Synthesis and Studies on Photophysical Properties of Rhodamine Derivatives for Bioimaging Applications

Article abstract of DOI:10.1002/bkcs.11733

Rhodamine derivatives have been persistently used as fluorescent/solvatochromic dyes in various areas. For example, they are used as a labeling agent in biotechnology and a chemosensor. Also, rhodamine derivatives are used for cell imaging experiment. Her

Full text of DOI:10.1002/bkcs.11733

Article
DOI: 10.1002/bkcs.11733
BULLETIN OF THE
B. D. Vanjare et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Studies on Photophysical Properties of Rhodamine
Derivatives for Bioimaging Applications
Balasaheb D. Vanjare,† Prasad G. Mahajan,† Nilam C. Dige,‡ Abdul Rehman Phull,‡,§
Song Ja Kim,‡ and Ki Hwan Lee†,
*
†Department of Chemistry, Kongju National University, Gongju 32588, Republic of Korea.
*E-mail: khlee@kongju.ac.kr
‡Department of Biological Sciences, Kongju National University, Gongju 32588, Republic of Korea
§Department of Biochemistry, Shah Abdul Latif University, Khairpur 66020, Pakistan
Received January 30, 2019, Accepted April 5, 2019
Rhodamine derivatives have been persistently used as fluorescent/solvatochromic dyes in various areas.
For example, they are used as a labeling agent in biotechnology and a chemosensor. Also, rhodamine
derivatives are used for cell imaging experiment. Herein, we report on the synthesis of fluorescent deriva-
tives based on rhodamine B (RHB1, RHB2, and RHB3). We confirmed the structures and purities of the
1
synthesized probes i.e., RHB1, RHB2, and RHB3 by using analytical technique’s viz. H NMR, 13C
NMR, Mass and FT-IR followed by exploring spectroscopic properties by using fluorescence/UV absorp-
tion spectroscopy. The effect of solvents on the fluorescence and absorption properties of the synthesized
compounds has been comprehensively studied using a series of solvent. In addition, synthesized RHB1,
RHB2, and RHB3 were found to be non-toxic against the cancer cell (MDA-MB-231). It is clear from
the result of the conventional fluorescence microscopy that probe RHB2 exhibits significant optical prop-
erties as compared with those of RHB1 and RHB3.
Keywords: Rhodamine derivatives, Photophysical properties, Cell imaging, Cytotoxicity.
Introduction
as
(MMP).15
In the last decade, numerous research groups were
a marker for mitochondrial membrane potential
During the past decade, fluorescent probes are used for the
selective detection of chemical species in the environmental
contaminants and labels of targeting molecules. For the
qualifying conditions in the selection of organic dyes as a
fluorescent probe, it must display the finest fluorescence
response when it irradiated in the visible range and less
lethal to the living cells and tissues.1–3 The common struc-
tural feature in several fluorophores is a conjugated π- sys-
tem and having both electrons donating and withdrawing
group.4–6 Rhodamine and its derivatives form a family of
multipurpose functional dyes, which have been extensively
used due to their excellent photophysical properties.7–10
Additionally, Rhodamine derivatives go through two forms
i.e. spirocyclic and open rings form. The spirocyclic forms
(non-fluorescent, colorless) exhibit limited π bond conju-
gated arrangement, whereas the open ring form (fluores-
cent, pink) shows exhaustive signals in the absorption and
fluorescence spectroscopy due to extended large
π-conjugated systems.11–14 Rhodamine derivatives are mas-
sively used in the field of the bioimaging and biosensors
because of their outstanding optical properties such as,
quantum yield, molar absorption coefficient and photo-
stability. For instance, Rhodamine 123 has been selectively
designated for restraining the active mitochondria and used
engaged in development of the conjugated organic systems
possessing rhodamine core.16–18 Besides, for the construc-
tion of the extra conjugation by π system, the design of the
inflexible rings in the rhodamine derivatives can have more
probabilities of shifting the absorption/emission spectra to a
longer wavelength. However, most of the rhodamine deriv-
atives have absorption and emission wavelengths below
600 nm. Additionally, owing to the lack of molecular rota-
tion in the xanthene ring, it raises the rigidity of the mole-
cule, which increases the fluorescence quantum yield.19
In the present work, we have synthesized three different
derivatives of Rhodamine (RHB1, RHB2, and RHB3) and
its optical properties have been studied in different solvents.
The comparison syntax of the synthesized three dyes for
the photophysical properties in various solvent systems
indicates RHB2 with higher-extinction coefficient, quantum
yield and Stokes shift than those of other dyes i.e., RHB1
and RHB3. Moreover, these compounds were employed
for cell imaging experiment, which reveals that RHB2
shows superior fluorescence imaging against cancer cells
(MDA- MB-231) because of their structural variation, sig-
nificant optical properties, higher Stokes shift value, and
low cytotoxicity as compared to those of RHB1
and RHB3.
Bull. Korean Chem. Soc. 2019
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley Online Library
1

Article
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
Experimental Section
until the confluence of the cells reached approximately
65%. Subsequently, cells were treated with and without
10 μM of RHB1, RHB2, and RHB3 for 2 h in the pres-
ence of growth medium. The treated and untreated sample
cells were washed with cold PBS buffer two to three times.
Finally, fluorescence images of green light excited cells
were taken by using a fluorescence microscope (BX51,
Olympus, and Tokyo, Japan). In order to compare the fluo-
rescence intensities during imaging investigations, all set-
tings such as exposure time, contrast and brightness were
maintained at the same level. The experiment was repeated
three times and performed 37 ꢀC in a CO2 incubator.
Materials. Rhodamine B, hydrazine hydrate, 2-amino-
5-{4-bromophenyl}-1,3,4-thiadiazole, 4–5 diazafluoren-9-one,
4- amino benzaldehyde, 2-(1H-benzotriazol-1-yl)-1,1,3,3,
−tetramethyluronium hexafluorophosphate (HBTU), N, N-
diisopropylethylamine (DIPEA) and N,N-dimethyl formamide
(DMF) were obtained from Sigma Aldrich (Munich, Ger-
many). Ethanol, methanol (MeOH), ethyl acetate (EtOAc),
dichloromethane (DCM), acetonitrile (ACN), dimethyl sulf-
oxide (DMSO), and acetone used in these experiments were
purchased from Samchun Chemicals (Daejeon, South Korea)
until and unless mentioned.
Cytotoxicity Assessment by MTT Assay. The cytotoxic
effect of samples was investigated through MTT assay on
the viability of cancer (MDA-MB-231) cells. MDA-MB-
231 cells were plated at a density of 2.5 × 104 cells/well on
96-well plate. The cells were grown for 12 h and the
growth medium was changed. The cells grew until the con-
fluence of the cells reached up to 75%. Subsequently,
grown cells were exposed with and without RHB1, RHB2,
and RHB3 at the concentration of 10 μM for 2 and 12 h in
the existence of a growth medium. After the treatment of
specific experimental times i.e., 2 and 12 h, 10 μL of MTT
reagent 1 (MTT, 10 mg/mL) was added in each well and
subjected to incubate for 4 h. Subsequently, each well was
added with 100 μL of MTT reagent 2 or solubilization
buffer (DMSO and 10% SDS with 0.01 N HCl) and sub-
jected to incubate in similar conditions for 12 h. Finally,
absorbance was measured at 595 nm by using assay plate
reader (Molecular Devices, Sunnyvale, CA, USA).23,24 The
experiment was repeated three times and the non-treated
cell was considered 100% viable and used as the control.
Equipment. Fluorescence spectra and UV–Visible spectra
of all the samples were recorded at room temperature by
using FS-2 fluorescence spectrophotometer (Scinco, Seoul,
Korea) and Shimadzu spectrophotometer (Kyoto, Japan),
respectively. FT-IR measurements were accomplished on
Frontier IR (Perkin Elmer, Santa Clara, CA, USA) spec-
1
trometer using KBr pellets. H NMR (400 MHz) and 13C
NMR (125 MHz) were recorded on the Bruker Avance
400 and 100 MHz spectrophotometer (Billerica, MA, USA)
and TMS is used as an internal standard. The LC–MS mass
spectra were recorded on 2795/ZQ2000 (Waters, Milford,
MA, USA) spectrometer. All the melting points of the syn-
thesized compounds were measured on Fisher Scientific
(Pittsburgh, PA, USA) melting point apparatus.
Quantum Yield Measurements. In absorption and fluo-
rescence experiment, a stock solution of RHB1, RHB2,
and RHB3 were prepared using different spectroscopic
grade pure solvents at room temperature. 1 cm path length
of the cells used for the absorption and emission study. All
the fluorescence experiments were completed by keeping
slit width constant i.e., 5 × 5 nm. Each fluorescence and
absorption spectra were taken after 60 min. The fluores-
cence quantum yield was calculated by using Eq. (1), given
below.20,21,26
Synthesis and Spectral Characterization Techniques
Synthesis of 2-(5-(4-Bromophenyl)-1, 3, 4-thiadiazol-
2-yl)-30, 60-bis (diethyl amino) spiro [isoindoline-1,
90-xanthen]-3-one (RHB1).
Rhodamine
B
(0.5 g,
ODref × Asample × η2
ODsample × Aref × η2
sample
1.043 mmol), 2-amino-5-{4-bromophenyl}-1, 3, 4-thiadiazole
(0.30 g, 1.148 mmol), and HBTU (0.48 g, 1.25 mmol) was
taken in DMF (5 mL) and stirred the reaction mixture for
10 min at ambient temperature. Di-isopropylethylamine
(0.6 mL, 3.13 mmol) was added and continued the reaction
for 4 h. Synthesis route of RHB1 is as shown in Scheme 1.
The reaction mixture was quenched with ice-cold water and
extracted with ethyl acetate. The ethyl acetate layer was
washed with water/brine, distilled out the organic layer and
purified by using column chromatography DCM: methanol
(9:1) as an eluent to afford a light pink solid, Yield: 76%, M.
P: 225–229 ꢀC; Figure S1 (Supporting Information): 1H
NMR (DMSO‑d6, 400 MHz) δ (ppm): 8.07 (d, J = 7.5 Hz,
1H), 7.87–7.78 (m, 2H), 7.77–7.61 (m, 2H), 7.77–7.61 (m,
4H), 7.19 (d, J = 7.6 Hz, 1H), 6.41 (d, J = 2.5 Hz, 2H), 6.36
(d, J = 8.9 Hz, 2H), 6.23 (dd, J = 8.9, 2.6 Hz, 2H) 3.29
(t, 8H), 1.07 (q, 12H); Figure S2: 13C NMR (DMSO‑d6,
Φsample = Φref
×
ð1Þ
ref
where A signifies the integrated area of the fluorescence
spectrum and OD signifies the optical density of the probe
at the excitation wavelength, η is the refractive index of the
solvent used for the experiment. Quinine sulphate in 0.1 M
sulfuric acid was used as the reference standard (Φf)
= 0.546.22
Culturing of Cancer Cells (MDA-MB-231) and Cell
Imaging Studies. The cancer cells were maintained and
grown in DMEM medium supplemented with FBS (10%),
antibiotics (streptomycin, 50 μg/mL and penicillin,
50 units/mL). Furthermore, previously cultured and
maintained cells were seeded in 35 mm culture plate at the
density of 1 × 105 per plate. Following day, the medium
was replaced with fresh medium and cells were left to grow
Bull. Korean Chem. Soc. 2019
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
2

BULLETIN OF THE
Article Synthesis and Studies on Photophysical Properties of Rhodamine Derivatives KOREAN CHEMICAL SOCIETY
1
ꢀ
125 MHz) δ (ppm): 165, 162, 154, 156, 148, 145, 149, 136,
133, 125, 124, 108, 106, 97, 68, 44, 12; Figure S3: IR (KBr)
v/cm: 2966.98, 2927.43, 2293.87, 1709.67, 1634.95,
1613.95, 913.55, 903.98, 817.52; Figure S4: LC–MS:
682.4 m/z (M + 2).
yellow solid, yield: 72%, M.P: 213–215 C; Figure S9: H
NMR (DMSO‑d6, 400 MHz) δ (ppm): 1.07 (t, 12H,
-NCH2CH3), 3.30 (q, 8H, -NCH2CH3), 4.26 (s, 2H, Ar-
NH2), 6.0–7.0 (m, 6H, xanthene-H), 6.3–6.5 (m, 4H, ArH),
7.78 (dd,1H, ArH), 7.5 (s, 1H, –CH=N), 7.4–7.5 (m, 3H,
ArH). Figure S10: 13C NMR (DMSO‑d6, 125 MHz) δ
(ppm): 166, 155, 152, 149, 133, 131, 128, 129, 126, 124,
108, 106, 98, 65, 44, 13. Figure S11: IR (KBr) v/cm:
3022.11, 2969.56, 2891.16, 2343.56, 2300.80, 1692.09,
1633.94, 1613.84, 1548.27, 1396.01, 967.13. Figure S12:
LC–MS: 560.5 m/z (M + 1).
Synthesis of 2-(9H-4-5 Diazafluorene)-30, 60-bis (diethyl
amino) spiro [isoindoline-1, 90-xanthen]-3-one (RHB2).
As demonstrated in Scheme 1, RHB2 compound was syn-
thesized by reaction of rhodamine hydrazide B with 4–5
diazafloren-9-one. Previously reported synthetic route has
been employed for the preparation of rhodamine B hydra-
zide.25 Rhodamine B hydrazide (0.5 g, 1.09 mmol) and 4–5
diazafluoren-9-one (0.19 g, 1.09 mmol) were dissolved in
DMF (4 mL), stirred for 12 h at ambient temperature (mon-
itored by TLC). The ice-cold water was added and stirred
for 10–15 min. The received solid was filtered and washed
with ice-cold water. Obtained solid was dried and subse-
quently purified by silica gel chromatography using DCM:
methanol (0–3%) as the eluent to afford RHB2 as an off-
Results and Discussions
UV Visible Absorption and Fluorescence Spectroscopy.
The influence on UV–Vis absorption and fluorescence
emission spectra of 1 × 10−4 mol/L solution of compounds
RHB1, RHB2, and RHB3 in a variety of solvents at room
temperature by considering an extensive range of polarity
including non-polar, polar, polar protic, and polar aprotic
solvents are presented in Figures 1–2, and 3, showing the
resultant statistics given in Table 1. The absorption of both
RHB1 (Figure 1(a)) and RHB3 (Figure 3(a)) were found
to be very less penetrating for the variation in the polarity
of the solvents as compared to that of RHB2. For all-
different polarity of solvents, two prominent absorption
band was observed in the UV region.26,35 The absorption
maximum of RHB1 changes from 560 nm (DCM) to
562 nm (methanol), except in polar aprotic solvents (ace-
tone) other solvents do not show an absorption band. The
absorption maximum of RHB2 ranges from 560 nm
1
ꢀ
white solid, yield: 85%, M.P: 148–150 C; Figure S5: H
NMR (DMSO‑d6, 400 MHz) δ (ppm): 8.9–8.51 (2H, m),
8.30–8.07 (2H, m), 7.76 (1H, s), 7.6–7.5 (5H, m), 7.10–6.9
(2H, m), 6.56–6.1 (4H, m), 3.5–3.23 (8H, q), 1.08 (12H, t).
Figure S6: 13C NMR (DMSO‑d6, 125 MHz) δ (ppm):
164, 162, 157, 155, 150, 133, 132, 130, 128, 1126,
122, 110, 108, 99, 64, 44, 12; Figure S7: IR (KBr) v/cm:
2940.85, 2292.49, 1694.51, 1460.94, 1429.32, 903.90,
809.07. Figure S8: LC–MS: 621.5 m/z (M + 1).
Synthesis of 2-(4-Aminobenzylideneamino)-30, 60-bis
(diethyl amino) spiro [isoindoline-1, 90-xanthen]-3-one
(RHB3). Initially, 0.5 g (1.09 mmol) of Rhodamine B
hydrazide and 0.13 g (1.09 mmol) of 4-amino benzalde-
hyde was dissolved in 6 mL ethanol by stirring for 12 h at
ambient temperature (monitored by TLC). The solid was
obtained by filtering and subjected to wash with cold etha-
nol. The crude solid was purified by using column chroma-
tography using (DCM:methanol) as the eluent to afford a
Figure 1. The absorption (a) and fluorescence (b) spectra of the
RHB1 at the concentration of 1 × 10−4 mol/L in different
solvents.
Scheme 1. Reagents and conditions: (a) 2-amino-
5-{4-bromophenyl}-1, 3, 4-thiadiazole, HBTU, DIEPA, DMF,
RT/4 h. (b) Hydrazine hydrate, ethanol, reflux (c) 4–5
diazafluoren-9-one, ethanol, ambient temperature, 12 h.
(d) 4-Amino benzaldehyde, ethanol, ambient temperature, 12 h.
Figure 2. The absorption (a) and fluorescence (b) spectra of the
RHB2 at the concentration of 1 × 10−4 mol/L in different
solvents.
Bull. Korean Chem. Soc. 2019
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
3

BULLETIN OF THE
Article Synthesis and Studies on Photophysical Properties of Rhodamine Derivatives KOREAN CHEMICAL SOCIETY
change in the little spectral shift by 2–20 nm for all the
compounds.27–30
Table 1 shows the photophysical parameters including
absorption and fluorescence maxima, molar extinction
coefficients, Stokes shifts and fluorescence quantum yields
for each rhodamine derivatives. The absorption spectra dis-
play broad band in the range from 540 to 620 nm for all
these three compounds. Moreover, a high molar absorption
coefficient ranges from 80 to 360 M−1 cm−1, 40 to 1000
Figure 3. The absorption (a) and fluorescence (b) spectra of the
RHB3 at the concentration of 1 × 10−4 mol/L in different
M−1 cm−1 and 30 to 630 M−1 cm−1 was observed for the
solvents.
compounds RHB1, RHB2, and RHB3, respectively. The
review of the data in Table 1 displays that the location of
(DCM) to 568 nm (DMF), polar protic solvents have no
consequence on the absorption of RHB2. The absorption
maximum of RHB3 compound in DMF and DCM solvents
is around 560 nm, but it is slightly blue shifted in polar
protic solvents i.e., 554 nm in methanol. The excellent
fluorescence properties are the main feature of the present
synthesized compounds. Figures 1(b), 2(b), and 3(b) illus-
trate the emission spectrums of the RHB1, RHB2, and
RHB3, respectively. The fluorescence emission bands are
broad in nature and shows wide spectral visible region
from 500 to 650 nm for RHB1, from 520 to 650 nm for
RHB2, and from 540 to 650 nm for RHB3 in a variety of
solvent. This band assigned to the S1 ! S0 electronic tran-
sition. In all three compounds, the effect of the solvent
polarity on the emission spectra was more prominent than
absorption spectrum, it is due to the intramolecular charge
transfer (ICT) effect.28,35 The change in the polarity of sol-
vent used for fluorescence emission study induces the
the π–π* absorption band considerably depends on the
polarity of the solvents.3
Moreover, the fluorescence band is affected by changing
the solvent polarity.31–34 While relating these three deriva-
tives and its fluorescence emission response against series
of solvent reveals that, RHB2 derivative shows maximum
fluorescence emission intensity as compared with RHB1
and RHB3, except in DCM solvent as shown in Figure 2.
Furthermore, the compound RHB1 shows very less fluores-
cence emission intensity in EtOAc, ACN, DMF, and ace-
tone as shown in Figure 1. Similarly, for the compound
RHB3 which shows low fluorescence intensity in EtOAc,
DMSO, and acetone, respectively as shown in Figure 3.
RHB1 compound was synthesized by the reaction of the
rhodamine B and amine (2-amino-5-{4-bromophenyl}-1,
3, 4-thiadiazole) which contains phenyl ring with para
directing electron donating group i.e., bromo ( Br) as a
substituent. Besides, the RHB2 and RHB3 compounds
Table 1. Photophysical parameters of the RHB1, RHB2, and RHB3 in different solvents.
Compound
Solvent
λex (nm)
λem (nm)
Stokes shift (cm−1
)
ε (M−1 cm−1
)
Φ
RHB1
DCM
EtOAc
ACN
560
—
577
—
526.12
—
360
—
0.39
—
—
—
—
—
—
DMF
—
—
—
—
—
DMSO
MeOH
Acetone
DCM
EtOAc
ACN
565
550
—
585
572
—
605.09
699.30
—
80
230
—
0.15
0.12
—
RHB2
RHB3
560
560
558
568
567
552
560
560
—
578
575
578
592
578
576
579
581
556.10
465.83
620.10
713.74
335.64
754.83
585.98
645.43
—
40
70
0.5
0.072
0.19
0.08
0.38
0.46
0.72
0.78
—
210
1000
100
890
40
DMF
DMSO
MeOH
Acetone
DCM
EtOAc
ACN
630
—
562
—
581
—
581.89
—
30
—
0.93
—
DMF
DMSO
MeOH
Acetone
—
—
—
—
—
554
—
574
—
628.93
—
290
—
0.30
—
Bull. Korean Chem. Soc. 2019
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
4

BULLETIN OF THE
Article Synthesis and Studies on Photophysical Properties of Rhodamine Derivatives KOREAN CHEMICAL SOCIETY
the different solvents are summarized in Table 1. Remark-
able trends for RHB1, RHB2, and RHB3 were observed.
The quantum yield of RHB1 reached a maximum of 0.39
in DCM and decreased from 0.15 to 0.12 in DMSO and
methanol, respectively. The quantum yield of RHB2
reached a maximum of 0.5 in dichloromethane but
decreased to 0.08, 0.38 in DMF and DMSO, respectively.
Figure 4. Fluorescence imaging studies of live MDA-MB-231
cells. Bright field images of control (a), RHB1 (b), RHB2 (c),
RHB3 (d), and fluorescence images of control (e), RHB1 (f),
Similarly, for RHB3, the quantum yield was 0.78 in DCM
and 0.93 in ACN decreased to 0.32 in methanol. This spec-
tacle can be explained by positive solvatokinetic effect,
RHB2 (g), RHB3 (h) treated cells are shown. Cells without expo-
when the energy level of the excited state reduces in the
sure of samples were considered as control.
polar solvent and may cause the reduction in the energy
gap and increasing non-radiative transitions, simulta-
were prepared by the reaction of the rhodamine B hydra-
neously. Therefore, we conclude that positive solvatokinetic
zide with 4–5 diazafluoren-9-one and 4-amino benzalde-
effect might be the plausible reason for the decrease in fluo-
hyde, respectively. RHB1 and RHB3 both compounds
rescence quantum yield with increase in the polarity of the
contain an electron donating group attached to phenyl ring
solvent.34–36
such as, Br and -NH2, respectively. RHB2 shows higher
Cell Imaging. The cancer cells were treated with RHB1,
fluorescence response than those of RHB1 and RHB3. It is
RHB2, and RHB3 samples and fluorescence study of each
well-known that rhodamine derivatives can suffer between
treated sample was performed for MDA-MB-231 cancer
spirocyclic (non-fluorescent) and open ring (fluorescent)
cells. Whereas, fluorescence and bright field images of sam-
forms.11,12,15 The reason for the variation in fluorescence
ple was correlated with control (C). The fluorescence inves-
properties of RHB1, RHB2, and RHB3 lies in the spi-
tigations were recorded at a wavelength of 532 nm.
rolactam ring opening mechanism. It might be because of
Experimental results are shown in Figure 4 which indicate
the presence of an electron donating group in the RHB1
that RHB2 exhibited superior fluorescence in comparison
and RHB3 which opposed the ring opening mechanism.
to other samples i.e., RHB1 and RHB3.
While, in case of RHB2, strong fluorescence attributes to a
Cytotoxicity. The cytotoxic effects of RHB1, RHB2, and
more conjugated fused ring of 4–5 diazafluoren-9-one. In
RHB3 were evaluated on cancer cells by using MTT assay.
addition to the fluorescence response, the synthesized deriv-
The RHB1, RHB2, and RHB3 samples were found non-
atives further examined for fluorescent cell imaging study
toxic, as they showed no significant toxic effect on cancer
in order to categorize the fluorescent response of the
cells. Results of the MTT assay are shown in Figure 5. The
respective compounds. Pleasingly, only RHB2 shows
results obtained in the cytotoxicity study revealed that syn-
excellent fluorescence image output when it was treated
thesized rhodamine derivatives could be explored as fluo-
with MDA-MB-231. While other compounds RHB1 and
rescent biomarker to trigger the MDA-MB-21 cell due to
RHB3 failed to produce good fluorescent image, it is
its non-toxic nature towards a cell line.
because of the structural variations and may fails to open
spirolactam ring in rhodamine core. Additionally, the fluo-
rescence quantum yield of RHB1, RHB2, and RHB3 in
Conclusion
In summary, we synthesized fluorescent dyes based on rho-
damine, which displays excellent spectroscopic properties
for RHB2 as compared to those of RHB1 and RHB3. The
fluorescence variations of the dyes are well correlated with
the polarity of the solvent, which can be used as a resource-
ful chemo-sensing platform for designing new ratiometric
fluorescent chemosensor. The variation in spectroscopic
characteristics posed by synthesized compounds was dis-
cussed in detail about the polarity of solvent, functional
group attached to rhodamine core and five membered spi-
rolactam ring opening mechanism. Considering their good
photophysical properties, we used these dyes (RHB1,
RHB2, and RHB3) for cell imaging and cytotoxicity exper-
iment against the MDA-MB-231 cancer cell. Furthermore,
the fluorescent cell imaging experiment reveals that RHB2
shows excellent fluorescent triggering responses as com-
pared to RHB1 and RHB3. Thus, noteworthy properties of
synthesized derivatives can be explored as attractive
Figure 5. Cytotoxicity assessment (MTT assay). Cancer cells
(MDA-MB-231 cells) were untreated (Control) and treated with
RHB1, RHB2, and RHB3 at 10 μM in DMSO for 2 and 12 h.
Bull. Korean Chem. Soc. 2019
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
5

BULLETIN OF THE
Article Synthesis and Studies on Photophysical Properties of Rhodamine Derivatives KOREAN CHEMICAL SOCIETY
15. J. Chen, W. Liu, B. Zhou, G. Niu, H. Zhang, J. Wu,
Y. Wang, W. Ju, P. Wang, J. Org. Chem. 2013, 78, 6121.
16. H. J. Chen, X. B. Zhou, A. L. Wang, B. Y. Zheng,
C. K. Yeh, J. D. Huang, Eur. J. Med. Chem. 2018, 145, 86.
17. X. Chen, T. Pradhan, F. Wang, J. S. Kim, J. Yoon, Chem.
Rev. 2012, 112, 1910.
applications as ecologically sensitive fluorescent probes and
labels in biology.
Supporting Information. Additional supporting informa-
tion is available in the online version of this article.
18. V. P. Boyarskiy, V. N. Belov, R. Medda, B. Hein, M. Bossi,
S. W. Hell, Chem. Eur. J. 2008, 14, 1784.
References
19. J. Liu, Z. Diwu, W. Y. Leung, Y. Lu, B. Patch,
R. P. Haugland, Tetrahedron Lett. 2003, 44, 4355.
20. S. Pal, M. Mukherjee, B. Sen, S. Lohar, P. Chattopadhyay,
RSC Adv. 2014, 4, 21608.
21. P. G. Mahajan, N. C. Dige, B. D. Vanjare, H. Raza,
M. Hassan, S. Y. Seo, S. K. Hong, K. H. Lee, J. Fluoresc.
2018, 28, 1305.
22. A. M. Brouwer, Pure Appl. Chem. 2011, 83, 2213.
23. A. R. Phull, M. Majid, I. U. Haq, M. R. Khan, S. J. Kim, Int.
J. Biol. Macromol. 2017, 97, 468.
1. X. Zhang, Y. Xiao, X. Qian, Angew. Chem. Int. Ed. 2008,
47, 8025.
2. (a) P. G. Mahajan, N. C. Dige, B. D. Vanjare, Y. Han,
S. J. Kim, S. K. Hong, K. H. Lee, Sensors Actuators B 2018,
267, 119. (b) P. G. Mahajan, N. C. Dige, B. D. Vanjare,
S. H. Eo, S. J. Kim, K. H. Lee, Spectrochimica Acta A: Mol.
Biomol. Spect. 2019, 216, 105.
3. P. Krawczyk, B. Jedrzejewska, M. Pietrzak, T. Janek,
J. Photochem. Photobiol. B 2016, 166, 74.
4. K. T. Wong, R. T. Chen, F. C. Fang, C. C. Wu, Y. T. Lin,
Org. Lett. 2005, 7, 1979.
24. A. R. Phull, S. H. Eo, S. J. Kim, Cell. Mol. Biol. 2017,
63, 12.
5. W. Z. Yuan, Y. Gong, S. Chen, X. Y. Shen, J. W. Y. Lam,
P. Lu, Y. Lu, Z. Wang, R. Hu, N. Xie, H. S. Kwok,
Y. Zhang, J. Z. Sun, B. Z. Tang, Chem. Mater. 2012,
24, 1518.
6. E. Pusztai, I. S. Toulokhonova, N. Temple, H. Albright,
U. I. Zakai, S. Guo, I. A. Guzei, R. Hu, R. West, Organome-
tallics 2013, 32, 2529.
25. Y. Xiang, L. Mei, N. Li, A. Tong, Anal. Chim. Acta 2007,
581, 132.
26. B. D. Vanjare, P. G. Mahajan, M. Hassan, H. Raza,
S. Y. Seo, S. K. Hong, K. H. Lee, J. Fluoresc. 2018,
28, 1295.
27. H. Kim, S. Wang, Y. A. Son, Textile Color Finish 2012,
24, 153.
7. P. S. Beaumont, D. G. Johnson, B. J. Parsons, J. Chem. Soc.
Faraday Trans. 1993, 89, 4185.
28. E. Bozkurt, H. I. Gul, E. Mete, J. Photochem. Photobio. A:
Chem. 2018, 352, 35.
8. J. Fan, M. Hu, P. Zhan, X. Peng, Chem. Soc. Rev. 2012,
42, 29.
29. J. Papadopoulos, K. Merkens, T. J. J. Meller, Chem. Eur. J.
2018, 24, 974.
9. P. Pal, H. Zeng, G. Durocher, D. Girand, T. Li, A. K. Gupta,
R. Giasson, L. Blanchard, L. Gaboury, A. Balassy,
C. Turmel, A. Laperriere, L. Villeneuve, Photochem. Photo-
biol. 1996, 63, 161.
10. M. Cizkova, L. Cattiaux, J. M. Mallet, E. Labbe, O. Buriez,
Electrochim. Acta 2018, 260, 589.
11. Z. Yang, M. She, J. Zhang, X. Chen, Y. Huang, H. Zhu,
P. Liu, J. Li, Z. Shi, Sensors Actuators B 2013, 176, 482.
12. Z. Xu, L. Zhang, R. Guo, T. Xiang, C. Wu, Z. Zheng,
F. Yang, Sensors Actuators B 2011, 156, 546.
13. Y. A. Son, J. Park, Textile Color Finish 2012, 24, 158.
14. J. Zhang, L. Zhang, Y. Zhou, T. Ma, J. Niu, Microchim. Acta
2014, 180, 211.
30. A. B. Ormond, H. S. Freeman, Dyes Pigments 2013, 96, 440.
31. S. Meng, Z. Xu, G. Hong, L. Zhao, Z. Zhao, J. Guo, H. Ji,
T. Liu, Eur. J. Med. Chem. 2015, 92, 35.
32. R. Payal, M. K. Saroj, N. Sharma, R. C. Rastogi, J. Lumin.
2018, 198, 92.
33. S. Ghosh, N. Roy, T. S. Singh, N. Chattopadhyay, Spe-
ctrochim. Acta A: Mol. Biomol. Spectro. 2018, 188, 252.
34. H. Homocianu, A. Airinei, D. O. Dorohoi, J. Adv. Res. Phys.
2011, 2, 1.
35. B. Varghese, S. N. Al-Busafi, F. O. Suliman, S. M. Z. Al-
Kindy, J. Lumin. 2015, 159, 9.
36. Z. Zhang, P. Xue, P. Gong, G. Zhang, J. Peng, R. Lu, Mater.
Chem. C 2014, 2, 9543.
Bull. Korean Chem. Soc. 2019
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
6