Synthesis, spectroscopic properties, and biological applications of eight novel chlorinated fluorescent proteins-labeling probes

Article abstract of DOI:10.1007/s10895-014-1351-x

Eight novel chlorinated fluorescent proteins-labeling probes with a linker and reactive group were prepared in 7 steps by the reaction of chlorinated resorcinols with 3, 6-dichloro-4-carboxyphthalic anhydride in the presence of methanesulfonic acid. Structures of target compounds and intermediates were determined via IR, MS, ¹H NMR and element analysis. The spectral properties of the chlorinated fluoresceins were studied. These fluorescent probes showed absorbance peaks at 508-536 nm and fluorescence peaks at 524-550 nm. It was found that they have absorption and emission maxima at long wavelengths and high fluorescence quantum yields. Emission spectra of chlorinated fluoresceins shifted towards long wavelength with increase in chlorine. The probes were used for fluorescence imaging of cells in order to investigate whether they can conjugate to cells. The fluorescence imaging of living cells showed that they were localized in cell nucleus. However, they were localized in cytosol of chemically fixed cells. These probes will be useful reagents for the preparation of stable fluorescent conjugates.

Full text of DOI:10.1007/s10895-014-1351-x

J Fluoresc
DOI 10.1007/s10895-014-1351-x
ORIGINAL PAPER
Synthesis, Spectroscopic Properties, and Biological Applications
of Eight Novel Chlorinated Fluorescent Proteins-labeling Probes
Xianglong Wu & Min Tian & Wutu Fan & Ya l e i P a n &
Yuankun Zhai & Yinbo Niu & Chenrui Li & Tingli Lu &
Qibing Mei
Received: 12 September 2013 /Accepted: 27 January 2014
#
Springer Science+Business Media New York 2014
Abstract Eight novel chlorinated fluorescent proteins-
labeling probes with a linker and reactive group were prepared
in 7 steps by the reaction of chlorinated resorcinols with 3, 6-
dichloro-4-carboxyphthalic anhydride in the presence of
methanesulfonic acid. Structures of target compounds and
Introduction
For applications in imaging and microscopy, the most impor-
tant characteristics of a fluorescent dye and its bio-conjugates
are: the position, form, fluorescence quantum yield, and life-
time of the excited state of a free dye and its bio-conjugates
[1]. Carboxy-functionalized fluorescein and rhodamine dyes
are useful reagents for the preparation of hydrolytically stable
fluorescent conjugates [2]. They are powerful tools for explor-
ing cellular biology owing to their brightness, high quantum
yields, low-energy excitation and emission wavelengths, and
biocompatibility [3].
A wide variety of fluorescein derivatives have been pre-
pared and used as fluorescent detection reagents. Fluorescent-
ly labeled biologically active molecules have many important
analytical and biochemical applications. Many fluorescein
derivatives have functional groups that are suitable for reac-
tion with other molecules, and can therefore serve as labels in
a variety of analytical applications ranging from probing cell
functions to monitoring the level of drugs in human fluids via
immunoassays [4]. Fluorescein derivatives are particularly
suitable for, but not limited to, biological experiments in
which fluorescein is covalently attached to materies such as
peptides, proteins, nucleotides, oligonucleotides, drugs, hor-
mones, lipids, and other biomolecules [5–10].
1
intermediates were determined via IR, MS, H NMR and
element analysis. The spectral properties of the chlorinated
fluoresceins were studied. These fluorescent probes showed
absorbance peaks at 508–536 nm and fluorescence peaks at
524–550 nm. It was found that they have absorption and
emission maxima at long wavelengths and high fluorescence
quantum yields. Emission spectra of chlorinated fluoresceins
shifted towards long wavelength with increase in chlorine.
The probes were used for fluorescence imaging of cells in
order to investigate whether they can conjugate to cells. The
fluorescence imaging of living cells showed that they were
localized in cell nucleus. However, they were localized in
cytosol of chemically fixed cells. These probes will be useful
reagents for the preparation of stable fluorescent conjugates.
.
.
.
Keywords Fluorescent dye Probe dye Cell labeled
.
Spectroscopic properties Synthesis
Although these dyes have a same xanthene-based skeleton,
different substituents can be made to cause marked differences
in absorbance and fluorescence emission wavelengths. Selec-
tive substitution of chlorine for aromatic hydrogen has been
seen to increase fluorescence efficiency and to narrow absor-
bance and emission maxima compared with fluorescein. The-
se characteristics are very useful for multi-color imaging [11].
The selective substitution of chlorine for aromatic hydro-
gen in organic compounds results in profound changed in their
photophysical properties. Therefore, it is very important to
study the fluorescein derivatives substituted by chlorine [12].
:
:
:
:
:
:
:
X. Wu W. Fan Y. Pan Y. Zhai Y. Niu C. Li T. Lu
Q. Mei (*)
Key Laboratory for Space Bioscience and Biotechnology, School of
Life Sciences, Northwestern Polytechnical University, Xi’an 710072,
Peoples Republic of China
e-mail: gahpyudx@163.com
M. Tian
School of Materials and Chemical Engineering, Xi’an Technological
University, Xi’an 710021, Peoples Republic of China

J Fluoresc
Several chlorinated fluoresceins such as 4, 5, 6, 7-
tetrachlorofluorescein, 2′, 7′-dichloro-4, 5, 6, 7-
tetrachlorofluorescein, 4′, 5′-dichloro-4, 5, 6, 7-
tetrachlorofluorescein, and 2′, 4′, 5′, 7′-tetrachloro-4, 5, 6, 7-
tetrachlorofluorescein have been synthesized by our group.
They were found to have absorption and emission maxima at
long wavelengths and high fluorescence quantum yields [13].
In an effort to facilitate and improve conjugations, we have
added a spacer linker between the chlorinated fluorophore and
the reactive group. 4-Aminobutanoic acid or 3-amiopropanoic
acid can be used as a spacer linker. This allows us to prepare
succinimidyl ester derivatives, the most amine-selective and
stable reactive group for labeling proteins.
However, to our knowledge, the preparation and spectro-
scopic properties of chlorinated fluorescein have not been
reported detailedly and systematically. Four probes were re-
ported briefly in a form of letter by our group [14, 15].
The present work details, for the first time, the preparation
of eight novel chlorinated fluorescent proteins-labeling
probes. Spectroscopic properties and biological applications
of these novel chlorinated fluorescent proteins-labeling probes
are also described in this work.
with 10 % fetal calf serum, 1 % penicillin, and 1 % strepto-
mycin at 37 °C in an atmosphere of 95 % oxygen and 5 % CO2
[16–18].
Cells were plated at a cell density of 1×104 cells/well on
glass coverslips placed at the bottom of 24-well plates and
incubated at 37 °C (5 % CO2) overnight.
Preparation of Living Cell for Fluorescent Imaging
After incubation overnight to allow the cells to attach on the
plate, the culture media was discarded, and 0.1 mL of the
probe solution (1×10−5 mol/L in DMEM) was added to each
well, followed by incubation at 37 °C for 45 min. The super-
natant was abandoned. Then, the cells were washed gently
twice with sterilized PBS to remove excess dye before plate
sealing and imaging.
Preparation of Fixed Cell for Fluorescent Imaging
After incubation overnight to allow the cells to attach on the
plate, the culture media was discarded, and incubate the cells
with 4 % PFA in PBS for 10 min at rt. Rinsed the fixed cells
three times in PBS, and 0.1 mL of the probe solution (1×
10−5 mol/L in PBS) was added to each well. Then, stained the
cells at rt for 30 min. Rinsed cells gently with PBS several
times to remove excess dye before plate sealing and imaging.
Images were taken using a Nikon Eclipse 80i microscope
equipped for epifluorescence using the following filter sets:
DAPI(Blue: DAPI channel); FITC (Oregon Green channel);
For experiments that evaluated the intracellular distribution of
fluorescent probes, the microscope parameters, including
fluorescence intensity and exposure, were optimized for each
fluorophore.
Experimental Procedures
Materials Unless mentioned otherwise, all chemicals, re-
agents and solvents for the synthesis of the compounds were
analytically pure grade and purchased from commercial
sources. All organic solvents were anhydrous grade. 3, 6-
dichloro-4-carboxy-phthalic anhydride was prepared by our
group.
Instrumentation All fluorescence measurements were carried
out at room temperature on a Hitachi F-4500 spectrofluorim-
eter equipped with a xenon lamp source and a 10 mm quartz
cell. Absorption spectra were recorded on a Hitachi U-3310
Synthesis
1
UV-vis spectrophotometer using a 10 mm quartz cell. H
Synthesis of 5(6)-carboxy-4, 7-dichlorofluorescein (1a)
NMR spectra were recorded on a Varian Inova-400 spectrom-
eter operating at 400 MHz using either CDCl3 or DMSO-d6 as
solvents. MALDI-TOF mass spectra were recorded using an
AXIMA-CFR™ plus mass spectrometer. For MALDI-TOF
analysis, samples were prepared by dissolving in methanol
and spotting 1 μL of the solution. Infrared spectra were
obtained as KBr pellets on an EQUINOX-55 FTIR
spectrometer.
The mixture of 3, 6-dichloro-4-carboxyphthalic anhydride
(2.61 g, 10 mmol) and resorcinol (2.53 g, 23 mmol) in
methanesulfonic acid (50 mL) was heated and stirred under
nitrogen at 50 °C for 4 h then at 110 °C for 24 h. After cooling
to room temperature, the mixture was poured into the ice-
water. The precipitation was filtered and washed several times
with 0.1 M HCl. The solid was dried in air overnight or in
vacuum to constant weight. The mixture was separated by
column chromatography on silica with the eluent of 1:10:80
glacial acetic acid/methanol/chloroform. The compound 1a
(3.74 g) was obtained as a green black powder. C21H10Cl2O7,
yield: 84 %, 1H NMR (DMSO-d6, 400 MHz) δ: 10.15(s, 1H, -
Cell Culture MC3T3-E1 murine calvarial preosteoblasts were
obtained from the American Type Culture Collection. The
MC3T3-E1 cells were cultured in Dulbecco’s modified ea-
gle’s medium (Gibco, Grand Island, NY, USA), supplemented

J Fluoresc
COOH), 6.85(d, 2H 1′ and 8′-ArH), 6.66(s, 2H, 4′and 5′-
ArH), 6.55(d, 2H, 2′ and 7′-ArH). MALDI-TOF MS, m/z:
445.39(Calcd: 445.21). FT-IR(KBr), υ/cm−1: 3476, 1765,
1609, 1487, 1433.
stirring, ether (50 mL) was added and the aqueous layer
was removed. The organic layer was washed with phos-
phate buffer (3×50 mL, 1.4 M, pH 7.0), aqueous HCl
(50 mL, 1 M), and saturated NaCl and dried with
MgSO4. The solvent was removed by evaporation, and
resulting yellow syrup was dissolved in anhydrous eth-
anol (50 mL). Diisopropylamine (5.0 mL, 36 mmol)
was added, and the mixture was cooed to −20 °C in
freezer overnight. The resulting solid was removed by
filtration and washed with cold ethanol and acetone to
give white powder 4.23 g (39.5 %) of compound 2a,
which had a isomeric purity >95 % as determined by
Synthesis of 2′, 7′-dichloro-5(6)-carboxy-4,
7-dichlorofluorescein (1b)
This compound was prepared from 3, 6-dichloro-4-
carboxyphthalic anhydride (2.61 g, 10 mmol) and 4-
chlororesorcinol (3.32 g, 23 mmol) by the procedure utilized
for compound 1a. Compound (1b) (4.01 g) was obtained as a tan
powder. C21H8Cl4O7, yield: 78 %, 1H NMR (400 MHz, DMSO-
d6) δ: 11.12(s, 1H, -COOH), 7.21(s, 2H, 4′ and 5′ -ArH), 6.87(s,
2H, 1′ and 8′ -ArH). MALDI-TOF MS, m/z: 515.55(Calcd:
514.10). FT-IR(KBr), υ/cm−1: 3421, 1759, 1608, 1434, 1175.
1
HPLC analysis. H NMR (400 MHz, CDCl3) δ: 6.84(d,
2H, 1′ and 8′-ArH), 6.81(d, 2H, 2′ and 7′-ArH), 7.71(s,
1H, 5-ArH), 3.40(q, 2H, -CH=), 1.36(s, 18H, -tBuH),
1.25(s, 12H, i-propyl H). MALDI-TOF MS, m/z: 714.41
(Calcd: 714.63). FT-IR(KBr), υ/cm−1: 3423, 2873, 2723,
1777, 1610, 1583, 1497, 1392, 1225, 1119, 894.
Synthesis of 4′, 5′-dichloro-5(6)-carboxy-4,
7-dichlorofluorescein (1c)
Synthesis of 2′, 7′-dichloro-6-carboxy-4, 7-dichlorofluorescein
dipivalate diisopropylamine salt (2b)
This compound was prepared from 3, 6-dichloro-4-
carboxyphthalic anhydride (2.61 g, 10 mmol) and 2-
chlororesorcinol (3.32 g, 23 mmol) by the procedure utilized
for compound 1a. Compound (1c) (4.06 g) was obtained as an
orange powder. C21H8Cl4O7, yield: 79 %, 1H NMR (400 MHz,
DMSO-d6) δ: 11.05(s, 1H, -COOH), 6.93(d, 2H, 1′ and 8′-ArH),
6.82(d, 2H, 2′ and 7′-ArH). MALDI-TOF MS, m/z: 514.76
(Calcd: 514.10). FT-IR(KBr), υ/cm−1: 3432, 1760, 1605, 1433,
1213.
This compound was prepared from compound 1b according
to the procedure described above for compounds 2a. The
compound 2b (3.68, 36 %) was obtained as a white powder.
1H NMR (400 MHz, DMSO-d6) δ: 8.40(s, 1H) 7.52 (s, 2H),
7.49(s, 2H), 3.44(a quintet, 2H), 1.34(s, 18H), 1.19(d, 12H).
MALDI-TOF MS, m/z: 783.10 (calcd: 783.52). FT-IR(KBr),
υ/cm−1: 3440, 2979, 1767, 1640, 1094; Anal. Calcd. for
C39H39NO2Cl4: C 56.71, H 5.02, N 1.79; found: C 56.73, H
5.05, N 1.74.
Synthesis of 2′, 4′, 5′, 7′-tetrachloro-5(6)-carboxy-4,
7-dichlorofluorescein (1d)
Synthesis of 4′, 5′-dichloro-6-carboxy-4, 7-dichlorofluorescein
dipivalate diisopropylamine salt (2c)
This compound was prepared from 3, 6-dichloro-4-
carboxyphthalic anhydride (2.61 g, 10 mmol) and 2, 4-
dichlororesorcinol (4.12 g, 23 mmol) by the procedure utilized
for compound 1a. Compound (1d) (4.26 g) was obtained as a
brick red powder. C21H6Cl6O7, yield: 73 %, 1H NMR
(400 MHz, DMSO-d6) δ: 11.15(s, 1H, -COOH), 8.54(s, 1H,
ArOH), 8.22(s, 1H, 5-ArH), 6.86 (s, 2H, 1′ and 8′-ArH).
MALDI-TOF MS, m/z: 581.90 (Calcd: 582.99). FT-
IR(KBr), υ/cm−1: 3480, 1768, 1709, 1478, 1433, 1212, 997.
This compound was prepared from compound 1c according to
the procedure described above for compounds 2a. The com-
1
pound 2c (4.09, 40 %) was obtained as a white powder. H
NMR (DMSO-d6, 400 MHz) δ: 8.40 (s, 1H), 7.38(d, 2H),
7.26(d, 2H), 3.44(a quintet, 2H), 1.36 (s, 18H), 1.19(d, 12H).
MALDI-TOF MS, m/z: 783.41 (calcd: 783.52). FT-IR(KBr),
υ/cm−1: 3424, 2977, 1781, 1640, 1424, 1092. Anal. Calcd. for
C39H39NO2Cl4: C 56.71, H 5.02, N 1.79; found: C 56.93, H
5.08, N 1.83.
Synthesis of 6-carboxy-4, 7-dichlorofluorescein dipivalate
diisopropylamine salt (2a)
Synthesis of 2′, 4′, 5′, 7′-tetrachloro-6-carboxy-4,
5(6)-carboxy-4, 7-dichlorofluorescein (1a) (6.68 g,
15 mmol) was refluxed in trimethylacetic anhydride
(28 mL) for 2 h. The reaction mixture was cooled to
room temperature and diluted with tetrahydrofuran (THF
40 mL) and water (40 mL). After 2 h of vigorous
7-dichlorofluorescein dipivalate diisopropylamine salt (2d)
This compound was prepared from compound 1d according to
the procedure described above for compounds 2a. The com-
1
pound 2d (4.94, 38.6 %) was obtained as a white powder. H

J Fluoresc
NMR (CDCl3, 400 MHz) δ: 6.87 (s, 2H, 1′ and 8′-ArH), 7.81 (s,
1H, 5-ArH), 3.25 (s, 2H, –NH2), 1.44 (s, 18H, t-BuH), 1.24 (s,
12H, i-propyl H). MALDI-TOF MS, m/z: 852.52, (calcd:
852.41). FT-IR(KBr), υ/cm−1: 2979, 2874, 1775, 1642, 1425,
1214, 1075, 682.
Synthesis of 4, 7-dichloro-dipivaloyl-6-carboxyfluorescein
N-hydroxysuccinimidyl ester (4a)
Anhydrous pyridine (16 mL) and N-hydroxysuccinimidyl
trifluoroacetate (NHS-TFA) (8.44 g, 40 mmol) were add to a
solution of compound 3a (3.07 g, 5 mmol) in anhydrous
CH2Cl2 under vigorous stirring. The resultant mixture was
stirred at room temperature for 5 h. TLC (CHCl3/ MeOH =
10:1) was used to confirm the completion of the reaction. The
mixture was washed with 4 % HCl (2×100 mL) and saturated
saline (2×50 mL), dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure, and residue was dried in
vacuum to yield compound 4a (2.84 g, 80 %) as an off-white
Synthesis of 6-carboxy-4, 7-dichlorofluorescein dipivalate
(3a)
Carboxyfluorescein salt 2a (2.89 g, 4 mmol) was dis-
solved in CH2Cl2 (50 mL) and extracted with aqueous
HCl (1 M, 3×50 mL). The organic layer was dried with
Na2SO4, and the solvent was evaporated to give 2.21 g
1
1
solid. H NMR (400 MHz, CDCl3) δ: 8.16(s, 1H, 5-ArH),
of compound 3a. Yield 90 %, white powder, H NMR
7.09(s, 2H, 4′ and 5′-ArH), 6.88(d, 2H, 1′ and 8′-ArH), 6.84(d,
2H, 2′ and 7′-ArH), 2.90(s, 4H, -CH2CH2-), 1.37(s, 18H, -
tBuH). MALDI-TOF MS, m/z: 710.51, 613.34 (Calcd:
710.51). FT-IR(KBr), υ/cm−1: 3442, 2975, 1784, 1757,
1612, 1496, 1424, 1371, 1157, 1116, 1028, 993, 795, 651.
(400 MHz, CDCl3) δ: 6.87(s, 2H, 1′ and 8′-ArH),
6.84(d, 2H, 2′ and 7′-ArH), 8.11(s, 1H, 5-ArH),
1.37(s, 18H, -tBuH). MALDI-TOF MS, m/z: 613.30,
(Calcd: 613.44). FT-IR(KBr), υ/cm−1: 3445, 2975,
1784, 1612, 1496, 1423, 1398, 1157,1116, 1027, 877.
Synthesis of 2′, 7′, 4, 7-tetrachloro-dipivaloyl-6-
carboxyfluorescein N-hydroxysuccinimidyl ester (4b)
Synthesis of 2′, 7′-dichloro-6-carboxy-4, 7-dichlorofluorescein
dipivalate (3b)
Compound (4b) was prepared from compound 3b according
to the procedure described above. Yield, 81.5 %, white solid,
1H NMR (400 MHz, CDCl3) δ: 7.15(s, 2H), 7.13(s, 1H),
6.93(s, 2H), 2.90(s, 4H), 1.45(s, 18H). FT-IR(KBr), υ/cm−1:
3513, 2977, 1744, 1479, 1408, 1092. Anal. Calcd. for
C35H27NO11Cl4: C 53.93, H 3.49, N 1.80; Found: C 54.13,
H 3.69, N 1.90.
This compound was prepared from compound 2b according
to the procedure described above for compound 3a. 1H NMR
(400 MHz, CDCl3) δ: 8.18(s, 1H), 7.13(s, 2H), 6.91 (s,
2H),1.40(s, 18H). MALDI-TOF MS, m/z: 682.89 (calcd:
682.33). FT-IR(KBr), υ/cm−1: 2976, 1765, 1478, 1408,
1093. Anal. Calcd. for C31H24O9Cl4: C 52.59, H 3.10; Found:
C 52.73, H 3.05.
Synthesis of 4′, 5′, 4, 7-tetrachloro-dipivaloyl-6-
carboxyfluorescein N-hydroxysuccinimidyl ester (4c)
Synthesis of 4′, 5′-dichloro-6-carboxy-4, 7-dichlorofluorescein
dipivalate (3c)
Compound (4c) was prepared from compound 3c according to
the procedure described above. Yield, 82.3 %, white solid, 1H
NMR (400 MHz, CDCl3) δ: 8.27(s, 1H), 6.96(d, 2H), 6.80 (d,
2H), 2.90(s, 4H), 1.42(s, 18H). FT-IR(KBr), υ/cm−1: 3513,
2977, 1744, 1479, 1408, 1092. Anal. Calcd. for
C35H27NO11Cl4: C 53.93, H 3.49, N 1.80; Found: C 54.03,
H 3.23, N 1.74.
This compound was prepared from compound 2c according to
the procedure described above for compound 3a. Yield,
89.5 %. 1H NMR (400 MHz, CDCl3) δ: 8.13(s, 1H) 6.94(d,
2H), 6.81 (d, 2H), 1.42(s, 18H). MALDI-TOF MS, m/z:
682.45(calcd: 682.33). FT-IR(KBr), υ/cm−1: 2976, 1765,
1478, 1425, 1092. Anal. Calcd. for C31H24O9Cl4: C 52.59,
H 3.10; Found: C 52.63, H 3.25.
Synthesis of 2′, 4′, 5′, 7′-tetrachloro-6-carboxy-4,
7-dichlorofluorescein dipivalate (3d)
Synthesis of 2′, 4′, 5′, 7′, 4, 7-hexachloro-dipivaloyl-6-
carboxyfluorescein N-hydroxysuccinimidyl ester (4d)
This compound was prepared from compound 2d according
to the procedure described above for compound 3a. Yield,
88 %. 1H NMR (400 MHz, CDCl3) δ: 6.87(s, 2H, 1′ and 8′-
ArH), 8.20(s, 1H, 5-ArH), 1.44(s, 18H, -tBuH). FT-IR(KBr),
υ/cm−1: 2977, 1778, 1589, 1454, 1425, 1369, 1212, 1078,
873. Anal. Calcd for C31H22Cl6O9: C 49.56, H 2.95; Found: C
49.52, H 2.94.
Compound (4d) was prepared from compound 3d according
to the procedure described above. Yield, 85 %.1H NMR
(400 MHz, CDCl3) δ: 8.29(s, 1H, 5-ArH), 6.88(s, 2H, 1′ and
8′-ArH), 2.94(t, 4H, -CH2CH2-), 1.45(s, 18H, t-BuH).
MALDI-TOF MS, m/z: 848.25, 751.86 (Calcd: 848.29). FT-
IR(KBr), υ/cm−1: 3365, 2947, 1780, 1739, 1647, 1436, 1373,
1210, 1065, 647.

J Fluoresc
Synthesis of 4, 7-dichloro-dipivaloyl-6-
2.77(t, 2H, -CH2-), 1.39(s, 18H, -tBuH). FT-IR(KBr), υ/cm−1:
(3-carboxypropylaminocarbonyl) fluorescein (5at)
3132, 2978, 1767, 1705, 1477, 1409, 1091, 614.
To compound 4a (4.62 g, 6.5 mmol) in 50 mL anhydrous
dichloromethane was added dropwise a solution of 4-
aminobutenoic acid (6.5×1.2 mmol, 0.80 g) in 20 mL
CH2Cl2 under stirring. The resultant mixture was stirred at
room temperature for 1–2 h. TLC (CHCl3 / MeOH = 10:1)
indicated the completion of the reaction. The solvent was
removed under reduced pressure, and the residual crude
product was purified by flash column chromatography on
silica gel eluted with CHCl3/EtOAc = 4/1, 3/1, 2/1 to yield
Synthesis of 4′, 5′, 4, 7-tetrachloro-dipivaloyl-6-
(3-carboxypropylaminocarbonyl) fluorescein (5ct)
Compound (5ct) was prepared from compound 4c and 4-
aminobutenoic acid by the procedure utilized for compound
5at. Compound 5ct, white solid, yield, 83.4 %, H NMR
(400 MHz, CDCl3) δ: 6.84(s, 2H, 2′ and 7′-ArH), 6.94(d,
2H, 1′ and 8′-ArH), 7.77(s, 1H, 5-ArH), 3.41(t, 2H, -CH2-),
2.44(t, 2H, -CH2-), 1.85–1.87(m, 2H, -CH2-), 1.41(s, 18H, -
tBuH). FT-IR(KBr), υ/cm−1: 3386, 3215, 2975, 2875, 1786,
1609, 1641, 1567, 1478, 1425, 1212, 1092, 1058, 881, 728.
1
1
compound 5at (3.84, 84.5 %)as a white solid. H NMR
(400 MHz, CDCl3) δ: 7.06(s, 2H, 4′ and 5′-ArH), 6.86(d,
2H, 1′ and 8′-ArH), 6.81(d, 2H, 2′ and 7′-ArH), 7.76(s, 1H,
5-ArH), 3.55(t, 2H, -CH2-), 2.49(t, 2H, -CH2-), 1.95–
1.99(m, 2H, -CH2-), 1.36(s, 18H, -tBuH). FT-IR(KBr),
υ/cm−1: 3380, 2974, 2935, 2875, 1780, 1758, 1652, 1611,
1547, 1496, 1423, 1373, 1218, 1157, 1115, 1027, 992, 896.
Synthesis of 4′, 5′, 4, 7-tetrachloro-dipivaloyl-6-
(2-carboxyethylaminocarbonyl) fluorescein (5cp)
Synthesis of 4, 7-dichloro-dipivaloyl-6-
Compound (5cp) was prepared from compound 4c and 3-
(2-carboxyethylaminocarbonyl) fluorescein (5ap)
aminopropanoic acid by the procedure utilized for compound
5at. Compound 5cp, white solid, yield, 84.2 %, H NMR
1
Compound (5ap) was prepared from compound 4a and 3-
aminopropanoic acid by the procedure utilized for com-
pound 5 at. Compound 5ap (3.69, 83 %) was obtained as
(400 MHz, CDCl3) δ: 6.82(s, 2H, 2′ and 7′-ArH), 6.93(d, 2H,
1′ and 8′-ArH), 7.82(s, 1H, 5-ArH), 3.78(t, 2H, -CH2-), 2.80(t,
2H, -CH2-), 1.41(s, 18H, -tBuH). FT-IR(KBr), υ/cm−1: 3424,
2977, 1767, 1600, 1477, 1425, 1398, 1096, 1052, 666.
1
white solid. H NMR (400 MHz, CDCl3) δ: 7.07(s, 2H, 4′
and 5′-ArH), 6.87(d, 2H, 1′ and 8′-ArH), 6.82(d, 2H, 2′ and
7′-ArH), 7.79(s, 1H, 5-ArH), 3.78(t, 2H, -CH2-), 2.78(t, 2H,
-CH2-),1.36(s, 18H, -tBuH). FT-IR(KBr), υ/cm−1: 2976,
1758, 1612, 1495, 1424, 1157, 1115, 730.
Synthesis of 2′, 4′, 5′, 7′, 4, 7-hexachloro-dipivaloyl-6-
(3-carboxypropylaminocarbonyl) fluorescein (5dt)
Synthesis of 2′, 7′, 4, 7-tetrachloro-dipivaloyl-6-
(3-carboxypropylaminocarbonyl) fluorescein (5bt)
Compound (5dt) was prepared from compound 4d and 4-
aminobutenoic acid by the procedure utilized for compound
1
5at. Compound 5dt, white solid, yield, 80 %, H NMR
Compound (5bt) was prepared from compound 4b and 4-
aminobutenoic acid by the procedure utilized for compound
5at. Compound 5bt (4.09 g, 82 %) was obtained as white
solid. 1H NMR (400 MHz, CDCl3) δ: 7.05(s, 2H, 4′ and 5′-
ArH), 6.95(d, 2H, 1′ and 8′-ArH), 7.72(s, 1H, 5-ArH), 3.47(t,
2H, -CH2-), 2.52(t, 2H, -CH2-), 1.20–1.25(m, 2H, -CH2-),
1.35(s, 18H, -tBuH). FT-IR(KBr), υ/cm−1: 3417, 2975,
2935, 2874, 1786, 1609, 1573, 1477, 1409, 1201, 1168,
1093, 1028, 896, 685.
(400 MHz, CDCl3) δ: 6.87(s, 2H, 1′ and 8′ -ArH), 7.83(s,
1H, 5-ArH), 3.57(t, 2H, -CH2-), 1.96–2.00(m, 2H, -CH2-),
2.53(t, 2H, -CH2-), 1.44(s, 18H, -tBuH). FT-IR(KBr), υ/cm−1:
3371, 2977, 1778, 1657, 1547, 1454, 1424, 1373, 1211, 1079,
1026, 754. Anal. Calcd for C35H29Cl6NO10: C 50.26, H 3.50,
N 1.67; Found: C 50.28, H 3.51, N 1.66.
Synthesis of 2′, 4′, 5′, 7′, 4, 7-hexachloro-dipivaloyl-6-
(2-carboxyethylaminocarbonyl) fluorescein (5dp)
Synthesis of 2′, 7′, 4, 7-tetrachloro-dipivaloyl-6-
(2-carboxyethylaminocarbonyl) fluorescein (5bp)
Compound (5dp) was prepared from compound 4d and 3-
aminopropanoic acid by the procedure utilized for compound
1
Compound (5bp) was prepared from compound 4b and 3-
5at. Compound 5dp, yield, 84.2 %, H NMR (400 MHz,
aminopropanoic acid by the procedure utilized for compound
5at. Compound 5bp, white solid, yield, 86 %, H NMR
(400 MHz, CDCl3) δ: 7.13 (s, 2H, 4′ and 5′-ArH), 6.93 (d,
2H, 1′ and 8′-ArH), 7.84(s, 1H, 5-ArH), 3.78(t, 2H, -CH2-),
CDCl3) δ: 6.89(s, 2H, 1′ and 8′ -ArH), 7.87(s, 1H, 5-ArH),
3.82(t, 2H, -CH2-), 2.79(t, 2H, -CH2-), 1.45(s, 18H, -tBuH).
FT-IR(KBr), υ/cm−1: 2976, 1775, 1453, 1426, 1261, 1211,
1081, 874.
1

J Fluoresc
Compound 4, 7-dichloro -6-(3-carboxypropylaminocarbonyl)
IR(KBr), υ/cm−1: 3420, 1740, 1606, 1489, 1433, 1376, 1267,
fluorescein (6at)
1171, 1138, 953, 867, 690.
Compound 5at (4.19 g, 6 mmol) was dissolved in 50 mL of
CH2Cl2. To the solution was added 10 mL of NH4OH (28 %),
and the mixture was stirred for 2 h. The reaction mixture was
diluted with 60 mL of water. The water layer was acidified
with 10 % HCl to pH 2. The resulting precipitate was collect-
ed, washed with cold water (3×15 mL), and dried in vacuum.
6at as an orange solid was obtained in 78 % yield (2.48 g). 1H
NMR (400 MHz, DMSO-d6) δ: 10.19(s, 1H, -COOH), 6.77(s,
2H, 4′ and 5′-ArH), 6.67(d, 2H, 1′ and 8′-ArH), 6.57(d, 2H, 2′
and 7′-ArH), 7.92(s, 1H, 5-ArH), 3.29(t, 2H, -CH2-), 2.32(t,
2H, -CH2-), 1.72–1.79(m, 2H, -CH2-). MALDI-TOF MS,
m/z: 530.87 (Calcd:530.31). FT-IR(KBr), υ/cm−1: 3379,
2960, 1732, 1687, 1609, 1560, 1505, 1449, 1377, 1224,
1177, 1138, 1112, 992, 923, 844, 664.
Compound 4′, 5′, 4, 7-tetrachloro-6-
(3-carboxypropylaminocarbonyl) fluorescein (6ct)
Starting from compound 5ct and using a similar procedure as
described above, compound (6ct) was obtained in 82 % yield
1
(2.94 g). H NMR (400 MHz, DMSO-d6) δ: 11.08(s, H, -
COOH), 6.84(s, 2H, 2′ and 7′ -ArH), 6.87(s, 2H, 1′ and 8′-
ArH), 7.90(s, 1H, 5-ArH), 3.19(t, 2H, -CH2-), 2.22(t, 2H, -
CH2-), 1.62–1.69(m, 2H, -CH2-). MALDI-TOF MS, m/z:
599.40 (Calcd: 599.2). FT-IR(KBr), υ/cm−1: 3427, 3065, 1737,
1629, 1610, 1503, 1432, 1265, 1178, 1121, 1046, 876, 744.
Compound 4′, 5′, 4, 7-tetrachloro-6-
Compound 4, 7-dichloro -6-(2-carboxyethylaminocarbonyl)
(2-carboxyethylaminocarbonyl) fluorescein (6cp)
fluorescein (6ap)
Starting from compound 5cp and using a similar procedure as
described above, compound (6cp) was obtained in 78 % yield
(2.74 g). H NMR (400 MHz, DMSO-d6) δ: 10.99(s, 1H, -
COOH), 6.83(s, 2H, 2′ and 7′ -ArH), 6.92(s, 2H, 1′ and 8′-
ArH), 7.89(s, 1H, 5-ArH), 3.46(t, 2H, -CH2-), 2.48(t, 2H, -
CH2-), 1.62–1.69(m, 2H, -CH2-). MALDI-TOF MS, m/z:
588.44 (Calcd: 585.17).
Starting from compound 5ap and using a similar procedure as
described above, compound (6ap) was obtained in 75 % yield
(2.32 g). H NMR (400 MHz, DMSO-d6) δ: 10.21(s, H, -
COOH), 6.66(s, 2H, 4′ and 5′-ArH), 6.76(d, 2H, 1′ and 8′-
ArH), 6.55(d, 2H, 2′ and 7′-ArH), 7.84(s, 1H, 5-ArH), 3.40(t,
2H, -CH2-), 2.54(t, 2H, -CH2-). MALDI-TOF MS, m/z:
516.92 (Calcd:516.28). FT-IR(KBr), υ/cm−1: 3343, 3080,
1741, 1608, 1506, 1450, 1376, 1227, 1180, 1140, 1113, 994,
930, 846, 813, 665.
1
1
Compound 2′, 4′, 5′, 7′, 4, 7-hexachloro-6-
(3-carboxypropylaminocarbonyl) fluorescein (6dt)
Compound 2′, 7′, 4, 7-tetrachloro-6-
(3-carboxypropylaminocarbonyl) fluorescein (6bt)
Starting from compound 5dt and using a similar procedure as
described above, compound (6dt) was obtained in 76 % yield
(3.04 g). 1H NMR (400 MHz, DMSO-d6) δ: 7.11(s, 2H, 1′ and
8′ -ArH), 7.95(s, 1H, 5-ArH), 3.45(t, 2H, -CH2-), 1.72-
1.80(m, 2H, -CH2-), 2.35(t, 2H, -CH2-). MALDI-TOF MS,
m/z: 669.00, (Calcd: 668.09). FT-IR(KBr), υ/cm−1: 3579,
3333, 1772, 1646, 1559, 1436, 1198, 1150, 1092, 749. Anal.
Calcd for C25H13Cl6NO8: C 44.94, H 1.96, N 2.10; Found: C
44.95, H 1.95, N 2.09.
Starting from compound 5bt and using a similar procedure as
described above, compound (6bt) was obtained in 83 % yield
1
(2.98 g). H NMR (400 MHz, DMSO-d6) δ: 11.11(s, 1H, -
COOH), 7.21(s, 2H, 4′ and 5′-ArH), 6.86(s, 2H, 1′ and 8′-
ArH), 8.04(s, 1H, 5-ArH), 3.17(t, 2H, -CH2-), 2.23(t, 2H, -
CH2-), 1.64–1.74(m, 2H, -CH2-). MALDI-TOF MS, m/z:
600.47 (Calcd: 599.2). FT-IR(KBr), υ/cm−1: 3419, 3302,
1752, 1705, 1656, 1602, 1558, 1432, 1310, 1231, 1179,
1131, 1056, 1032, 918, 810, 725, 663.
Compound 2′, 4′, 5′, 7′, 4, 7-hexachloro-6-
Compound 2′, 7′, 4, 7-tetrachloro-6-
(2-carboxyethylaminocarbonyl) fluorescein (6dp)
(2-carboxyethylaminocarbonyl) fluorescein (6bp)
Starting from compound 5dp and using a similar procedure as
described above, compound (6dp) was obtained in 76 % yield
(2.98 g). H NMR (400 MHz, DMSO-d6) δ: 10.99(s, 1H, -
COOH), 7.26(s, 2H, 1′ and 8′ -ArH), 7.87(s, 1H, 5-ArH),
3.47(t, 2H, -CH2-), 2.54(t, 2H, -CH2-). MALDI-TOF MS,
m/z: 654.28, (Calcd: 654.06). FT-IR(KBr), υ/cm−1: 3410,
1769, 1642, 1476, 1433, 1213, 1141, 101, 863.
Starting from compound 5bp and using a similar procedure as
described above, compound (6bp) was obtained in 77 % yield
(2.70 g). H NMR (400 MHz, DMSO-d6) δ: 11.13(s, 1H, -
COOH), 7.23(s, 2H, 4′ and 5′-ArH), 7.10(s, 2H, 1′ and 8′-
ArH), 8.12(s, 1H, 5-ArH), 3.62(t, 2H, -CH2-), 2.56(t, 2H, -
CH2-). MALDI-TOF MS, m/z: 585.60 (Calcd: 585.17). FT-
1
1

J Fluoresc
1
Synthesis of 4-(4, 7-dichlorofluorescein-6-carboxamido)
orange solid in 75 % yield (2.56 g). HNMR (400 MHz,
-butanoic acid succinimidyl ester (7at)
DMSO-d6) δ: 11.14(s, H, -COOH), 7.10(s, 2H, 4′ and 5′-
ArH), 6.88(d, 2H, 1′ and 8′-ArH), 7.89(s, 1H, 5-ArH),
3.60(t, 2H, -CH2-), 3.04(t, 2H, -CH2-), 2.82(t, 4H, -
CH2CH2-). MALDI-TOF MS, m/z: 683.08, (Calcd: 682.25).
FT-IR (KBr), υ/cm−1: 3378, 2939, 1814, 1772, 1735, 1612,
1506, 1450, 1376, 1215, 1179, 1067, 850, 650.
To compound 6at (2.65 g, 5 mmol) in 50 mL anhydrous
dichloromethane was added anhydrous pyridine (16 mL)
and N-hydroxysuccinimidyl trifluoroacetate (NHS-TFA)
(8.44 g, 40 mmol) under vigorous stirring. The resultant
mixture was stirred at room temperature for 6 h. TLC (CHCl3/
MeOH=5:1) was used to confirm the completion of the reac-
tion. The mixture was washed with 4 % HCl (2×100 mL) and
saturated saline (2×50 mL), dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure, and residue
was dried in vacuum to yield compound 7at as a yellow solid
Compound 4-(4′, 5′, 4, 7-tetrachlorofluorescein-6-
carboxamido)-butanoic acid succinimidyl ester (7ct)
Starting from compound 6ct and using a similar procedure as
described above, compound (7ct) was obtained as a salmon
pink solid in 78 % yield (2.72 g). 1HNMR (400 MHz, DMSO-
d6) δ: 11.01(s, 1H, -COOH), 6.87(s, 2H, 2′ and 7′-ArH),
6.82(d, 2H, 1′ and 8′-ArH), 7.96(s, 1H, 5-ArH), 3.26(t, 2H, -
CH2-), 2.72(t, 2H, -CH2-), 1.79–1.83(m, 2H, -CH2-), 2.80(t,
4H, -CH2CH2-). MALDI-TOF MS, m/z: 697.59, (Calcd:
696.27). FT-IR(KBr), υ/cm-1: 3410, 3310, 1739, 1658,
1601, 1563, 1433, 1371, 1205, 1132, 1059, 645.
1
in 80 % yield (2.51 g). H NMR (400 MHz, DMSO-d6) δ:
10.19(s, 1H, -COOH), 6,78(s, 2H, 4′ and 5′-ArH), 6.67(d, 2H,
1′ and 8′-ArH), 6.57(d, 2H, 2′ and 7′-ArH), 7.99(s, 1H, 5-
ArH), 3.34(t, 2H, -CH2-), 2.59(t, 2H, -CH2-), 1.87–1.90(m,
2H, -CH2-), 2.82(t, 4H, -CH2CH2-). MALDI-TOF MS, m/z:
627.33, (Calcd: 627.38). FT-IR(KBr), υ/cm−1: 3408, 2938,
1812, 1768, 1734, 1613, 1507, 1449, 1376, 1216, 1181, 1070,
994, 668.
Compound 3-(4′, 5′, 4, 7-tetrachlorofluorescein-6-
carboxamido)-propanoic acid succinimidyl ester (7cp)
Compound 3-(4, 7-dichlorofluorescein-6-carboxamido)
-propanoic acid succinimidyl ester (7ap)
Starting from compound 6cp and using a similar procedure as
described above, compound (7cp) was obtained as a salmon
pink solid in 76 % yield (2.59 g). 1HNMR (400 MHz, DMSO-
d6) δ: 11.04(s, 1H, -COOH), 7.17(d, 2H, 2′ and 7′-ArH),
6.92(d, 2H, 1′ and 8′-ArH), 7.95(s, 1H, 5-ArH), 3.45(t, 2H, -
CH2-), 2.60(t, 2H, -CH2-), 2.81(t, 4H, -CH2CH2-). MALDI-
TOF MS, m/z: 683.65, (Calcd: 682.25). FT-IR(KBr), υ/cm−1:
3401, 1773, 1738, 1708, 1605, 1434, 1217, 1060.
Starting from compound 6ap and using a similar procedure as
described above, compound (7ap) was obtained as a yellow
solid in 78 % yield (2.39 g). 1H NMR (400 MHz, DMSO-d6)
δ: 10.20(s, 1H, -COOH), 6,78(s, 2H, 4′ and 5′-ArH), 6.67(d,
2H, 1′ and 8′-ArH), 6.56(d, 2H, 2′ and 7′-ArH), 7.90(s, 1H, 5-
ArH), 3.59(t, 2H, -CH2-), 3.05 (t, 2H, -CH2-), 2.82(t, 4H, -
CH2CH2-). MALDI-TOF MS, m/z: 613.54, (Calcd: 613.36).
FT-IR(KBr), υ/cm−1: 3383, 1814, 1769, 1735, 1612, 1507,
1450, 1375, 1214, 1181, 1072, 993, 850.
Compound 4-(2′, 4′, 5′, 7′, 4, 7-hexachlorofluorescein-6-
carboxamido)-butanoic acid succinimidyl ester (7dt)
Compound 4-(2′, 7′, 4, 7-tetrachlorofluorescein-6-
carboxamido)-butanoic acid succinimidyl ester (7bt)
Starting from compound 6dt and using a similar procedure as
described above, compound (7dt) was obtained as a red solid
in 80 % yield (3.06 g). H NMR (400 MHz, DMSO-d6) δ:
11.22(s, 1H, -COOH), 8.78(s, 1H, ArOH), 8.00(s, 1H, 5-
ArH), 7.29(s, 2H, 1′ and 8′ -ArH), 3.44(t, 2H, -CH2-), 1.88–
1.91(m, 2H, -CH2-), 2.73(t, 2H, -CH2-), 2.82(t, 4H, -
CH2CH2-). MALDI-TOF MS, m/z: 766.36, (Calcd: 765.16).
FT-IR(KBr), υ/cm−1: 3318, 2981, 1785, 1732, 1649, 1600,
1430, 1207, 1064, 747, 868, 649.
1
Starting from compound 6bt and using a similar procedure as
described above, compound (7bt) was obtained as a deep orange
solid in 75 % yield (2.61 g). 1HNMR (400 MHz, DMSO-d6) δ:
11.03(s, 1H, -COOH), 6.18(s, 2H, 4′ and 5′-ArH), 6.87(d, 2H, 1′
and 8′-ArH), 7.92(s, 1H, 5-ArH), 3.42(t, 2H, -CH2-), 2.59(t, 2H, -
CH2-), 1.81–1.84(m, 2H, -CH2-), 2.81(t, 4H, -CH2CH2-).
MALDI-TOF MS, m/z: 697.42, (Calcd: 696.27). FT-IR (KBr),
υ/cm−1: 3429, 2929, 1812, 1764, 1734, 1630, 1608, 1530, 1435,
1379, 1204, 1141, 1066, 960, 681.
Compound 3-(2′, 4′, 5′, 7′, 4, 7-hexachlorofluorescein-6-
carboxamido)-propanoic acid succinimidyl ester (7dp)
Compound 3-(2′, 7′, 4, 7-tetrachlorofluorescein-6-
carboxamido)-propanoic acid succinimidyl ester (7bp)
Starting from compound 6dp and using a similar procedure as
described above, compound (7dp) was obtained as a red solid
in 79 % yield (2.97 g). H NMR (400 MHz, DMSO-d6) δ:
1
Starting from compound 6bp and using a similar procedure as
described above, compound (7bp) was obtained as a deep
11.21(s, 1H, -COOH), 8.73(s, 1H, ArOH), 7.90(s, 1H, 5-

J Fluoresc
ArH), 7.29(s, 2H, 1′ and 8′ -ArH), 3.51(t, 2H, -CH2-), 2.59(t,
2H, -CH2-), 2.81(t, 4H, -CH2CH2-). MALDI-TOF MS, m/z:
751.65, (Calcd: 751.14). FT-IR (KBr), υ/cm−1: 3339, 1816,
1707, 1430, 1216, 1080, 1001, 867, 813.
wavelength was 470 nm. For determination of the fluores-
cence quantum yields, fluorescein in 0.1 M NaOH (ΦF=0.79)
was used as a standard [19]. Fluorescence quantum yields
were obtained from multiple measurements (N=3) with the
following equation [20–24]
h
i
X
Preparation of Working Solution
AbsðstandardÞ
FðsampleÞ
ϕðsampleÞ ¼ ϕðstandardÞ
h
i
X
The 1.25×10−3 mol L−1 sample stock solutions were prepared
by dissolving the sample in dimethyl sulfoxide (DMSO) and
further diluted with distilled water to result in 1.25×
10−5 mol L−1 stock solutions of sample containing 1 % DMSO.
Working solutions, which were used to study spectroscopic
properties and measure fluorescence quantum yields, were
prepared by diluting with 0.1 M NaOH. The solution concen-
tration of these solutions was 5.0×10−7 mol L−1. All working
solutions were prepared immediately before the experiment.
AbsðsampleÞ
FðstandardÞ
Where ΣF denotes integrated fluorescence intensity which
is the area of the fluorescence spectrum from the fully
corrected fluorescence spectra. In the equation, Abs denotes
absorbance at the excitation wavelength (470 nm). The Abs
was obtained from the absorption spectra.
Results and Discussion
Fluorescence Properties and Fluorescence Quantum Yields
Synthesis and Chemical Properties of Probes
Fluorescence spectroscopic studies were performed on a
Hitachi F-4500 spectrofluorimeter. The slit width was 10 nm
for excitation and 5 nm for emission. The excitation
These chlorinated fluorescent probes were synthesized
according to the synthetic procedure shown in Scheme 1.
R1
R1
R1
R1
CH3
O
H3C
O
R1
R1
OH
R2
CH3
O
H3C
O
H3C
H3C
CH3
CH3
HO
R2
O
H3C
H3C
CH3
CH3
O
OH
O
O
O
O
O
R2
R1
O
O
O
R2
Cl
O
R2
R2
R2
OH
O
O
1M HCl
1. PiV2O
Cl
O
Cl
O
Cl
CH3
O
O
Cl
COOH
2. DIPA, EtOH
MeSO3H, N2
H3C
H3C
O
NH2
CH3
Cl
Cl
Cl
HOOC
HOOC
1a-1d
3a-3d
2a-2d
R1
Cl
R1
R1
R1
R1
R1
CH3
O
H3C
O
O
HO
R2
O
R2
H3C
H3C
CH3
HO
R2
O
O
O
CH3
O
R2
O
R2
R2
O
COOH
NHS-TFA
Pyridine
Cl
1. NH3 H2O
2. HCl
COOH
Cl
O
O
O
Cl
O
Cl
Cl
COOH
R1
Cl
R1
CH3
O
H3C
H3C
H3C
CH3
CH3
HN
7at-7dt
O
HN
O
O
O
HN
COOH
6at-6dt
COOH
O N
O
O
O
R2
R2
5at-5dt
H2N
NHS-TFA
Pyridine
O
O
O
Cl
O
H2N
O
O
N
R1
R1
R1
Cl
R1
R1
Cl
R1
CH3
O
COOH
H3C
O
O
HO
R2
H3C
H3C
CH3
CH3
O
O
HO
R2
O
O
4a-4d
O
O
R2
R2
R2
R2
O
Cl
COOH
NHS-TFA
Pyridine
1 .NH3 H2O
2. HCl
COOH
Cl
O
O
O
Cl
O
Cl
COOH
O
O
HN
HN
HN
O
N
COOH
6ap-6dp
O
5ap-5dp
7ap-7dp
1a, 2a, 3a, 4a, 5at, 6at, 7at, 5ap, 6ap, 7ap: R1=H, R2=H
1b, 2b, 3b, 4b, 5bt, 6bt, 7bt, 5bp, 6bp, 7bp: R1=H, R2=Cl
1c, 2c, 3c, 4c, 5ct, 6ct, 7ct, 5cp, 6cp, 7cp: R1=Cl, R2=H
1d, 2d, 3d, 4d, 5dt, 6dt, 7dt, 5dp, 6dp, 7dp: R1=Cl, R2=Cl
Scheme 1 Synthesis of eight novel fluorescent proteins-labeling probes

J Fluoresc
Ta b l e 1 Spectral properties of eight probes
Compounds
em(nm)
7at
7ap
7bt
7bp
7ct
7cp
7dt
7dp
λ
524
508
508
16
524
508
508
16
534
534
539
522
522
17
539
522
522
17
550
550
λex(nm)
521
521
536
536
λab(nm)
521
521
536
536
Stokes shift(nm)
Abs(pH=13)
ε(M−1 cm−1×104)
13
13
14
14
0.067
8.43
0.72
0.044
8.33
0.72
0.022
14.99
0.71
0.041
14.23
0.71
0.074
8.05
0.68
0.086
8.10
0.68
0.051
15.66
0.65
0.053
15.92
0.65
a
ΦF (pH=13)
a
Fluorescent was used as a standard (ΦF=0.79 in 0.1 M NaOH), see reference [19]
There were two potential approaches to the synthesis of
Carboxyfluorescein was prepared and commonly used as
chlorinated fluoresceins. One method was chlorination
of the building blocks of fluorescein and then synthesis
of chlorinated fluoresceins; the other was the directed
chlorination of fluorescein. There were several active
positions on the fluorescein that are readily available
for direct chlorination by electrophilic chlorination re-
agents. However, the isolation of pure products from
chlorinated fluoresceins was a very difficult problem.
Therefore, we selected the first method in this work.
In this paper, chlorinated fluoresceins were synthesized by
condensation of the appropriate chlorinated resorcinol and 3,
6-dichloro-4-carboxyphthalic anhydride in the presence of
methanesulfonic acid. Methanesulfonic acid as a catalyzer
was reported in many references. It was found that
methanesulfonic acid served as both a Lewis acid catalyst
and a suitable solvent in the dye-forming reaction, giving a
good procedure with higher yields of products under milder
conditions.5 The high yield was obtained by gradual increase
of temperature. The better procedure was that the reaction
mixture was warmed and stirred under nitrogen at 50 °C for
4 h then at 110 °C for 24 h.
mixture of two isomers, namely 5- and 6-carboxyfluorescein.
A few reports suggested that two isomers have some differ-
ences in behavior and subsequent binding assays [25, 26].
These two isomers had almost identical spectroscopic proper-
ties, making a direct comparison of lectin-binding properties
possible. Working with single isomers also significantly sim-
plified chromatographic separations and subsequent charac-
terizations by NMR [27].
The literature[2, 28] reported that the 5(6)-isomers
can be separated at this point by selective crystallization
of the 6-isomer as its diisopropylamine salt from etha-
nol, and the 5-isomer can be recovered from the super-
natant and recrystallized from nitromethane. We success-
fully separated the 6-isomer as its diisopropylamine salt from
ethanol. However, we were never successful in obtaining the
5-isomer in pure form via this route.
When carboxyfluorescein was refluxed in pivalic anhy-
dride, make sure it was completely converted to its dipivalae.
Reflux time should be more than two hours and the pivalic
anhydride should be overdose. After diisopropylamine was
added, the mixture should be shaken several times and then
Fig. 1 Excitation and emission spectra of compounds 7ap and 7at.
Fig. 2 Fluorescent emission spectra of compounds 7ap, 7bp, 7cp and
7dp
Concentration of all solutions was 5.0×10−7 mol L−1

J Fluoresc
Fig. 3 Fluorescent microscopic
images of the living MC3T3-E1
cells directly labeled by 7ap in the
blue channels
the mixture was cooed to −20 °C in freezer overnight. Small-
scale test showed that the solubility of 5-isomer carboxyfluo-
rescein diisopropylamine salt is much better in ethanol at
room temperature than 6-isomer. If the 6-isomer carboxyflu-
orescein diisopropylamine salt is not very pure, pure 6-isomer
can be afforded by the treatment of it with ethanol (sonicated
for 10 min) several times.
fluorophore and succinimidyl ester group. The spacer
linker was designed to reduce potential interactions be-
tween the probe dye and the protein. 4-Aminobutanoic
acid or 3-amiopropanoic acid was used as a spacer
linker. This allowed us to prepare succinimidyl ester
derivatives, the most amine-selective and stable reactive
group for labeling proteins.
N-hydroxysuccinimide (NHS) esters are among the
most commonly used reagents for modification of amine
groups. These reagents have intermediate reactivity to-
ward amines, with high selectivity toward aliphatic
amines. However, their reaction rate with aromatic
amines, phenols (tyrosine), alcohols, and histidine is
relatively low [29]. In this work, our chlorinated fluo-
rescent probes were designed by introducing N-
hydroxysuccinimide as reactive group, which is the
most amine-selective and stable reactive group for la-
beling proteins, peptides, and other amines. The aliphat-
ic amide products are very stable, which are formed by
reacting NHS ester of the probe with the terminal amine
group. Although the NHS esters are slowly hydrolyzed
by water, they are stable to storage if kept well desic-
cated. Virtually any molecule that contains a carboxylic
acid or that can be chemically modified to contain a
carboxylic acid can be converted into its NHS ester,
making these reagents among the most powerful
protein-modification reagents available.
Spectral Properties of Eight Probes
The wavelength of fluorescence emission maxima, the wave-
length of fluorescence excitation maxima, the wavelength of
absorption maxima, Stokes shift, fluorescence quantum yields
and molar extinction coefficient are summarized in Table 1 for
these probes. All of the compounds showed in Table 1 had
excitation maxima in the range 508–536 nm, and their molar
extinction coefficients were high ((8.05–15.92) ×
104 M−1 cm−1). They exhibited interesting fluorescence prop-
erties at room temperature. Their fluorescence maxima were
located at 524–550 nm. Fluorescent quantum yields for these
compounds were all reasonably high for the fluorescent probes.
The chlorine fluorescein structure can be divided into
two parts, the benzene moiety and the fluorophore [30].
Compounds 7ap and 7at, which have the same
fluorophore but different benzene moieties, almost had
the same Ex / Em maximum (See Table 1 and Fig. 1).
Compounds 7bp and 7bt had the same phenomenon.
From Fig. 2, the chlorinated fluorescent probes were
found to shift the emission spectra towards long
In order to facilitate and improve conjugations, we
have added a spacer linker between the chlorinated
Fig. 4 Fluorescent microscopic
images of fixed MC3T3-E1 cells
directly labeled by 7ct in the
green channels

J Fluoresc
wavelength, and shift of the spectra toward long wave-
length increased with increase in chlorine. For example,
3-(2′, 4′, 5′, 7′, 4, 7-hexachlorofluorescein-6-
carboxamido)-propanoic acid succinimidyl ester (7dp)
with four chlorine substitutions on fluorophore had
emission maxima at 550 nm, however, 3-(4, 7-
dichlorofluorescein-6-carboxamido)-propanoic acid
succinimidyl ester (7ap) without chlorine atoms on
fluorophore had emission maxima at 524 nm. A red
shift of 26 nm was observed between compounds 7dp
and 7ap. The reason for this phenomenon might be the
electron-withdrawing ability of the chlorine substituted
on fluorophore. The structure of fluorophore played an
important role in fluorescent excitation and emission
spectra. However, the benzene moiety almost had no
effect on excitation and emission.
preparation of stable fluorescent conjugates. If these probes
are conjugated to anti-IgG antibodies, they can also be used in
immunofluorescent histochemistry.
Acknowledgments The project was supported by the National Natural
Science Foundation of China (No.21202130, 81073037, and 81202457),
the China Postdoctoral Science Foundation funded project (grant No.
2012M521806).
Competing financial interest Xianglong Wu and Min Tian are co-first
authors. The authors declare no competing financial interest.
Author contributions The manuscript was written through contribu-
tions of all authors. All authors have given approval to the final version of
the manuscript.
We chose compounds 7ap and 7ct for labeling ex-
periments. Firstly, we incubated the living MC3T3-E1
cells grown on a coverslip with 7ap at 25 °C for
45 min. Figure 3 showed that the probe is localized in
the nucleus. This was attributed to the fact that the
probe dye attached on the cell membrane was taken
up by the MC3T3-E1 cells and transferred into the cell
nucleus, while the cells maintain their characteristic
nematomorphic morphology and well-spread pattern.
These observations further indicated that the incubation
of MC3T3-E1 cells with the fluorescent 7ap causes
almost no harmful effect to the essential activities of
MC3T3-E1 cells, such as cell attachment and spread.
This indicated that these chlorinate fluorescent dyes
could be a favorable intercellular imaging probe with
the ability of cell penetration. We also stained the
chemical fixed MC3T3-E1 with the compound 7ct (see
Fig. 4). To our surprise, we found that the dye is
localized in cytosol instead of nucleus. Certainly, under
the circumstances the dye might be only conjugates to
the cell surface.
In conclusion, we have synthesized eight novel chlorinated
fluorescent proteins-labeling probes with a linker and reactive
group in 7 steps. Thirty-two intermediate compounds were
also prepared. To our knowledge, most of compounds have
not been reported.
The spectral properties of the chlorinated fluoresceins were
studied in detail. These fluorescent probes showed absorbance
peaks at 508–536 nm and emission peaks at 524–550 nm with
high quantum yields. We also confirmed that red shift of emis-
sion, 26 nm, occurs depending on the increase of chlorine atom.
The probes were used for fluorescence imaging of cells in
order to investigate whether they can conjugate to cells. The
fluorescence imaging of living cells showed that probes are
localized in cell nucleus. However, the imaging of fixed cells
showed that probes are localized in cytosol of chemically
fixed cells. These probes will be useful reagents for the
References
1. Sednev MV, Wurm CA, Belov VN, Hell SW (2013) Carborhodol: a
new hybrid fluorophore obtained by combination of fluorescein and
carbopyronine dye cores. Bioconjug Chem 24:690–700
2. Rossi FM, Kao JPY (1997) Practical method for the multigram
separation of the 5- and 6-isomers of carboxyfluorescein.
Bioconjug Chem 8:495–497
3. Woodroofe CC, Lim MH, Bu W, Lippard SJ (2005) Synthesis of
isomerically pure carboxylate- and sulfonate-substituted xanthene
fluorophores. Tetrahedron 61:3097–3105
4. Mattingly PG (1992) Preparation of 5- and 6-(aminomethy1)fluorescein.
Bioconjug Chem 3:430–431
5. Sun W-C, Gee KR, Klaubert DH, Haugland RP (1997) Synthesis of
fluorinated fluoresceins. J Org Chem 62:6469–6475
6. Murgia S, Bonacchi S, Falchi AM, Lampis S, Lippolis V, Meli W,
Monduzzi M, Prodi L, Schmidt J, Talmon Y, Caltgairone C (2013)
Drug loaded fluorescent cubosomes: versatile nanoparticles for po-
tential theranostic applications. Langmuir 29:6673–6679
7. Zhao Y, Hammoudeh D, Lin W, Das S, Yun MK, Li Z, Griffith E,
Chem T, White SW, Lee RE (2011) Development of a pterin-based
fluorescent probe for screening dihydropteroate synthase. Bioconjug
Chem 22:2110–2117
8. Marchand P, Becerril-Ortega J, Mony L, Bouteiller C, Paoletti P,
Nicole O, Barré L, Buisson A, Perrio C (2011) Confocal microscopy
imaging of NR2B-containing NMDA receptors based on fluorescent
ifenprodil-like conjugates. Bioconjug Chem 23:21–26
9. Sakabe M, Asanuma D, Kamiya M, Iwatate RJ, Hanaoka K,
Terai T, Nagano T, Urano Y (2012) Rational design of highly
sensitive fluorescence probes for protease and glycosidase
based on precisely controlled spirocyclization. J Am Chem
Soc 135:409–414
10. Gao HZ, Yang KW, Wu XL, Liu JY, Feng L, Xiao JM, Zhou LS, Jia
C, Shi Z (2011) Novel conjugation of norvancomycin - fluorescein
for photodynamic inactivation of bacillus subtilis. Bioconjug Chem
22:2217–2221
11. Lyttle MH, Carter TG, Cook RM (2001) Improved synthetic proce-
dures for 4, 7, 2′, 7′-tetrachloro- and 4′, 5′-dichloro-2′, 7′-dimethoxy-
5(and 6)-carboxyfluoresceins. Org Process Res Dev 5:45–49
12. Ge FY, Chen LG (2008) pH Fluorescent probes: chlorinated fluores-
ceins. J Fluoresc 18:741–747
13. Wu XL, Jin XL, Wang YX, Mei QB, Li JL, Shi Z (2011) Synthesis
and spectral properties of novel chlorinated pH fluorescent probes. J
Lumin 131:776–780

J Fluoresc
14. Tian M, Wu XL, Zhang B, Li JL, Shi Z (2008) Synthesis of chlori-
nated fluoresceins for labeling proteins. Bioorg Med Chem Lett 18:
1977–1979
15. Wu XL, Tian M, He HZ, Sun W, Li JL, Shi Z (2009) Synthesis and
biological applications of two novel fluorescent proteins-labeling
probes. Bioorg Med Chem Lett 19:2957–2959
16. Tang CH, Yang RS, Liou HC, Fu WM (2003) Enhancement of
fibronectin synthesis and fibrillogenesis by BMP - 4 in cultured rat
osteoblast. J Bone Miner Res 18:502–511
17. Tang CH, Yang RS, Chien MY, Chen CC, Fu WM (2008)
Enhancement of bone morphogenetic protein - 2 expression
and bone formation by coumarin derivatives via p38 and
ERK-dependent pathway in osteoblasts. Eur J Pharmacol
579:40–49
23. Miura T, Urano Y, Tanaka K, Nagano T, Ohkubo K, Fukuzumi S
(2003) Rational design principle for modulating fluorescence prop-
erties of fluorescein-based probes by photoinduced electron transfer.
J Am Chem Soc 125:8666–8671
24. Bandichhor R, Petrescu AD, Vespa A, Kier AB, Schroeder F,
Burgess K (2006) Synthesis of a new water-soluble rhodamine
derivative and application to protein labeling and intracellular imag-
ing. Bioconjug Chem 17:1219–1225
25. Ajtai K, Ilich PJ, Ringler A, Sedarous SS, Toft DJ, Burghardt TP
(1992) Stereospecific reaction of muscle fiber proteins with the 5′ or
6′ isomer of (iodoacetamido) tetramethylrhodamine. Biochemistry
31:12431–12440
26. Buolamwini JK, Craik JD, Wiley JS, Robins MJ, Gati WP, Cass CE,
Paterson ARP (1994) Conjugates of fluorescein and SAENTA (5′-S-
(2-aminoethyl) N6- (4-nitrobenzyl)-5′-thioadenosine): flow cytome-
try probes for the ES nucleoside transport elements of the plasma
membrane. Nucleosides Nucleotides 13:737–751
18. Yewle JN, Puleo DA, Bachas LG (2011) Enhanced affinity bifunc-
tional bisphosphonates for targeted delivery of therapeutic agents to
bone. Bioconjug Chem 22:2496–2506
19. Umberger JQ, LaMer VK (1945) The kinetics of diffusion controlled
molecular and Ionic reactions in solution as determined by
measurements of the quenching of fluorescence. J Am Chem
Soc 67:1099–1109
20. Li L, Han J, Nguyen B, Burgess K (2008) Syntheses and spectral
properties of functionalized, water-soluble BODIPY derivatives. J
Org Chem 73:1963–1970
21. Lee H, Mason JC, Achilefu S (2006) Heptamethine cyanine dyes
with a robust C–C bond at the central position of the chromophore. J
Org Chem 71:7862–7865
22. Tanaka K, Miura T, Umezawa N, Urano Y, Kikuchi K, Higuchi T,
Nagano T (2001) Rational design of fluorescein-based fluorescence
probes. Mechanism-based design of a maximum fluorescence probe
for singlet oxygen. J Am Chem Soc 123:2530–2536
27. Öberg CT, Carlsson S, Fillion E, Leffler H, Nilsson UJ (2003)
Efficient and expedient two-step pyranose-retaining fluorescein con-
jugation of complex reducing oligosaccharides: galectin oligosaccha-
ride specificity studies in a fluorescence polarization assay.
Bioconjug Chem 14:1289–1297
28. Adamczyk M, Chan CM, Fino JR, Mattingly PG (2000) Synthesis of
5-and 6-hydroxymethylfluorescein phosphoramidites. J Org Chem
65:596–601
29. Brinkley M (1992) A brief survey of methods for preparing protein
conjugates with dyes, haptens and crosslinking reagents. Bioconjug
Chem 3:2–13
30. Urano Y, Kamiya M, Kanda K, Ueno T, Hirose K, Nagano T (2005)
Evolution of fluorescein as a platform for finely tunable fluorescence
probes. J Am Chem Soc 127:4888–4894