Novel Rhodamine B and 2H-benzo[b][1,4]oxazin-3(4H)-one-derived Fluorescent Sensor for Low pH Value Detection

Article abstract of DOI:10.1002/bkcs.11819

A new fluorescent probe L fusing the rhodamine B scaffold with 2H-benzo[b][1,4]oxazin-3(4H)-one moiety has been developed and applied as an acidic pH sensor. The ultraviolet (UV) and fluorescence spectra of the probe at different pH values were investigat

Full text of DOI:10.1002/bkcs.11819

Article
DOI: 10.1002/bkcs.11819
BULLETIN OF THE
M. Chen et al.
KOREAN CHEMICAL SOCIETY
Novel Rhodamine B and 2H-benzo[b][1,4]oxazin-3(4H)-one-derived
Fluorescent Sensor for Low pH Value Detection
†
‡
‡
§
‡,
¶,*
*
Mingjun Chen, Xueke Bai, Zhenping Qi, Shoubo Xiang, Hua Zuo, and Dong-Soo Shin
†
College of Teacher Education, Chongqing Three Gorges University, Chongqing 404000, China
‡
College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
*
E-mail: zuohua@swu.edu.cn
The West China College of Medicine, Sichuan University, Sichuan 610041, China
Department of Chemistry, Changwon National University, Changwon 641-773, South Korea.
E-mail: dsshin@changwon.ac.kr
Received February 18, 2019, Accepted May 25, 2019
§
¶
*
A new fluorescent probe L fusing the rhodamine B scaffold with 2H-benzo[b][1,4]oxazin-3(4H)-one moi-
ety has been developed and applied as an acidic pH sensor. The ultraviolet (UV) and fluorescence spectra
of the probe at different pH values were investigated in B–R buffer/MeOH (v: v = 3: 7). This probe
showed increase of both the UV–Vis absorption and fluorescence emission from alkaline/neutral to acidic
condition, reaching a strongest intensity at pH 2.13, but the intensities decreased when the pH decreased
to 1.83. The color variation along the pH changes proved that the probe L can be used as a low-cost opti-
cal sensor for the naked eye detection of proton in the present buffer.
Keywords: Rhodamine B, 2H-benzo[b][1,4]oxazin-3(4H)-one, Proton detection, Fluorescence, UV–Vis
absorption
Introduction
visible region, high quantum yields, large extinction coeffi-
cients and great photostability, herein, a new rhodamine
analogue was designed, synthesized and used for sensing
pH sensors are essentially required in the field of chemistry,
biology, and industry, such as chemical and biological
strong acidic condition according to rhodamine-based pro-
1,2
1,3
29,30
analyses,
environmental monitoring and protection,
bes for low pH value detection reported.
The new
4
and chemical process control. A variety of different
methods have been reported for pH sensing. Potentiometric
structure was constructed from rhodamine hydrazide and a
ketone with a 2H-benzo[b][1,4]oxazin-3(4H)-one moiety.
This probe exhibited high fluorescent intensity working
under strong acidic conditions with pH ranging from 4.00
to 1.83, which allows us to precisely calculate the pH value
of any given sample at pH 1.83–4.00.
3
5
methods and proton nuclear magnetic resonance (NMR)
allow the pH determination of typical samples with high
accuracy. Fluorescent analysis is recognized as a conve-
nient and highly sensitive technique for the detection of pH
values and has been proven to be one of the best methods
for the analysis of trace biological compounds in complex
Experimental Section
6
biological samples. Compared with other detection
methods, the noninvasive fluorescent probes have advan-
Chemicals and Apparatus. All chemicals and reagents
used in the present study were of analytical reagent
(AR) grade from commercial suppliers and used without
further purification. Deionized water was used throughout
the experiments. Britton–Robinson (B–R) buffer was a
mixture of 40 mM acetic acid, boric acid and phosphoric
acid. Dilute hydrochloric acid or sodium hydroxide was
used for tuning pH values.
tages such as high selectivity and the ability for real-time
7,8
detection. There are numerous classes of fluorescent sen-
9
,10
sors.
The newly developed fluorescent pH chemosensors
1
1
12
14,15
4
include aminophthalimide, cyanine, aminophthalimide,
benzoxanthenes, BODIPY dyes,
derivatives.
Rhodamine is a promising scaffold for the design of
reversible and selective probes and its derivatives have
13
fluorescein and it
16,17
1
8
Melting point (uncorrected) was determined on a micro
melting point apparatus (Shanghai Shenguang Instrument
19–24
been widely used as “turn-on” fluorescence probes.
1
13
The detection toward specific chemical species is based on
the equilibrium between spirolactam form (nonfluorescent)
Co., Ltd., Shanghai, China). H and C NMR (nuclear
magnetic resonance) spectra (at 400 and 100 MHz, respec-
25–28
and ring-open amide structure (highly fluorescent).
tively) were recorded in CDCl with tetramethylsilane as
3
Due to their excellent photophysical properties including
relatively long absorption and emission wavelengths in the
internal reference on a Bruker Advance 500 FT spectrome-
ter (Billerica, MA, USA). Chemical shifts were reported in
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parts per million. Mass spectra (MS) were measured by the
electrospray ionization (ESI) method on an Agilent 6510
NMR (400 MHz, CDCl ) δ: 1.14 (t, J = 7.1 Hz, 12H), 2.19
3
(s, 3H), 2.81 (t, J = 6.9 Hz, 2H), 3.32 (q, J = 7.2 Hz, 8H),
3.59 (t, J = 6.6 Hz, 2H), 4.49 (s, 2H), 6.29 (dd, J = 8.9,
2.6 Hz, 2H)，6.40 (m, 2H), 6.58 (d, J = 8.8 Hz, 2H), 6.78
(dd, J = 7.9, 1.6 Hz, 1H), 6.89 (t, J = 7.7 Hz, 1H), 7.05 (d,
J = 8.0 Hz, 2H), 7.15 (m, 1H), 7.21 (dd, J = 5.8, 2.9 Hz,
Q-TOF mass spectrometer. CDCl delivered from Adamas
3
Co., Ltd. (Shanghai, China) was used. Silica gel (200–300
mesh) was used for flash column chromatography. All reac-
tions were monitored by thin-layer chromatography (TLC)
using 0.25 mm silica gel plates with a UV light indicator
1
3
H), 7.30 (dd, J = 8.0, 1.6 Hz, 1H), 7.49 (dd, J = 5.6,
.2 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.94 (m, 1H) ppm;
(
Shanghai Jiapeng Technology Co., Ltd., Shanghai, China).
UV–Vis spectra were recorded on a UV-2550 spectrometry
Hitachi, Tokyo, Japan). Fluorescence was measured on an
1
3
C NMR (100 MHz, CDCl ) δ: 12.64, 18.23, 35.26,
3
39.88, 44.33, 66.96, 67.92, 97.95, 106.88, 107.83, 113.56,
122.82, 122.86, 122.98, 124.03, 127.68, 127.99, 128.19,
128.28, 128.47, 130.52, 131.24, 132.23, 136.30, 140.31,
148.68, 151.44, 152.60, 153.76, 160.51, 167.55, 168.59 ppm.
(
F-4500 FL spectrophotometer (Hitachi). The pH measure-
ments were performed on a PHS-3C digital pH meter
(Mettler Toledo, Greifensee, Switzerland).
Synthesis of Probe L. The synthetic route for probe L is
shown in Scheme 1. 2-Phenyl ethylamine (3) (13 mmol,
Result and Discussions
1.0 equiv) was first acylated by chloroacetyl chloride
(
15.5 mmol, 1.2 equiv) assisted by K CO (19 mmol, 1.5
2
3
Synthesis of Probe L. As depicted in Scheme 1, the acety-
lation reaction of 4-phenethyl-2H-benzo[b] [1, 4] oxazin-3
equiv), and then reacted with o-chlorophenol (10.4 mmol,
.8 equiv) and NaH (12.5 mmol, 1.2 equiv) to give
cyclized product 4-phenethyl-2H-benzo[b][1,4]oxazin-3
0
(4H)-one (4) with acetic anhydride afforded the 6-acetyl-
4
-phenethyl-2H-benzo[b][1,4]oxazin-3(4H)-one (5). Its
3
1
(
4H)-one (4) via Smiles rearrangement. The acetylation
condensation reaction with rhodamine hydrazide 2 generated
of 2H-benzo[b][1,4]oxazin-3(4H)-one 4 (0.56 mmol, 1.0
equiv) with acetic anhydride (0.56 mmol, 1.0 equiv) and
AlCl (2.8 mmol, 5.0 equiv) gave 6-acetyl-4-phenethyl-2H-
benzo[b][1,4]oxazin-3(4H)-one (5), which was further con-
densed with rhodamine hydrazide 2 (0.56 mmol, 1.0
1
the probe L, which was further characterized by H NMR
13
and C NMR. The chemical shift for the protons of
3
CH CH , CH O and CH from 6-acetyl-4-phenethyl-2H-
2
2
2
3
benzo[b][1,4]oxazin-3(4H)-one (5) appeared at δ 2.81,
1
3
.59, 4.49 and 2.19 in the H NMR spectrum of probe
32
equiv) in refluxing ethanol to afford the probe L. The
reaction was monitored by TLC. When the reaction was
completed, the solvent was removed under vacuum and to
the residue was added water. The crude product was
extracted by ethyl acetate for three times. The combined
organic layer was dried and filtered, and the filtrate was
evaporated to give the crude product. It was further purified
through column chromatography on silica gel (200–300
L (Figure S2, Supporting Information), and the new peaks
at δ 160.5–168.6 from the two carbonyl groups and C N
in C NMR spectrum (Figure S3) confirmed the fusion of
rhodamine hydrazide 2 and 6-acetyl-4-phenethyl-2H-
benzo[b][1,4]oxazin-3(4H)-one (5) and the formation of
probe L.
1
3
1
Spectroscopic Properties and Optical Responses to pH.
After pH value slightly decreased from 11.04 to 4.00, the
fluorescence emission of probe L exhibited a faint intensity
and remained unchanged. However, it began to dramati-
cally rise under strongly acidic condition (pH < 4.0). The
probe at pH 2.13 induced the strongest fluorescence
enhancement at 584 nm, with the peak intensity arising at
nearly 3000 (Figure 1). We also found that the probe did
not give fluorescence in alkaline and neutral conditions, but
gave strong pink fluorescence under acidic conditions at
mesh) to generate probe L as a solid in 60% yield. H
Synthesis of probe L
3
65 nm (Figure 1).
Although the fluorescence intensity sharply increased
from pH 4.00 to 2.13, a dramatic decrease was observed at
83 nm when pH value decreased from 2.13 to 1.83
5
according to the fluorescent pH titration (pH 1.83–11.04,
λex = 560 nm, Figure 2). The relationship is in good accor-
dance with the Hendersone–Hasselbach-type mass action
equation pH = pKa + log[(Imax – I)/(I – Imin)], which allows
us to precisely calculate the pH value of any given sample
with pH ranging from 4.00 to 1.83. We therefore, assumed
that probe L might be an ideal probe for sensing proton by
fluorescence spectra.
Scheme 1. Synthesis of probe L. Reagents and conditions:
(
a) K
c) CH
2
CO
Cl
3
/CH
2
Cl
2
, r.t., 92%; (b) DMF/NaH, reflux, 89%;
ꢀ
(
2
2
, 0 C to reflux, 75%; (d) EtOH, reflux, 60%.
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BULLETIN OF THE
Article
A Novel Rhodamine B and 2H-benzo[b][1,4]oxazin-3(4H)-one-derived
Fluorescence Sensor for Low pH Values
KOREAN CHEMICAL SOCIETY
Figure 1. The fluorescent spectrum of probe L (20 μM) in B–R
Figure 3. The color change of probe L (20 μM, B–R buffer-
EtOH, v: v = 3: 7) at different pH.
buffer/MeOH (v: v = 3: 7) at different pH (excitation wavelength:
λ
ex = 560 nm). The inset showed the fluorescence color of L at
pH 2.13 (left) and 6.97 (right).
Figure 2. Fluorescence intensity at 584 nm for probe L (20 μM)
at different pH, λex = 560 nm.
Figure 4. The UV absorption spectrum of probe L (20 μM) in B–
R buffer/methanol (v: v = 3: 7) at different pH.
The probe L existed in conjugated form in the low pH
value and showed good color variation in the detection
buffer. The results showed that we can detect proton by
naked eye using the changes in color from colorless to
pink, and light pink when pH decreased from high to low,
and that the intensity of the pink color increases or
reached the maximum within 1 min, and remained constant
within 30 min (Figure 5), which indicated that the probe
L responded to low pH value very fast and the complex
thus formed possessed perfect stability.
+
decreases depending on the concentration of H (Figure 3).
Conclusion
It acts as a low-cost optical sensor for the naked eye detec-
tion of proton in the present buffer.
In summary, we have developed a novel fluorescent pH
probe L based on rhodamine B benzoxazinone derivatives
with high sensitivity and excellent stability to extremely
acidic conditions. The color variation along the pH changes
proved that the probe L can be used as a low-cost optical
sensor for the naked eye detection of proton in the present
buffer. Considering that rhodamine fluorescent probe has
the characteristics of low toxicity and good water solubility
and it is widely used in the biosensing, the acid fluorescent
probe is expected to be applied in the organism.
In agreement with the tendency in fluorescence intensity,
the absorption intensity increased significantly upon the
+
addition of H to the alkaline/neutral solution of probe L,
and then decreased when the pH value was lower than
2.13. An obvious absorption peak appeared at 560 nm, and
the strongest peak UV absorption reached when pH value
was at 2.13 (Figure 4).
The Effect of Time and pH Value on the Probe L. The
fluorescence intensity of the probe L (20 μM) at pH 2.13
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BULLETIN OF THE
Article
A Novel Rhodamine B and 2H-benzo[b][1,4]oxazin-3(4H)-one-derived
Fluorescence Sensor for Low pH Values
KOREAN CHEMICAL SOCIETY
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