Inorganic Chemistry
Article
Analysis and Measurements. Various spectroscopic techniques
2H, J = 7.4 Hz), 7.83 (dd, 2H, J = 1.7 and 8 Hz). 13C NMR (125
MHz, CDCl , 25 °C, Si(CH ) ) δ: 68.72, 115.91, 121.45, 125.70,
were employed to characterize the newly synthesized compounds.
3
3 4
1
13
Both H NMR (500 MHz) and C NMR (125 MHz) spectra of the
134.42, 151.96. Anal. Calcd for C H N O : C, 55.27; H, 3.98; N,
14 12 2 6
compounds were recorded on a JEOL spectrometer in CDCl with
9.21%. Found: C, 55.39; H, 4.05; N, 9.11%.
3
tetramethylsilane as the internal standard. The ESI-mass data were
obtained in methanol from a Waters Q-Tof Premier mass
spectrometer. UV−visible spectra were recorded on a Shimadzu
Synthesis of 3. To a solution of 2 (15 g, 49.34 mmol) in 200 mL of
ethyl acetate was added 10% Pd/C (1.5 g), and the reaction mixture
was heated under N2 atmosphere. To this solution was added
dropwise 15 mL of N H ·H O over 30 min. Once the addition was
2
450 UV−vis spectrophotometer in aqueous buffer solution at 298 K.
2
4
2
Steady-state fluorescence spectra were obtained using a PerkinElmer
LS 50B luminescence spectrometer at 298 K. The excitation
wavelength was 460 nm, and the spectra were recorded in the range
complete, the mixture was heated to reflux for 3 h. It was then filtered
under hot condition and washed with hot methanol. The combined
filtrate upon evaporation gave a solid, which was recrystallized from
methanol to obtain about 10.9 g of white crystalline solid. Yield ∼90%.
4
80−650 nm. Fluorescence quantum yields were determined by
1
comparing the corrected spectra with that of pure Rhodamine B in
ethanol taking the total area under the curve and using eq i
Mp: 132 °C. H NMR (500 MHz, CDCl , 25 °C, Si(CH ) ) δ: 3.67
3
3 4
16
13
(br, s, 4H), 4.36 (s, 4H), 6.72−6.74 (m, 4H), 6.82−6.87 (m, 4H). C
NMR (125 MHz, CDCl , 25 °C, Si(CH ) ) δ: 67.52, 112.56, 115.46,
3
3 4
2
Φ = Φ (F A /F A )(η /η )
118.48, 122.00, 136.89, 146.33. Anal. Calcd for C14
H N O : C, 68.83;
16 2 2
S
R
S
R
R
S
S
R
(i)
H, 6.60; N, 11.47%. Found: C, 68.94; H, 6.87; N, 11.36%.
Synthesis of 4. Pyridine-2-aldehyde (11.70 mL, 122.81 mmol) was
added to a solution of 3 (10 g, 40.93 mmol) in 150 mL of methanol,
and the solution was heated to reflux for 12 h. After the reaction
where Φ stands for quantum yield, F stands for area under the curve, A
stands for absorbance value, and η stands for the refractive index value.
The subscript “R” indicates the value of the parameter for reference
mixture was cooled to room temperature, excess NaBH was added
(
i.e., Rhodamine-B), and “S” subscript indicates value of the parameter
4
portionwise with stirring. After stirring for about 6 h at room
temperature, the solvent was evaporated and the residue dissolved in
dichloromethane. The organic layer was washed several times with
for the sample.
Cell Culture and Imaging. HeLa cell line was cultured in
Dulbeco’s modified Eagle’s medium (DMEM, WelGene) supple-
mented with 10% FBS (fetal bovine serum, WelGene), 1% penicillin
water and then dried over anhydrous Na SO . Finally it was
2
4
completely evaporated off to obtain a brown colored solid. The
solid was subjected to column chromatography using basic alumina
with dichloromethane:hexane (3:2 v/v) as the eluent to obtain
(
3
100 units/mL), and streptomycin (100 μg/mL). Cells were grown at
7 °C under humidified atmosphere in 5% CO . Before imaging, the
2
cells were placed for 48 h on Delta T dishes (Bioptechs). Cells were
incubated with 5 μM probe (1% DMSO buffer solution) for 30 min,
and probes not taken up by the cells were removed by washing three
compound 4 as a light yellow crystalline solid. Yield ∼70%. Mp: 135
1
°
C. H NMR (500 MHz, CDCl , 25 °C, Si(CH ) ) δ: 4.41 (s, 4H),
3
3 4
times with PBS solution. Aqueous solutions of Hg2 and Cd were
+
2+
4.47 (s, 4H), 6.48 (d, 2H, J = 8 Hz), 6.65 (t, 2H, J = 8 Hz), 6.80 (t,
H, J = 7.4 Hz), 6.87 (d, 2H, J = 7.4 Hz), 7.08 (t, 2H, J = 6.8 Hz), 7.20
d, 2H, J = 8 Hz), 7.47 (t, 2H, J = 7.4 Hz), 8.50 (d, 2H, J = 4.6 Hz).
2
(
treated for an additional 30 min. Cell images were obtained with a
confocal microscope (Leica TCS SP2 model) fitted with a 100× oil
lens (numerical aperture = 1.30). Fluorescence images were collected
at 480−650 nm range following excitation at 458 nm. Internal
photomultiplier tubes (PMTs) were used to collect the signals in 8-bit
unsigned 512 × 512 pixels at 400 Hz scan speed.
Synthesis. The synthesis of L can be achieved in multisteps as
outlined in Scheme 1.
Synthesis of 1. The BODIPY dye 1, [(8-(4-carboxyphenyl)-1,3,7,9-
13
C NMR (125 MHz, CDCl , 25 °C, Si(CH ) ) δ: 46.26, 67.64,
3
3 4
110.70, 111.79, 116.71, 121.17, 121.99, 122.17, 136.71, 138.53, 145.97,
+
+
149.29, 159.3. ESI-mass (m/z): calcd 427.2134 [M + H ] , found
427.2131 (100%). Anal. Calcd for C26 : C, 73.22; H, 6.14; N,
13.14%. Found: C, 73.14; H, 6.31; N, 13.07%.
H N O
26 4 2
Synthesis of 5. To a solution of 4 (10 g, 23.45 mmol) in 200 mL of
dichloroethane (DCE) at room temperature was added pyridine-2-
tetramethyl-BODIPY)], was synthesized following the reported
aldehyde (6.7 mL, 70.31 mmol). After 1 h stirring, NaB(OAc) H
3
17
procedure. 4-Formylbenzoic acid (1.6 g, 10.66 mmol) and 2,4-
dimethylpyrrole (2.2 mL, 21.37 mmol) were added to freshly purified
CH Cl (300 mL) purged with N , a few drops of TFA were added to
(14.90 g, 70.31 mmol) and an additional 50 mL of DCE were added.
After overnight stirring, the solvent was removed under reduced
pressure, 100 mL water was added, and the pH was adjusted between
8 and 9 with saturated bicarbonate solution. The product was extracted
with chloroform (60 mL × 3). The combined organic fractions were
2
2
2
the mixture, and stirring was continued for 8 h at room temperature.
After TLC showing complete consumption of pyrrole, DDQ was
added to the reaction mixture and stirring was continued for another 4
h. The resultant mixture was cooled to 5 °C and treated with
triethylamine (10 mL) followed by boron trifluoride etherate (10 mL).
After 3 h of stirring, the resulting solution was poured into water and
extracted with dichloromethane. The organic layer was dried over
Na SO , and the solvent was evaporated completely in a rotary
dried over anhydrous Na SO , and the solvent was removed. The
2 4
product was purified on basic alumina with ethyl acetate:hexane (1:1
v/v) as the eluent affording the desired compound as a white
1
crystalline solid. Yield ∼60%. Mp: 148 °C. H NMR (500 MHz,
CDCl , 25 °C, Si(CH ) ) δ: 4.09 (s, 4H), 4.48 (s, 8H), 6.66 (dd, 2H, J
3 3 4
= 1.5 and 8 Hz), 6.78 (t, 2H, J = 9.1 Hz), 6.82−6.86 (m, 10H), 6.94
(dd, 2H, J = 1.2 and 8 Hz), 7.67 (t, 4H, J = 4.6 Hz), 8.37 (t, 4H, J = 3.4
2
4
evaporator. The crude product was purified by column chromatog-
raphy using silica gel with hexane:ethyl acetate (9:1 v/v) as the eluent.
Hz). 13C NMR (125 MHz, CDCl
111.83, 119.93, 120.98, 121.78, 122.16, 136.49, 139.39, 148.79, 151.38,
, 25 °C, Si(CH
) ) δ: 58.95, 66.23,
3
3 4
Evaporation of the solvent afforded about 0.98 g of the desired
1
+ +
product as a red solid. Yield ∼25%. H NMR (500 MHz, DMSO-d ,
159.77. ESI-mass (m/z): calcd 609.2978 [M + H ] , found 609.2955
(100%). Anal. Calcd for C38 : C, 74.98; H, 5.96; N, 13.81%.
Found: C, 75.16; H, 6.09; N, 13.69%.
6
2
5 °C, TMS) δ: 1.29 (s, 6H), 2.42 (s, 6H), 6.16 (s, 2H), 7.49 (d, 2H, J
8.3 Hz), 8.06 (d, 2H, J = 8.3 Hz). ESI-mass (m/z): calcd 367.1430
H N O
36 6 2
=
+
+
[
M − H ] , found 367.1423.
Synthesis of 6. To a solution of 5 (1 g, 16.43 mmol) in 3 mL of
Synthesis of 2. To a preheated solution of 1,2-dibromoethane (6 g,
glacial acetic acid was added concentrated HNO (250 μL) followed
3
3
1.94 mmol) and K CO (9.5 g, 68.84 mmol) in 100 mL of anhydrous
by concentrated H SO4 (250 μL). The resulting brown reaction
2
3
2
DMF was added a solution of 2-nitrophenol (9.5 g, 68.34 mmol) in 40
mL of acetonitrile over a period of 15 min. The reaction mixture was
then heated to reflux for 6 h. After the nitrophenol was completely
consumed, the solvent was distilled off and the residue was stirred in
cold water, whereupon a solid substance precipitated out. It was
collected by filtration, washed several times with ice-cold water, and
mixture was stirred at room temperature for 30 min. Then it was
quenched carefully with saturated NaHCO solution and extracted
3
with dichloromethane. The organic layer was washed with brine, dried
over Na SO , and evaporated to a mustard yellow residue. Trituration
2
4
of this residue with methanol:ethyl acetate (1:1, v/v) afforded 6 as a
1
yellow solid. Yield ∼70%. H NMR (500 MHz, CDCl , 25 °C,
3
recrystallized from methanol to obtain a light yellow solid. Yield
Si(CH ) ) δ: 3.59 (s, 4H), 4.72 (s, 8H), 6.81(d, 2H, J = 9.1 Hz), 7.08
3
4
1
∼
78%. Mp: 157 °C. H NMR (500 MHz, CDCl , 25 °C, Si(CH ) ) δ:
(t, 4H, J = 6.3 Hz), 7.22 (d, 2H, J = 2.3 Hz), 7.38(d, 4H, J = 8.1 Hz),
7.52 (t, 4H, J = 8 Hz), 7.72 (dd, 2H, J = 2.3 and 9.1 Hz), 8.44 (d, 4H, J
3
3 4
4
.53 (s, 4H), 7.07 (t, 2H, J = 8.3 Hz), 7.23 (d, 2H, J = 8.3 Hz), 7.56 (t,
C
Inorg. Chem. XXXX, XXX, XXX−XXX