Cellular uptake of a fluorescent calix[4]arene derivative
Ruth LalorJournal of the American Chemical Society
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A rapid synthetic route to a nontoxic fluorescently labeled water-soluble calixarene has been developed. Investigation of the cellular uptake of the labeled calixarene, via confocal microscopy, through coincubation with uptake inhibitors demonstrates that uptake is not through the common clathrin coated pits or caveolae (lipid raft) endocytic pathways and that the calixarene derivative localizes within the cytoplasm and does not enter the nucleus. The study demonstrates the power of fluorescent labeling for investigation of interactions between calixarenes and biological systems and the potential for calixarene based intracellular imaging agents.
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Supporting Information
Cellular Uptake of a Fluorescent Calix[4]arene Derivative
Ruth Lalor,1 Hugo Baillie-Johnson,2 Carl Redshaw,1 Susan. E. Matthews*,1 Anja Mueller*1
1
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, UK.
2
Department of Oncology, Norwich and Norfolk University Hospital, Norwich, NR4 7UY
[email protected], [email protected]
Experimental Procedures
Chemical Syntheses Compound 3 To a stirring solution of 5,11,17,23-tetra-Boc-amino-25,26,27tripropoxy-28-aminopropoxycalix[4]arene1 (0.15 g, 0.14 mmol) in EtOAc (3 mL) was added 4-chloro7-nitrobenzofurazan (NBD chloride) (0.042 g, 0.21 mmol), and the resultant solution heated at 60 ºC
for 4 h. Water (3 mL) and dichloromethane (5 mL) were added, the organic phase separated, washed
with HCl (10 %), NaOH (10 %) and brine, and the solvent removed under reduced pressure. The
residue was purified over column chromatography, eluting with a gradient of hexane : dichloromethane
(1:1) to dichloromethane to dichloromethane : EtOAc (19:1), yielding the product 3 as an orange solid
(0.05 g, 30%). IR vNH=3315 cm-1, vNO2=1698 cm-1, vNH=1593 cm-1; 1H NMR (400 MHz, CDCl3);
8.47 (1H, d, J = 8.5 Hz, ArNBDH), 6.75 (3H, b s, ArH), 6.54 (3H, m, ArH), 6.29 (1H, s, ArH), 6.27 (1H,
s, ArH), 6.10 (1H, d, J = 8.5 Hz, ArNBDH), 4.34 (2H, d, J = 13.3 Hz, ArCH2Ar), 4.29 (2H, d, J = 13.3
Hz, ArCH2Ar), 4.05 (2H, t, J = 6.1 Hz, OCH2CH2CH2N), 3.81-3.62 (8H, m, OCH2CH2CH3,
OCH2CH2CH2NH2, coincident), 3.12 (2H, d, J = 13.3 Hz, ArCH2Ar), 3.09 (2H, d, J = 13.3 Hz,
ArCH2Ar), 2.36 (2H, m, OCH2CH2CH2N), 1.83 (6H, m, OCH2CH2CH3), 1.50 (18H, s, C(CH3)3), 1.46
(18H, s, C(CH3)3), 0.93 (9H, m,
OCH2CH2CH3); 13C NMR (100 MHz; CDCl3); 155.63, 153.7, 153.6, 153.5,136.6, 136.5, 135.7, 134.5,
132.3, 120.4, 120.3, 120.2, 120.1, 119.9, 80.3, 77.1, 71.4, 60.6, 31.4, 31.3, 31.2, 29.4, 28.6, 23.5, 23.2,
10.7, 10.6; M/z (ESI) 1248 [M+NH4]+
Compound 4 HCl(g) was bubbled for 15 min through a stirring solution of 3 (0.05 g, 0.04 mmol) in
dichloromethane(3 mL), after which time a precipitate formed. Bubbling of HCl(g) was continued for a
further 10 min. to ensure complete deprotection. The solvent was removed under reduced pressure,
yielding 4 as a dark red solid (quantitative). IR vNH=3375 cm-1, 3180 cm-1, vNO2=1616 cm-1,
vNH=1567 cm-1; 1H NMR (400 MHz, CDCl3); 8.45 (1H, d, J = 8.5 Hz, ArNBDH), 6.78 (8H, m, ArH),
6.31 (1H, d, J = 8.5 Hz, ArNBDH), 4.45 (2H, d, J = 13.3 Hz, ArCH2Ar), 4.44 (2H, d, J = 13.3 Hz,
ArCH2Ar), 4.09 (2H, t, J = 6.9 Hz, OCH2CH2CH2N), 3.83 (8H, m, OCH2CH2CH3, OCH2CH2CH2NH2,
coincident), 3.26 (2H, d, J = 13.3 Hz, ArCH2Ar), 3.25 (2H, d, J = 13.3 Hz, ArCH2Ar), 2.37 (2H, m,
OCH2CH2CH2N), 1.85 (6H, m, OCH2CH2CH3), 0.90 (9H, m, OCH2CH2CH3); 13C NMR (100 MHz;
CDCl3);156.1, 155.9, 135.9, 135.8, 135.7, 135.6, 124.4, 124.1, 122.8, 122.7, 122.6, 76.8, 76.6, 72.5,
29.6, 22.4, 22.3, 8.7, 8.6; M/z (ESI) Calc’d for free amine C46H55N8O7 831.4188, observed 831.4189
Biological Methods. Cell Viability Studies. MTS assays were performed using a CellTiter 96® AQueous
Non-Radioactive Cell Proliferation Assay (Promega). All cell lines used have been described earlier.2
All cells were incubated at 37 ˚C. Freshly harvested CHO cells were suspended in DMEM (Invitrogen)
S1
with 10% Foetal Calf serum and 2mM glutamine, HL-60 cells in RPMI (Invitrogen) with 10% Foetal
Calf serum and 2mM glutamine, at a concentration of 1x105 cells /mL. 50 µL of the cell solution was
incubated with 30 µL of each of the calixarenes 4 and 5 in buffer, at concentrations of 150mM, 15mM,
1.5mM, 0.15mM, 0.015mM or with a mixture of 15mM calixarene 4 and increasing concentration of βcyclodextrin. CHO cell viability was measured by MTS assay at 72 h. HL-60 cell viability was
measured by MTS assay at 84h.
Image acquisition. CHO cells were grown on coverslips overnight, incubated with NBD, 4 or 5 for
different time points at 37°C. Cells were washed three times in ice-cold PBS and either mounted on
glass-slides or used for counter stain with a monoclonal antibody that binds to a receptor in the cell
membrane. The immunofluorescence procedure has been described previously.2 Briefly cells were
incubated with saturating amounts of antibody HEK/1/85a/7a for 1 hour at 4°C, washed three times
with PBS, incubated for 1 hour with a corresponding TRITC conjugated antibody, washed with PBS
three times and mounted in glycerol on glass slides. Pictures were acquired using either a Zeiss
Axiovison 2 fluorescence microscope or a Leica TCS SP2 UV Confocal Imaging system.
Results and Discussion
Figure S1. Images of CHO cells (a) NBD control (500 mM) (b) after 20 minutes pre-incubation with 4
(150 mM) (c) after 30 minutes (d) after 60 minutes. Images were acquired using a Zeiss Axiovision 2
microscope.
Figure S2: Control fluorescence with NBD fluorophore on its own. Images of CHO cells 1 hour after
pre-incubation with inhibitors with NBD added for 10 min at 300 mM concentration (a) NBD (b) NBD
and filipin (5µg/ml) (c) NBD and sucrose (0.4 M) (d) NBD and β-cyclodextrin (0.1 M)
S2
Figure S3. Images of CHO cells 1 hour after pre-incubation with inhibitors with 4 added for 10 min at
15 mM concentration (a) 4 (b) 4 and filipin (5µg/ml) (c) 4 and sucrose (0.4 M) (d) 4 and β-cyclodextrin
(0.1 M)
a)
absorbance
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.01
control
4
5
0.1
1
10
100
1000 10000
concentration [mM]
b)
1.4
absorbance
1.2
1.0
0.8
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0.2
control
4
4 + cyclodextrin
0.0
0.001 0.01
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Figure S4 Representative cell viability assays (CHO cells tested for proliferation after 72 hours using
an MTS assay) (a) comparison of 4 and 5 (b) comparison of 4, 4 (15mM) and increasing concentration
of β-cyclodextrin. Phosphate Buffered Saline used as a control in all studies.
S3
Figure S5 Absorbance spectrum of 4 (5.12 x 10-8 M) [black full line] and 5 (5.12 x 10-6 M) [black
dotted line]. Emission spectrum of 4 (5.12 x 10-8 M) [red full line] and 5 (5.12 x 10-6 M) [red dotted
line] following excitation at 488 nm
References
(1)
(2)
Lalor, R.; DiGesso, J. L.; Mueller, A.; Matthews, S. E. Chem. Commun. 2007, 4907-4909
Mueller A., Kelly E., Strange P. G., P. Blood 2007, 99, 785-791.
S4
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