A general strategy to increase emission shift of two-photon ratiometric pH probes using a reversible intramolecular reaction of spiro-oxazolidine

Article abstract of DOI:10.1016/j.saa.2020.119035

Fluorescent pH probes have been served as powerful tools in biological and pathological studies in recent years due to the important roles of pH values in various physiological processes. Although plenty of pH probes have been delivered, development of two-photon ratiometric pH probes with large emission shift for detecting the variation of intracellular pH values is still a greatly challenging task. To address this concern, in this work, we have discovered a general strategy designing pH probes by means of a pH-dependent reversible intramolecular reaction of spiro-oxazolidine which can efficiently change their conjugation length and the electronic effect concurrently. To display the generality of the strategy, we have synthesized six pH probes, and all these probes exhibit short emission in basic conditions and dramatically red-shifted emission in acid environments. The emission shift of the six probes is more than 150 nm and even up to 210 nm, much larger than shift of all commercial and reported pH probes. The chemical sensing mechanism of intramolecular ring opening/closing reaction of spiro-oxazolidine has been confirmed with ¹H NMR spectra and density functional theory (DFT) calculations. Finally, we have used one of six with one- and two-photon properties to successfully image lysosomal pH changes under confocal and two-photon microscopes in a ratiometric manner. We believed that this spiro-oxazolidine strategy can serve as a general and powerful platform for the design of ideal pH probes.

Full text of DOI:10.1016/j.saa.2020.119035

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119035
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A general strategy to increase emission shift of two-photon ratiometric
pH probes using a reversible intramolecular reaction of spiro-oxazolidine
a
a
a
b,
a,
Huamiao Zhang , Minggang Tian , Rui Yang , Xiuquan He ⁎, Xiaoqiang Yu ⁎
a
Center of Bio & Micro/Nano Functional Materials, State Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, PR China
Department of Anatomy and Histoembryology, School of Basic Medical Sciences, Shandong University, Jinan 250012, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2020
Received in revised form 28 September 2020
Accepted 28 September 2020
Available online 04 October 2020
Fluorescent pH probes have been served as powerful tools in biological and pathological studies in recent years
due to the important roles of pH values in various physiological processes. Although plenty of pH probes have
been delivered, development of two-photon ratiometric pH probes with large emission shift for detecting the
variation of intracellular pH values is still a greatly challenging task. To address this concern, in this work, we
have discovered a general strategy designing pH probes by means of a pH-dependent reversible intramolecular
reaction of spiro-oxazolidine which can efficiently change their conjugation length and the electronic effect con-
currently. To display the generality of the strategy, we have synthesized six pH probes, and all these probes ex-
hibit short emission in basic conditions and dramatically red-shifted emission in acid environments. The
emission shift of the six probes is more than 150 nm and even up to 210 nm, much larger than shift of all com-
mercial and reported pH probes. The chemical sensing mechanism of intramolecular ring opening/closing reac-
Keywords:
Fluorescent probe
pH value
Ratiometric
Cyclization reaction
1
tion of spiro-oxazolidine has been confirmed with H NMR spectra and density functional theory (DFT)
calculations. Finally, we have used one of six with one- and two-photon properties to successfully image lyso-
somal pH changes under confocal and two-photon microscopes in a ratiometric manner. We believed that this
spiro-oxazolidine strategy can serve as a general and powerful platform for the design of ideal pH probes.
©
2020 Elsevier B.V. All rights reserved.
1
. Introduction
time visualization in living cells and tissues [11,12]. Amongst, fluores-
cent ratiometric probes have gained particular interest in recent years,
due to their superior advantages in bioimaging applications [13,14]. By
means of a ratiometric probe, fluorescent images with a pH scale plate
can be obtained via calculation of the fluorescent intensity ratio in the
two channels by pixels. Through this method, the errors resulted from
inhomogenous staining, fluctuation of laser power, and many other fac-
tors can be efficiently avoided [15]. Due to the importance, ratiometric
pH probes have been developed with different mechanisms, including
Fӧrster resonance energy transfer (FRET) and intramolecular charge
transfer (ICT) mechanisms [16–20]. For example, Tian and co-workers
have developed a probe for lysosomal pH values based on FRET mecha-
nism, by linking pH-inert coumarin to a pH-sensitive green emission
fluorophore [16]. Ma's group has presented a ratiometric pH probe
based on ICT mechanism with a phenolic hydroxyl group as the recog-
nition group [17]. In basic conditions, the hydroxyl group could donate
a proton and product phenolate with enhanced electron-donating prop-
erty, and result in red shift of the emission spectra. Our group has repre-
sented a ratiometric pH probe with an emission shift of 84 nm that
contained a pyridine unit and a carbazole core. As the protonation of
the pyridine N atom, its pH-dependent absorption and fluorescence
properties were shown with a pKa of 5.47 matching the pH range of in-
tracellular acidic compartments.
All the biological substances including various ions, amino acids, nu-
cleotides, lipids, proteins, and nucleic acids are drifted in an aqueous en-
vironment with certain pH value. Consequently, intracellular pH value is
a key parameter that would directly affect various biological activities
[
1–3], such as enzymatic reaction, maintaining mitochondrial mem-
brane potential, and tuning protein conformations. As a result, minor
deviation of cytoplasm or organelle pH values would greatly influence
numerous biological functions [4,5]. Particularly, many diseases are
closely related to the abnormal of cellular pH values, including neuronal
ceroid lipofuscinoses, Alzheimer's disease, and even cancer [6–8].
Therefore, in situ and real-time observation of intracellular pH values
is an important task in biological and pathological researches.
At present, various methods including pH meter and colorimetric pH
indicators could be used to monitor environmental pH values [9,10].
Compared with these methods, fluorescent imaging method assisted
with pH sensitive probes displays great advantages, including ignorable
injury to biosamples, facile operation, and capability of in situ and real-
⁎
Corresponding authors.
E-mail addresses: hxq3000@sdu.edu.cn (X. He), yuxq@sdu.edu.cn, zqliu@sdu.edu.cn
(X. Yu).
https://doi.org/10.1016/j.saa.2020.119035
386-1425/© 2020 Elsevier B.V. All rights reserved.
1

H. Zhang, M. Tian, R. Yang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119035
Although plenty of elegant works have been devoted to develop
ratiometric pH probes, shortcomings are still existed. One of the serious
defects is that the shift of emission spectra is modest (no larger than
[23]. This intramolecular reaction can cause large changes in their ab-
sorption properties and they have recently been used in water-jet
rewritable paper [24]. Very importantly, the intramolecular reactions
in these spiro-oxazolidine compounds were also reported to be affected
by environmental acidity [25], but have rarely been applied as fluores-
cent pH probes. Thus, it is very possible that the intramolecular reaction
of a fluorophore with well-designed spiro-oxazolidine structure will
bring dramatic changes to the compound in both the conjugation length
and the electronic effect simultaneously. According to the literatures
above, the intramolecular reaction of spiro-oxazolidine could be in-
duced by light irradiation in solid state, organic solvents, or low-
temperature environments. Most recently, we have found that in
aqueous solutions at room temperature, the ratio of ring opening/
closing states some spiro-oxazolidine fluorophores is determined
only by environmental pH values (Scheme 1b), and not by light
irradiation, which has been strictly confirmed in the following text.
Consequently, in this work, we have presented a general strategy
to increase the emission shift of ratiometric pH probes by means of
the unique pH-dependent intramolecular reaction of spiro-
oxazolidine, and constructed six probes. Due to that the ring open-
ing/closing states from such a reaction could simultaneously alter
the conjugation length and the electronic effect, all the six probes
display extremely large emission shift (150–210 nm) upon the
variation of intracellular pH values. To our best knowledge, no
ratiometric pH probes with emission shift up to 210 nm have been
reported yet. Finally, probe 3-C was selected to image lysosomal pH
variation in living cells under one- and two-photon microscope in a
ratiometric manner.
130 nm), which may bring limitations in ratiometric cell imaging appli-
cations. To obtain reliable ratiometric images, large spectra separation
of fluorescence signals in two channels is essential. Thus, development
of novel fluorescent pH probes with large emission shift is an essential
and urgent task for the relative applications.
The extent of emission shift is intrinsically limited by the current de-
sign strategies of probes. Up to now, ratiometric pH probes were gener-
ally designed based on FRET and ICT mechanisms, as shown in Scheme
1a. Due to the prerequisite of spectra overlap between donor emission
and acceptor absorption for FRET probes, the emission shift is limited
by the Stokes shift of the acceptor fluorogens. On the other hand, ICT
probes also display modest emission shift, because the protonation or
deprotonation of the recognition group can only change the electronic
effect, which could bring limited spectra change. In fact, the most direct
method to shift the emission spectra of a fluorophore is to change its
conjugation length. For example, in cyanine dyes frequently used in
fluorescence imaging applications, Cy3 exhibits yellow emission peaked
at 570 nm [21], while Cy7 with two additional double bonds displays
near-infrared emission peaked 767 nm [22], the emission separation is
as high as 197 nm. Therefore, it should be the best strategy to develop
ratiometric pH probes with large emission shift based on a pH-
dependent and only pH-dependent reaction that can alter the conjuga-
tion length.
To realize the idea above, we noticed a spiro-oxazolidine compound
in which an intramolecular ring opening/closing reaction can take place
Scheme 1. The sensing mechanism based on FRET and ICT (a); the sensing mechanism based on intramolecular reaction of spiro-oxazolidine (b); chemical structures of the six pH probes
designed based on new mechanism.
2

H. Zhang, M. Tian, R. Yang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119035
2
. Experimental section
yellow powder, which could be obtained after filtered. The crude
product was recrystallized in methanol to obtain final product as
2.1. Materials
yellow powder, in 67% yield. 1H NMR (400 MHz, DMSO‑d
6
), δ
ppm): 8.35 (s, 1H), 8.18 (d, J = 8 Hz, 1H), 7.67–7.57 (m, 3H), 7.44
t, J = 7.6 Hz, 1H), 7.22–6.86 (m, 6H), 6.35 (d, J = 15.6 Hz, 1H),
(
(
4
All chemicals used are in analytical grade, salts, HCl, NaOH, NH Cl,
NaHS, 1-pyrenecarboxaldehyde, 2,3,3-trimethylindolenine, 2-
indoethanol, and pyrrolidine were purchased from J&K Chemical Co.
Ltd. GSH, amino acids and PBS were purchased from Seikagaku Corpora-
tion (Japan). The solvents used in the spectral measurement are all in
chromatographic grade.
4
3
.55 (t, J = 10.4 Hz, 2H), 3.75–3.33 (m, 8H), 1.41 (s, 3H), 1.13 (s,
H), 0.96 (t, J = 7 Hz, 3H). 13C NMR (100 MHz, DMSO‑d
), δ
6
(
ppm) = 151.33, 141.14, 140.61, 139.79, 132.68, 127.85, 127.79,
1
1
26.20, 125.16, 123.08, 122.82, 122.65, 121.54, 120.79, 119.41,
19.24, 112.43, 110.15, 110.09, 68.61, 66.09, 63.43, 50.11, 47.69,
−
1
2.2. Instrumentations
43.19, 28.79, 20.75, 15.46. IR (cm ): 3047.6, 2963.0, 2928.8,
2
1
1
8
868.5, 1647.3, 1626.7, 1598.0, 1575.1, 1489.1, 1461.3, 1435.6,
381.8, 1363.7, 1351.8, 1324.2, 1294.3, 1252.5, 1215.7, 1169.8,
148.4, 1111.7, 1064.9, 1041.5, 1019.1, 1003.2, 979.3, 944.3, 870.5,
The UV–visible-near-IR absorption spectra of dilute solutions were
recorded on a U2910 spectrophotometer using a quartz cuvette having
cm path length. IR spectra were measured on Nexus 670. One-photon
fluorescence spectra of dilute solutions were obtained on a HITACH F-
700 spectrofluorimeter equipped with a 450-W Xe lamp. Two-
1
+
05.8, 768.6, 740.9, 729.8, 720.9, 684.9. (HRMS, m/z): [M+H]
+
calcd. for C30
H
33
N
2
O
2
, 453.2537; found, 453.2583.
2
photon fluorescence was measured on a SpectraPro300 and the pump
laser beam came from a mode-locked Ti:sapphire laser system with
the pulse duration of 200 fs and the repetition rate of 76 MHz (Coherent
Mira900-D). Nuclear magnetic resonance spectra ( H and C) were ob-
tained on a Bruker Avanace 400 spectrometer. The following abbrevia-
tions were used to explain the multiplicities: s = singlet; d =
doublet; t = triplet; m = multiplet. The HRMS spectra were recorded
on Agilent Technologies 6510 Q-TOF LC/MS.
Probe 2-C, 3-C, 4-C, 5-C and 6-C were synthesized with the same
steps to 1-C.
1
13
Probe 2-C ((E)-10a-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)-11,11-di-
methyl-8,9,10a,11-tetrahydrobenzo[e]oxazolo[3,2-a]indole) in 61%
1
yield. H NMR (400 MHz, DMSO‑d
6
), δ (ppm): 8.39 (s, 1H), 8.20 (d,
J = 7.6 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H),
7
7
.80 (d, J = 8.8 Hz, 1H), 7.72–7.70 (m, 1H), 7.62–7.58 (m, 2H),
.48–7.44 (m, 2H), 7.33–7.28 (m, 2H), 7.23–7.19 (m, 1H), 7.02 (d,
2.3. Methods
J = 16.0 Hz, 1H), 6.45 (d, J = 16.0 Hz, 1H), 4.45 (t, J = 14.0 Hz,
2H), 3.96 (s, 1H), 3.80–3.78 (m, 1H), 3.53–3.45 (m, 2H), 1.75 (s,
The geometries of the acid and basic forms of the probe molecule
were optimized at the B3LYP/6-31G level using Gaussian 03 program
package. The HOMO and LUMO of the acid and basic forms were then
obtained by single-point calculation, which was performed at B3LYP/
13
3H), 1.35–1.29 (m, 6H).
6
C NMR (100 MHz, DMSO‑d ), δ
(ppm) = 148.49, 140.45, 139.85, 133.07, 130.63, 130.22, 129.69,
129.48, 129.21, 127.78, 126.79, 126.33, 125.34, 122.91, 122.77,
6
-311g (2d, p) level.
1
6
1
1
1
8
22.22, 121.00, 119.48, 119.35, 114.83, 110.49, 109.71, 109.66,
3.32, 50.07, 49.24, 37.50, 26.73, 21.80, 14.14. IR (cm ): 2965.2,
2
.4. Cell culture and staining
−1
649.3, 1622.4, 1591.7, 1518.0, 1490.6, 1471.8, 1458.9, 1439.0,
369.4, 1344.6, 1323.1, 1277.5, 1233.2, 1207.6, 1169.3, 1145.9,
123.9, 1112.7, 1085.8, 1044.4, 1010.6, 974.8, 954.2, 940.6, 913.6,
81.7, 859.7, 819.3, 800.0, 787.5, 752.3, 743.1, 730.7, 705.9. HRMS
PBS buffer solutions were initially prepared with pH value of 7.4. A
series of buffer solutions with different pH values was then tuned
with 1.0 mol/L of HCl and NaOH solutions adjusted by a pH meter. For
reversibility studies, the pH values were tuned repeatedly with drops
of 1.0 mol/L of HCl and NaOH solutions, adjusted with the pH meter.
HeLa cells were grown in H-DMEM (Dulbecco's Modified Eagle's
+
+
31 2
(m/z): [M+H] calcd. for C32H N O , 459.2431; found, 459.2419.
Probe
3-C
(9,9-dimethyl-9a-(2-(pyren-4-yl)vinyl)-2,3,9,9a-
1
Medium, High Glucose) supplemented with 10% FBS (Fetal Bovine
Serum) in a 5% CO
tetrahydrooxazolo[3,2-a]indole) in 45% yield. H NMR (400 MHz,
DMSO‑d ), δ (ppm): 8.44–8.06 (m, 9H), 7.88 (d, J = 15.6 Hz, 1H),
5
2
incubator at 37 °C. Cells (1 × 10 /mL) were placed
6
on glass coverslips and allowed to adhere for 24 h. For living cells imag-
ing experiment of the probes, the culture medium surrounding the cells
were firstly removed, and the cells were washed with PBS twice. The
cells were the incubated with 5 μM of the probe in 1 mL PBS for
7
1
.19–6.90 (m, 4H), 6.60 (d, J = 16.0 Hz, 1H), 3.90–3.52 (m, 4H),
.49 (s, 3H), 1.21 (s, 3H). C NMR (100 MHz, DMSO‑d ), δ
6
13
(
ppm) = 151.24, 139.69, 131.47, 131.16, 130.95, 130.86, 130.26,
1
1
1
28.73, 128.39, 128.06, 127.96, 127.88, 127.79, 126.85, 125.96,
25.72, 125.68, 124.85, 124.85, 124.60, 124.51, 123.28, 122.72,
21.72, 112.54, 110.08, 63.73, 55.37, 50.30, 47.85, 28.79, 20.87. IR
2
0 min. Cells were imaged immediately after rinsing with PBS three
times.
−
1
(
1
cm ): 3046.7, 3038.7, 2990.1, 2958.6, 2936.1, 2881.9, 2865.4,
599.4, 1590.9, 1475.1, 1453.5, 1438.9, 1336.3, 1312.1, 1293.9,
2.5. Probe synthesis
The synthesize procedure of compound 1–6 was presented in the
1280.9, 1270.1, 1237.7, 1208.3, 1188.0, 1180.5, 1150.4, 1112.3,
Supporting information.
1063.8, 1045.7, 1019.9, 1008.0, 971.5, 931.2, 840.3, 819.2, 754.9,
+
7
49.3, 714.7, 689.5, 679.8. HRMS (m/z): [M+H] calcd. for
Synthesis of (E)-9a-(2-(9-(2-ethoxyethyl)-9H-carbazol-3-yl)vinyl)-
C
30
H
26NO
+
, 416.2014; found, 416.2227.
((E)-11,11-dimethyl-10a-(2-(pyren-1-yl)vinyl)-
9
,9-dimethyl-2,3,9,9a-tetrahydrooxazolo[3,2-a]indole (1-C). 0.5 g
1.5 mmol) of compound 1 was firstly added into a 50 mL flask,
and then 0.4 g (1.5 mmol) of compound 5 was introduced. 5 mL
1.5 mmol) of ethanol was added and the system was stirred for
min. After that, 200 μL of pyrrolidine was added into the above
Probe
4-C
(
8,9,10a,11-tetrahydrobenzo[e]oxazolo[3,2-a]indole) in 65% yield.
1
6
H NMR (400 MHz, DMSO‑d ), δ (ppm): 8.54–8.41 (m, 2H),
(
5
8.34–8.27 (m, 4H), 8.21 (s, 2H), 8.12–8.04 (m, 2H), 7.95–7.82 (m,
2H), 7.84 (d, J = 8.4 Hz, 1H), 7.48 (t, J = 8.4 Hz, 1H), 7.38–7.30
(m, 2H), 6.71 (d, J = 15.6 Hz, 1H), 4.15–3.91 (m, 2H), 3.68–3.53
mixture, and the resulted system was stirred for 14 h at room tem-
perature to complete the reaction. Crude product participated as
(m, 2H), 1.82 (s, 3H), 1.41 (s, 3H). 13C NMR (100 MHz, DMSO‑d
6
),
3

H. Zhang, M. Tian, R. Yang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119035
δ (ppm) = 148.41, 131.47, 131.15, 130.98, 130.86, 130.71, 130.19,
basic emission. The emission shifts of 3-C and 4-C were 150 nm and
1
55 nm respectively. This is because they have the same fluorophore
1
1
1
2
1
1
4
30.05, 129.73, 129.39, 129.33, 129.07, 128.41, 128.09, 127.89,
27.82, 126.87, 125.98, 125.75, 125.70, 124.88, 124.60, 124.51,
23.31, 123.02, 122.24, 114.88, 110.46, 63.61, 50.22, 49.40, 26.71,
at basic condition and have the same basic emission, while 4-C have lon-
ger acidic emission because of the additional benzene ring. Similarly,
emission shift of 5-C was smaller than emission shift of 6-C. The emis-
sion shift of 5-C and 6-C were 200 nm and 210 nm, respectively. To
clearly and quantitatively understand the results, the absorption and
emission peaks of the six probes in acid and basic conditions have
been listed in Table 1. The six probes all show short-wavelength absorp-
tion and emission in basic conditions. In comparison, the absorption and
emission of these probes dramatically red-shifted to long wavelength in
acid environments. Notably, the emission shifts in two conditions of the
six probes are beyond 150 nm, and even up to 210 nm. An important
fact that should be indicated is that the change range of pH resulting
in the extremely large emission shift is just suitable for one of intracel-
lular pH. As known well, pH values of lysosomes, intracellular important
acidic compartments, are 4.0–6.0, and pH values of mitochondria, intra-
cellular important basic organelles, are 7.8–8.0 pH. These results prove
that it is an advanced strategy to design ratiometric pH probes based
on this intramolecular reaction of spiro-oxazolidine.
−1
1.89. IR (cm ): 3044.9, 2955.4, 1621.2, 1590.9, 1517.0, 1459.0,
438.5, 1369.7, 1350.7, 1311.9, 1208.9, 1178.1, 1146.7, 1123.7,
053.1, 1012.7, 978.1. HRMS (m/z): [M+H]+ calcd. for C34
H
28NO ,
+
66.2165; found, 466.2274.
Probe 5-C ((E)-4-(2-(9,9-dimethyl-2,3-dihydrooxazolo[3,2-a]indol-
1
9
a(9H)-yl)vinyl)-N,N-diphenylaniline) in 51% yield. H NMR
(
400 MHz, DMSO‑d
J = 8.0 Hz, 4H), 7.14–7.01 (m, 8H), 6.94 (d, J = 8.4, 2H), 6.89–6.85
m, 2H), 6.73 (d, J = 16.0 Hz, 1H), 6.22 (d, J = 15.6 Hz, 1H),
.71–3.67 (m, 2H), 3.51–3.32 (m, 4H), 1.36 (s, 3H), 1.06 (s, 3H).
6
), δ (ppm): 7.46 (d, J = 8.4 Hz, 2H), 7.31 (t,
(
3
13
6
C NMR (75 MHz, DMSO‑d ), δ (ppm) = 151.22, 147.49, 147.41,
1
1
2
1
1
1
39.68, 131.31, 131.01, 130.00, 128.39, 127.84, 124.73, 124.45,
23.71, 123.62, 122.62, 121.57, 112.40, 109.87, 63.42, 50.07, 47.67,
8.67, 20.71. IR (cm−1): 3036.3, 2958.0, 2879.5, 1649.6, 1592.5,
511.4, 1487.3, 1454.4, 1425.6, 1383.2, 1336.2, 1290.6, 1273.2,
206.1, 1177.2, 1152.0, 1111.5, 1076.9, 1062.2, 1038.8, 1020.1,
3
.3. The interferences from photo-induced ring opening reactions are
003.2, 988.6, 970.1, 839.1, 810.9, 767.4, 756.0, 746.6, 717.5, 698.6.
negligible
+
HRMS (m/z): [M+H]+ calcd. for C32
59.2455.
H
31
N
2
O , 459.2431; found,
4
According to literatures, the certain spiro-oxazolidine compounds
Probe 6-C ((E)-4-(2-(9,9-dimethyl-2,3-dihydrooxazolo[3,2-a]indol-
was reported to be able to occur photo-induced ring opening reactions
in solids, pure organic solvents, or under low temperature. To confirm
that the interferences from such photo-induced reactions in solutions
is negligible, the fluorescence spectra of 3-C solutions in mixed solvents
of EtOH/PBS (pH = 7.4, V/V = 1:1) were acquired at room temperature
with ceaseless light irradiation. As shown in Fig. S7, with the continuous
irradiation of 405 nm, the fluorescence intensity at 611 nm (ring open-
ing form) and 461 nm (ring closing form), as well as the intensity ratio
between the two wavelengths displayed little change in 60 min. Conse-
quently, in aqueous solutions at room temperature, photo-induced ring
opening reactions bring ignorable interferences.
1
9
a(9H)-yl)vinyl)-N,N-diphenylaniline) in 65% yield. H NMR
(
400 MHz, DMSO‑d
J = 8.0 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.51–7.43 (m, 3H),
.34–7.27 (m, 6H), 7.08–7.02 (m, 6H), 6.95 (d, J = 8.4, 2H), 6.79
d, J = 16.0 Hz, 1H), 6.31 (d, J = 16.0 Hz, 1H), 3.93–3.91 (m, 1H),
6
), δ (ppm): 7.99 (d, J = 8.4 Hz, 1H), 7.86 (d,
7
(
3
2
1
1
6
5
.50–3.38 (m, 2H), 1.70 (s, 3H), 1.27 (s, 3H). IR (cm−1): 2964.1,
881.4, 1623.3, 1591.4, 1510.4, 1492.9, 1463.6, 1384.4, 1369.8,
329.6, 1286.7, 1207.2, 1175.1, 1147.1, 1123.7, 1111.4, 1045.7,
025.0, 1004.7, 985.6, 945.3, 929.6, 840.4, 815.1, 756.9, 722.0,
+
+
99.0, 659.8. HRMS (m/z): [M+H] calcd. for C36
H
33
N
2
O ,
09.2587; found, 509.2626.
3.4. Mechanistic study of the acid-triggered ring-opening reaction
3
. Results and discussion
.1. Design and synthesis of the probes
In this work, we have designed the ratiometric pH probes with the
The acidichromism of spiro-oxazolidine derivatives are believed to
be resulted from the reversible cleavage/forming of C–O bond at the
junction of the indole and oxazolidine cycles.
3
(
Scheme 2). The absorption spectra of 3-C in the two conditions
Fig. S8) supported this statement. In Fig. S8, 3-C showed no absorption
at 500 nm in DMSO and low absorption peak at 360 nm in DMSO/
CF COOH, indicating that it was in basic and acid form in DMSO and
DMSO/CF COOH, respectively. In order to elucidate and conform the
mechanisms, H NMR spectra of 3-C in DMSO‑d
CF COOD were recorded and dramatic differences in the distribution
of the chemical shifts can be observed (Figs. 1 and S9).
N-hydroxylethyl-indolenine as the recognition group. As shown in
Scheme 1c, to achieve one-photon and two-photon ratiometric re-
sponses simultaneously and display the generality of the strategy, six
probes were designed by means of carbazole, pyrene, and
triphenylamine [26–28], which were frequently used to construct
one-photon, particular two-photon fluorescent dyes.
(
3
3
1
6
6
and DMSO‑d /
3
The six probes were synthesized following the synthetic routine in
Scheme S1. The experimental details were displayed in the Experimen-
1
Firstly, as shown in H NMR of 3-C in DMSO (Fig. 1a, above), there
are two sharp singlet peaks appear at δ 1.49 and 1.21 ppm, which can
1
13
tal section, and the characterization including H NMR spectra, C NMR
spectra, HR MS, and IR spectra were shown in Supporting Information.
Table 1
The absorption and emission peaks of the six probes in acid and basic conditions.
3.2. Emission shift of the six probes
Probe
Basic
absorption
Acid
absorption
Basic
emission
Acid
emission
Emission
shift
In order to testify whether the six probes can detect pH values, the
absorption and fluorescence spectra of these probes at basic and acid
conditions were firstly obtained, as shown in Figs. S1–S6. In Fig. S1,
probe 1-C displays two absorption peaks at 340 nm and 490 nm when
the pH value is 8.0, and the absorbance at 490 nm increases evidently
when the pH value changes to 4.0. At the same time, 1-C exhibits emis-
sion at 370 nm at the pH value of 8.0, which shifts to 580 nm when the
pH changes to 4.0. The emission shift is up to 210 nm. Different from 1-
C, emission shift of 2-C was reduced to 180 nm due to the red-shifted
1-C
340 nm
340 nm
360 nm
360 nm
340 nm
340 nm
490 nm
510 nm
510 nm
520 nm
540 nm
550 nm
370 nm
410 nm
465 nm
465 nm
415 nm
415 nm
580 nm
590 nm
615 nm
620 nm
615 nm
625 nm
210 nm
180 nm
150 nm
155 nm
200 nm
210 nm
2
3
4
5
-C
-C
-C
-C
6-C
The emission and absorption in basic conditions were obtained in the mixed solvent of
EtOH/PBS (V/V, 1:1, pH = 8.0). The emission and absorption in acid conditions were ob-
tained in the mixed solvent of EtOH/PBS (V/V, 1:1, pH = 4.0).
4

H. Zhang, M. Tian, R. Yang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119035
Scheme 2. The mechanism of 3-C for the reversible detection of pH values.
be assigned to the protons marked with H
on the indoline ring (Fig. 1a, above; Fig. 1b, left). Because the two methyl
groups containing H and H asymmetrically localize out of the pyrene
plane and almost in the same plane as the pyrene, respectively, H and
are chemically un-equivalent due to the ring current de-shielding ef-
fect of the pyrene part (Fig. 1b, left). This assignment has been further
confirmed by the geometrical optimization at the B3LYP/6-31G level
using Gaussian 03 program package [29]. On the other hand, in acid
a
and H
b
in two methyl groups
3
environment such as DMSO/CF COOD mixture, there is only a singlet
peak at δ 2.03 ppm (Fig. 1a, below). As displayed by the optimized ge-
ometry of molecule 3-O (Fig. 1b, right), the two methyl groups bearing
a
b
a
H
a
and H
the whole conjugated system, and thus the six protons (H
are chemically equivalent. The chemical shift of H and H
larger than that of H and H in 3-C because the conjugated interaction
b
symmetrically localize on the up and down sides of plane of
and H
in 3-O is
H
b
a
b
)
a
b
a
b
between pyrene and indole enhances the ring current and thereby the
de-shielding effect on the protons [30].
Secondly, H
shifts in 3-C and 3-O (Fig. 1a and b). H
cally affected by the de-shielding effect of the vinyl pyrene, thus present
as multiplet peaks. The chemical shift of H is smaller than that of H be-
cause the electronegativity of the adjacent nitrogen atom is lower than
that of oxygen. In comparison, H and H in 3-O shift to low field due to
c
and H
d
in two methylene also show different chemical
c
and H in 3-C were asymmetri-
d
c
d
c
d
the enhanced de-shielding effect, and present as standard triplet peaks
due to the symmetrical chemical environments (Fig. S9). In this condi-
tion, chemical shift of H
from the large conjugated system.
Thirdly, the two peaks appearing at δ 6.60 and δ 7.88 ppm with a
coupling constant (J) of 16.0 Hz in 3-C should be assigned to H and H
c d
is larger than H due to the de-shielding effect
e
f
with the resonances of the protons on the trans-configurational vinyl
bond (Fig. 1a, above and S10). In 3-O with large conjugated structure
(
e f
Fig. 1a, below, and S10), these two peaks of H and H shift to low-
field at around δ 8.13 and δ 9.44 ppm respectively, which can be de-
duced from the specific coupling constant for protons on trans-vinyl
bond. At the same time, the chemical shift of H on phenyl moiety
g
around δ 7.0 ppm in 3-C (Fig. 1a and S10) also obviously shifted to the
lower field in 3-O, although it is difficult to identify clearly in a complex
signal group.
Finally, the formation of the conjugated structure in molecule 3-O is
also simulated by the theoretical calculation performed at B3LYP/6-
3
11g (2d, p) level. As shown in Fig. 1c, both of the HOMO and LUMO
for 3-C populate on the pyrene moiety, while for 3-O the LUMO popu-
lates through the molecular skeleton. So, all the dramatic low-field
1
shift of 3-O relative to H NMR of 3-C, as mentioned above, should be as-
cribed to the pronouncedly extended conjugated system in 3-O form,
which will put the protons in the de-shielding environment by both of
pyrene and indoline macrocycles.
3.5. Emission behaviors of the fluorescent probe in ethanol/PBS buffer solu-
tions with different pH values
Amongst the six probes, in both basic and acid conditions, probe 3-C
displays strong dual-color emission under the excitation of 405 nm, a
usually equipped excitation source of confocal microscope.
Fig. 1. Mechanism investigations of 3-C responding to pH values. (a) The H NMR spectra of
3-C in DMSO and mixed solvents of DMSO/CF
3
COOH (V/V, 4:1); (b) the optimized
structure of 3-C and 3-O; (c) the molecular frontier orbits of 3-C and 3-O.
5

H. Zhang, M. Tian, R. Yang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119035
Consequently, 3-C should be a qualified probe for the imaging of intracel-
lular pH values in a ratiometric manner. Before cell imaging experiments,
the optical response of 3-C to pH value in ethanol/PBS buffer solutions was
firstly investigated. As shown in Fig. 2a, the absorption spectrum of 3-C
shows a maximum at 360 nm, which is corresponding to the absorption
peak of the vinyl pyrene chromophore in 3-C. The appearance of charac-
teristic absorption of pyrene indicates that pyrene is not conjugated
with the indoline unit. With the decrease of pH value, the absorption fea-
ture peaked at 360 nm becomes gradually weakened. Meanwhile, a pro-
nounced absorption peaked at 510 nm increases accordingly. The
emergence of this feature suggests the formation of new chromophore
that is distinct from 3-C. Based on the reaction mechanism shown in
Scheme 2, this new chromophore can be clearly assigned to the ring-
opening form of 3-C, that is, molecule 3-O. The evidently red-shifted ab-
sorption maximum consists with the formation of extended π-electron
system due to the conjugation between pyrene and indoline units.
The fluorescence emission behavior of molecule 3-C at different pH
values in ethanol/PBS buffer solutions have been examined simulta-
neously, and the results are shown in Fig. 2b. Upon excitation with
Fig. 3. Two-photon emission spectra of 10 μM of 3-C in mixed solvents of ethanol/PBS
buffer solutions (V/V, 1:1) with different pH values (4.0–8.0). λex = 800 nm.
adjusted by the addition of HCl and NaOH solution into the system.
The experimental results are shown in Fig. 2d, which indicate desirable
reversibility of the intensity ratio of I611/I461
.
4
4
05 nm light source, 3-C emits strong blue fluorescence peaked at
61 nm, which can be assigned to the vinyl pyrene fluorogen. With low-
As mentioned above, 3-O is expected to have two-photon absorp-
tion (TPA) property, thus we investigated the fluorescent responses of
ering the pH value, the emission intensity of this blue peak decreases
gradually. At the same time, a pronouncedly red-shifted emission peak
appears at around 611 nm and it enhances synchronously with the low-
ering of pH value. These spectral changes are in good agreement with
the absorption feature changes. The red fluorescence emission can be
associated with the whole conjugation system of 3-O. In Fig. 2c, the
curve of the intensity ratio of the emission intensity at 611 and
3
-C in mixed solvent with different pH values excited by 800 nm infra-
red laser beam. As demonstrated by the results in Fig. 3, the probe mol-
ecule displays dual emission peaks at around 450 and 630 nm. With the
increase of the pH value, the emission intensity of the blue peak bears
small change, while the emission peaked at 630 nm decreases evidently.
Specifically, the intensity of the red emission decays to almost zero
when pH value increases from 4.0 to 8.0, as shown in Fig. S9. These re-
sults suggest that 3-C is potential to serve as both one- and two-
photon fluorescent probe.
4
61 nm (I611/I461) versus pH value is plotted. The data clearly indicate
a ratiometric fluorescence response. Therefore, 3-C is potential to be
used as a fluorescent probe working in a ratiometric way.
According to literature and the results of mechanistic investigation,
the acid-triggered ring-opening reaction of oxazole is reversible [24].
This property is of importance for monitoring the changes of pH value
in proper media. We examined the reversibility of the fluorescent re-
sponse of 3-C to pH changes in ethanol/PBS buffer (1:1 by volume) so-
lution. The pH value of the system was set between 3.0 and 9.0 and
3.6. Estimation on the selectivity of the fluorescent probe
The mechanistic investigation has shown that the ratiometric fluo-
rescent response of probe 3-C to acid is a proton-triggered ring-
opening process. The reverse process in neutral and basic environment
Fig. 2. The spectra responses of 3-C to pH values. The absorption (a) and emission (b) spectra of 3-C in ethanol/PBS buffer solutions (V/V, 1:1, pH = 4.0–8.0); (c) the ratio between the
emission intensity at 611 nm to 461 nm of 3-C in ethanol/PBS buffer solutions (V/V, 1:1, pH = 4.0–8.0), inset: the bared-eye photos of 3-C in basic and acid solutions under daylight or
UV lamp; (d) reversibility study of 3-C to pH values;. Concentration of 3-C: 10 μM; λex = 405 nm.
6

H. Zhang, M. Tian, R. Yang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119035
is an in situ nucleophilic addition reaction between the hydroxyl and ac-
tivated C_N group (Scheme 2). Considering that the living cells have
rather complex constituents and various biological agents including
the nucleophiles of the hydroxyl groups on saccharides, SH- and thiols
on glutathione (GSH) and cysteine (Cys), as well as basic residuals on
amino acids and peptides may react with the activated C_N group in
pH value in a range of 4.0 to 8.0, and the sensitivity is scarcely influenced
by a variety of biological species. Before it is used in vivo to monitor the
lysosomal pH change, the targeting of 3-C to lysosome must be firstly
checked. To be practically useful, the probe should be incubated with
living cell in normal physiological condition. In other words, it should
be engaged in its 3-C form in neutral environment rather than in its 3-
O form in strong acidic condition (e.g., pH 4.0). Fortunately, it has
been widely accepted that weak basic compounds are capable of partial
protonation in a neutral environment thereby can be collected by lyso-
some with membrane-bound acid components [31]. But the positively
charged probe in 3-O form may be taken up by the mitochondria [32],
which possess negative potential in living cells.
Consequently, the co-localization experiments of 3-C with commer-
cialized both mitochondria probe (MTDR) and lysosome probe (LYR)
have been implemented, in order to clarify the localization of the
probe. Fig. 5a and b shows the fluorescence images of Hela cells co-
stained with 5 μM 3-C and 200 nM MTDR for 20 min, and the fluores-
cence was collected in red and deep-red channel, respectively. When
these two images are merged (Fig. 5c), the domains marked with red
color (containing 3-C) co-localize poorly with those marked with
green color (containing MTDR). Moreover, according to the emission
distribution profile shown in Fig. 5d, fluorescence signals from MTDR
and 3-C are poorly overlap. These results demonstrate that 3-C is not a
mitochondria-target probe. In contrast, as shown in Fig. 5e–g, the fluo-
rescent images for 3-C domains overlap very well with that of LYR,
and the co-localization co-efficient is as high 86%. Moreover, as shown
in Fig. 5h, the fluorescence signals of 3-C overlap with that of LYR per-
fectly. These results indicate that probe 3-C selectively targets lyso-
somes in living cells.
3
-O. Therefore, the selectivity of the probe has to be estimated before
it is convincingly used as a fluorescent probe to monitor the lysosomal
pH change.
We examined the fluorescent responses of the probe molecule to
different biological species in ethanol/PBS buffer solution (1:1 by vol-
ume, pH = 4.0) and the data are shown in Fig. 4a and b. At pH 4.0, the
probe is in an acidic conditions and 3-O form exists in the solution. As
indicated by the fluorescence spectrum of the control sample, there
are two emission peaks with maximum at 461 and 611 nm respectively.
The ratio of I611/I461 is 0.25, which is similar to that recorded in Fig. 2c.
The emission features of the probe solution have been little affected
by adding different reagents, no matter thiol-containing species,
amino acids, glucose or inorganic electrolytes. Meanwhile, the ratio of
I611/I461 keeps little change.
The effects of adding different reagents on the emission property of
the fluorescent probe at pH 7.0 were also checked, and the results are
presented in Fig. 4b. The experimental data indicate that the emission
origins exclusively from the pyrene fluorogen in 3-C and the ring open-
ing reaction cannot be triggered by the addition of various compounds.
Therefore, we conclude that the probe has high selectivity to pH change
in the window of 4.0 to 7.0.
3.7. Ratiometric fluorescent imaging pH change in lysosomes of living cells
In order to testify the performance of probe 3-C in fluorescent imag-
ing lysosomal pH change, HeLa cells were incubated with the probe and
the emission was collected in two channels under one-photon
Based on all above experimental results, molecule 3-C is suitable
used as a ratiometric fluorescent probe to respond to the variation of
Fig. 4. Selectivity of 3-C. Fluorescent spectra (a, c) and the ratio of I611/I461 (b, d) of 10 μM of 3-C in ethanol/buffer (V/V, 1:1, pH = 4.0 for a and b; pH = 7.0 for c and d) without and with
mM of various biological reagents. NaSH, NaCl, KCl, CaCl , NaBr, KI, GSH-glutathione, Cys-L-cysteine, Asn-L-asparagine, Asp-L-aspartic acid, Gln-L-glutamine, Glu-L-glutamic acid, Gly-L-
1
2
glycine, His-L-histidine, Phe-L-phenylalanine, Pro-L-proline, Thr-L-threonine, Tyr-L-tyrosine, Glc-L-glucose. λex = 405 nm.
7

H. Zhang, M. Tian, R. Yang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119035
Fig. 5. Co-localization investigations. (a–c) Fluorescent images of HeLa cells co-stained with 5 μM 3-C and 200 nM MTDR for 20 min; (e–g) Fluorescent images of HeLa cells co-stained with
5
6
μM 3-C and 200 nM LYR; (d, h) profile plot of two channels along the arrows in (c) and (g), respectively. (a, e) 3-C, λex = 405 nm, λem = 580–630 nm; (b) MTDR, λex = 635 nm, λem
55–700 nm; (f) LYR, λex = 561 nm, λem = 630–700 nm; (c, g) merged images; bar = 20 μm.
=
excitation at 405 nm (Fig. 6a–c). By calculating the intensity ratio of red
to blue channels by pixels, a ratiometric image shown in Fig. 6d was ob-
tained, which mapped the lysosomal pH distribution of living cells. The
yellow parts in Fig. 6d represent lysosomes with relatively lower pH
performed and the results are shown in Fig. 7. To obtain clear and highly
resolved digital image correlation (DIC) images, short incident light at
405 nm was used (Fig. 7a and e). With the excitation of 800 nm, bright
emission was found in both the blue and red channels, indicating good
two-photon absorption and emission properties of the probe in living
HeLa cells. Fig. 7d was obtained according to the pixels ratio of Fig. 7c
to b, and the data indicate that green to red pseudo colors are lysosomes
with lower pH values, while blue pseudo color represents high pH lyso-
somes. Similar to results obtained in the one-photon imaging experi-
ment, the pseudo colors in ratiometric images turn blue upon
(
4.5–5.5), while the blue ones are lysosomes with higher pH values.
These results mean that it is feasible to discriminate the difference of
pH values. To prove this notion, HeLa cells were pretreated with
NH
33]. Then the cells were stained with 3-C. As shown in Fig. 6e–h,
under the same experimental conditions, cells treated with NH Cl
4
Cl, a reagent that can cause elevation of lysosomal pH value (~6.0)
[
4
show decreased emission in red channel and slightly increased emission
in blue channel. The ratiometric images give blue pseudo color, which
represent higher pH values. Therefore, probe 3-C can successfully
point out the change of lysosomal pH values.
4
treatment with NH Cl, which means the alkalization of lysosomes in
the treated cells. Accordingly, probe 3-C can also detect the change of ly-
sosomal pH value in a ratiometric manner under two-photon micro-
scope mode.
Fluorescent images for HeLa cells stained with 3-C and excited with
two-photon light source (excitation at 800 nm) have also been
At last, we investigated the stability of these probes in H-DMEM
which contains 10% fetal bovine serum. As shown in Figs. S12, S13 and
4
Fig. 6. One-photon fluorescence images. Digital image correlation (DIC), fluorescence and ratiometric images of HeLa cells untreated (a–d) or pretreated (e–h) with 5 mM NH Cl for 30 min
then stained with 5 μM of 3-C for 20 min. (a, e) DIC images; (b, f) λex = 405 nm, λem = 430–490 nm; (c, g) λex = 405 nm, λem = 580–630 nm; (d, h) ratiometric images obtained according
to the pseudo color bar in (h); bar = 20 μm.
8

H. Zhang, M. Tian, R. Yang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119035
4
Fig. 7. Two-photon fluorescent images. DIC, two-photon fluorescent and ratiometric images of HeLa cells untreated (a–d) or pretreated (e–h) with 5 mM NH Cl for 30 min then stained
with 5 μM of 3-C for 20 min. (a, e) DIC images obtained with 405 nm; (b, f) λex = 800 nm, λem = 495–540 nm; (c, g) λex = 800 nm, λem = 575–630 nm; (d, h) ratiometric images obtained
according to the pseudo color bar in (h); bar = 20 μm.
S14, the absorbance spectra of these probes changes very little in 48 h
when dissolved in H-DMEM, which indicate that these probes are
chemically stable in H-DMEM. On the other hand, they maintain high
fluorescence intensity during 10 min continuous laser irradiation as
shown in Figs. S15, S16 and S17, which indicating their photostability
in H-DMEM.
therefore may find versatile applications in the fields of chemistry, ma-
terials science and biological investigations.
CRediT authorship contribution statement
Huamiao Zhang: Conceptualization, Methodology, Investigation,
Writing.
Minggang Tian: Validation, Analysis, Data curation.
Rui Yang: Validation, Analysis.
4. Conclusion
Xiuquan He: Resources, Writing: review & editing.
Xiaoqiang Yu: Writing: review & editing.
In summary, we have presented a general strategy to increase the
emission shift of ratiometric pH probes via a unique pH-dependent in-
tramolecular reaction of spiro-oxazolidine. Due to that the reaction
could change the conjugation length and electronic effect concurrently,
the emission shift of these probes were extremely large (150–210 nm).
Such a large emission shift could efficiently avoid interferences and
would be greatly favorable for ratiometric imaging applications.
Amongst, probe 3-C was an unprecedented two-photon ratiometric
probe that is suitable for living cell imaging applications. This probe in
ring-opened form (3-O) emits both red (λem = 611 nm) and blue
Declaration of competing interest
There are no conflicts of interest to this work.
We declare that we do not have any commercial or associative inter-
est that represents a conflict of interest in connection with the work
submitted.
Acknowledgements
(λem = 461 nm) fluorescence in acid environments, while in ring-
closed form (3-C) only emits blue light in neutral and basic environ-
ments. The intensity ratio of I611/I461 depends on the pH value in etha-
nol/buffer mixture solutions and the ratio at a specific pH value
The laser confocal scanning imaging was performed at the Micros-
copy Characterization Facility, Shandong University. For calculation
support, we thank Heng Zhang in School of Chemistry and Chemical En-
gineering, Shandong University. This work was financially supported by
National Natural Science Foundation of China (51773111, 21672130,
(
e.g., 4.0 and 7.0) is almost insusceptible to the existence of various bi-
ological species such as thiol containing amino (Cys) and GSH,
hydroxyl-containing glucose, and some other amino acids. These com-
prehensive properties bestow 3-C with great potentially to
ratiometrically detect the pH changes in the lysosome of living cells
with high sensitivity and selectivity. Cell imaging experiments con-
firmed this idea. Both one- and two-photon fluorescent images pro-
vided clear pictures showing pH variations in lysosomes of living HeLa
cells. Therefore, 3-C can be a powerful tool for lysosomal investigations.
More importantly, the strategy to develop ratiometric pH probes relying
on the reversible intramolecular cyclization/ring opening reaction in
mild condition is distinct from the ones based on proton acceptor/
donor design rationale. Furthermore, the chemical sensing mechanism
5
1273107 and 51273175), Natural Science Foundation of Shandong
Province (ZR2017ZC0227), Fundamental Research Fund of Shandong
University (2017JC011), Open Project of State Key Laboratory of Supra-
molecular Structure and Materials (SKLSSM201729).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2020.119035.
1
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tional theory (DFT) calculations. In a word, the present strategy based
on conjugation break/connection is helpful to achieve large spectral
separation for taking better fluorescent images via two channels, and
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