MS (ESI) m/z calculated for (M-H)- 427.10 found 427.02
Synthesis of 5
-1
(M-H)-. ncm (neat) 3397, 2931, 1689 cm-1.
Compound E (60 mg, 0.14 mmol), N-hydroxysuccinimide (81 mg,
0.7 mmol) and N,N’-diisopropylcarbodiimide (88 mg, 0.7 mmol)
were dissolved in dry DMF (2 ml) and stirred at 25 ◦C for 24 h. The
solvent was evaporated under reduced pressure and the residue
dissolved in EtOAc (5 mL) and washed with water (5 mL ¥ 3). The
organic layer was dried over MgSO4 and the solvent evaporated
to obtain a red colored material.
Synthesis of 3
2-Hydroxy diethyl Nile Red D (50.0 mg, 0.15 mmol) and K2CO3
(103.0 mg, 0.75 mmol) were dissolved in CH3CN (5 mL). 13
(162.0 mg, 0.18 mmol) in CH3CN (2 mL) was added dropwise
in 5 min to the above solution at 25 ◦C. The reaction mixture
was heated to 50 ◦C for 4 h. After completion of the reaction
the solvent was evaporated and the residue was subjected to
flash chromatography eluting with 30–40% acetone/EtOAc and
then with 10% MeOH–CH2Cl2 to afford 150.0 mg of red colored
material. Flash chromatography was performed to remove excess
13.
The above red material (60.0 mg, 0.06 mmol) was dissolved in
TFA/CH2Cl2 (1/1, 3 mL) and stirred at 25 ◦C for 1 h. The solvent
was removed under reduced pressure and the residual material
was dissolved in 5 mL of water. This solution was filtered to
remove solid impurities and the filtrate was purified by reverse
phase medium pressure liquid chromatography (MPLC) eluting
with 30% CH3CN–H2O to afford 3 as a dark red solid (35.0 mg,
58%).
The above red colored material was dissolved in DMF (2 ml)
along with tricarboxylic acid 15 (479 mg, 1.4 mmol), DMAP (1 mg,
0.01 mmol) and triethylamine (0.2 mL, 1.4 mmol). The reaction
mixture was stirred at 25 ◦C for 48 h. After removal of DMF
under reduced pressure the residue was dissolved in water (2 mL)
and washed with EtOAc (2 mL ¥ 3). The aqueous layer containing
crude product was loaded on to a reverse phase MPLC column
and purified using 3/2 CH3CN–H2O solvent mixture. The solvent
was evaporated to afford 5 as a dark purple solid (43 mg, 28%).
1H NMR (500 MHz, CD3OD) d 8.04 (br, 1H), 7.94 (br, 1H),
7.56 (br, 1H), 7.08 (br, 1H), 6.91 (s, 1H), 6.74 (s, 1H), 6.20 (s, 1H),
3.76 (br, 4H), 3.67 (br, 24H), 2.75 (br, 4H), 2.47 (s, 12H).
13C NMR (125 MHz, CD3OD) d 183.9, 175.6, 172.4, 161.0,
152.5, 151.3, 146.5, 139.1, 134.3, 130.8, 127.3, 125.1, 123.9, 118.0,
110.8, 108.4, 103.6, 96.9, 68.5 (2C), 67.2, 60.9, 35.4, 34.2.
MS (MALDI) m/z calculated for (M+3H)+ 1063.39 found
1063.35 (M+3H)+.
1H NMR (500 MHz, CD3OD) d 8.06 (d, 1H, J = 8.9 Hz), 8.01
(s, 1H), 7.97 (d, 1H, J = 3.0 Hz), 7.54 (d, 1H, J = 8.9 Hz), 7.17
(dd, 1H, J = 9.2, 3.0 Hz), 6.79 (dd, 1H, J = 9.2, 3.0 Hz), 6.54
(d, 1H, J = 3.0 Hz), 6.17 (s, 1H), 4.61 (s, 2H), 4.55 (t, 2H, J =
5.0 Hz), 4.32–4.29 (br, 2H), 3.93 (br, 2H), 3.89 (t, 2H, J = 5.0 Hz),
3.77–3.74 (br, 2H), 3.70–3.68 (br, 2H), 3.66–3.52 (m, 40H), 1.27
(t, 6H, J = 7.0 Hz).
ncm (neat) 2942, 1722, 1621 cm-1.
-1
Cell culture
Clone 9 cells (American Type Culture Collection) were cultured as
subconfluent monolayers on 75 cm2 culture flask with vent caps in
Ham’s medium supplemented with 10% fetal bovine serum (FBS)
in a humidified incubator at 37 ◦C with 5% CO2. Cells grown
to subconfluence were enzymatically dissociated from the surface
with trypsin and plated 2–3 days prior to the experiments in Lab-
Tek two-well chambered coverglass slides (Nunc).
13C NMR (75 MHz, CD3OD) d 183.7, 161.9, 152.8, 151.8, 147.1,
144.6, 143.8, 138.1, 134.3, 131.3, 127.3, 125.1, 124.7, 118.1, 110.7,
106.4, 103.7, 96.0, 72.5, 70.7, 70.5, 70.4, 70.3, 70.2, 70.1, 69.6, 69.2,
67.9, 63.8, 61.0, 50.2, 44.9, 11.8.
MS (ESI) m/z calculated for (M+H)+ 944.49 found 944.49
(M+H)+ MS (ESI) m/z calculated for (M+Na)+ 966.47 found
966.45 (M+Na)+; ncm (neat) 3435, 2925, 1641 cm-1.
-1
Fluorescence microscopy
Subcellular localization of Nile Red derivatives and BODIPY TR
ceramide complexed to BSA was measured on living Clone 9
cells using a Stallion Dual Detector Imaging System consisting
of an Axiovert 200M inverted fluorescence microscope, CoolSnap
HQ digital cameras and Intelligent Imaging Innovations (3I)
software. Digital images of Nile Red dyes, MITO tracker green
labeled mitochondria, BODIPY TR ceramide complexed to BSA
labeled Golgi, and LysoTracker Blue DND-22 were captured with
a C-APO 63X/1.2 W CORR D = 0.28 M27 objective with the
following filter sets: Exciter BP560/40, Dichroic FT 585, Emission
BP 630/75 for Nile Red derivatives and BODIPY TR ceramide
complexed to BSA; Exciter BP470/20, Dichroic FT 493, Emission
BP 505-530 for MITO tracker green; and Exciter G 365, Dichroic
FT 395, Emission BP 445/50 for LysoTracker Blue DND-22.
Synthesis of 4
Compound E (25 mg, 0.06 mmol) and activating agent EDCI
(28 mg, 0.24 mmol) were dissolved in pyridine (2 mL).
Tris(hydroxymethyl)aminomethane (43 mg, 0.36 mmol) was added
and the reaction was continued at 25 ◦C for 24 h. The solvent was
evaporated and the residue purified by reverse phase medium pres-
sure liquid chromatography (MPLC) eluting with 3/2 CH3CN–
H2O to afford 4 as a dark red solid (22 mg, 59%).
1H NMR (500 MHz, DMSO-d6) d 7.96 (d, 1H, J = 10.0 Hz),
7.87 (s, 1H), 7.61 (d, 1H, J = 10.0 Hz), 7.39 (s, 2H), 7.08 (dd, 1H,
J = 10.0, 5.0 Hz), 6.84 (dd, 1H, J = 10.0, 5.0 Hz), 6.68 (s, 1H),
6.19 (s, 1H), 4.73 (br, 6H), 3.65 (br, 4H), 3.53 (s, 12H), 2.51 (br,
4H).
13C NMR (125 MHz, DMSO-d6) d 182.4, 172.1, 152.2, 152.2,
151.3, 146.9, 140.1, 134.4, 131.4, 128.2, 124.8, 119.2, 110.8, 110.2,
109.2, 105.2, 97.4, 63.2, 61.2, 48.1, 34.6.
Cellular uptake of Nile Red derivatives
Clone 9 cells were incubated for 30 min to one hour at 37 ◦C
in ACAS with various concentration of probes (solution in PBS
or DMSO). After the incubation period, the cells were washed
MS (ESI) m/z calculated for (M+H)+ 629.25 found 629.23
-1
(M+H)+. ncm (neat) 3412, 3316, 1693 cm-1.
2058 | Org. Biomol. Chem., 2010, 8, 2052–2059
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The Royal Society of Chemistry 2010
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