90000
60000
30000
0
PH Value
(b)
(a)
1.0
0.8
0.6
0.4
0.2
1.57
2.03
3.71
4.89
5.52
6.84
7.42
8.15
9.38
10.07
11.17
12.03
pKa=5.12
2
4
6
8
10
12
350 400 450 500 550 600 650 700
Wavelength (nm)
pH value
Fig. 9. (a) The changes of PL intensity with different pH values of compound 1 (c = 2.07 ꢃ10ꢀ5 M) in CH3OH/H2O (1:9 v:v) mixtures. (b) The changes of relative PL intensity
versus pH values. The excitation wavelength is 307 nm.
specific action target between compound 1 and HepG2 cells will be
explored in our later works. In addition, normal morphology of
cells was kept and adhered very well to the cell plates in Fig. 11b,
which suggests that compound 1 possesses biocompatibility.
4. Conclusion
In conclusion, a series of berberine derivatives at 13-position
(compounds 1-5) has been designed and synthesized. AIEE
phenomenon happened when the substituent is hydroxy group
at 13-position, while others show weak fluorescence in nano-
aggregation state. Optical spectra of five kinds of derivatives were
obtained in aqueous suspension and 13-hydroxyberberine chloride
shows AIEE phenomenon owing to nanoaggregates formation. A
great deal of small globular nanoaggregates of compound 1 was
observed in SEM images and its particle sizes were obtained by DLS
method, which is in favor of fluorescence emission. Crystal
Fig.10. Cell viability of compound 1. HepG2 cells were incubated with 1–100
compound 1 for 8 and 24 h.
mM of
structure of compound
1 was achieved through the X-ray
diffraction analysis. Crystallographic data shows that J-aggregation
combined with molecular planarization are observed in the crystal,
which is the mechanism of fluorescence emission enhancement.
DFT calculation on HOMO and LUMO is consistent with the AIEE
property of compound 1. All results suggest that the aggregation-
induced emission enhancement property is influenced by substit-
uent. Moreover, compound 1 has excellent pH sensitivity, and the
LSM images showed that compound 1 can be easily internalized by
HepG2 cells, which implies its potential as pH fluorescence probe.
imaging application. As shown in Fig. 11, an intracellular
fluorescent was observed in the location of HepG2 cells after
compound 1 was incubated with the concentration of 100 mM for
3 h. The distribution of the probe within the cells was observed by
high resolution confocal fluorescent microscope. Compound 1
could be internalized by HepG2 cells and distributed in the whole
cells. Hence, it can be inferred that the compound 1 maybe locate
at a substance, which widely distributes in the cells. Therefore, the
Fig. 11. LSM images of HepG2 cells. Cells were incubated with 100 mM of compound 1 for 3 h. (a) Bright field, (b) Fluorescent image, (c) The overlap image of a and b. The
HepG2 cells were excitated with a laser with at 458 nm.

80
X. Tang et al. / Journal of Photochemistry and Photobiology A: Chemistry 337 (2017) 71–81
The structure of 13-hydroxyberberine will be modified to decrease
its effective concentration when applied to biological application
and its specific action target between compound 1 and HepG2 cells
will be explored in our following work.
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