Preparation and use of Coppersensor-1, a synthetic fluorophore for live-cell copper imaging.

Preparation and use of Coppersensor-1, a synthetic
fluorophore for live-cell copper imaging
Evan W Miller, Li Zeng, Dylan W Domaille & Christopher J Chang
Department of Chemistry, University of California, Berkeley, California 94720, USA. Correspondence should be addressed to C.J.C. (chrischang@berkeley.edu).
Published online 27 July 2006; doi:10.1038/nprot.2006.140
Coppersensor-1 (CS1) is a small-molecule, membrane-permeable fluorescent dye for imaging labile copper pools in biological
samples, including live cells. This probe, comprising a boron dipyrromethene (BODIPY) chromophore coupled to a thioether-rich
receptor, has a picomolar affinity for Cu+ with high selectivity over competing cellular metal ions. CS1 fluorescence increases up
to 10-fold on binding to Cu+. In this protocol we describe the synthesis of CS1 and how to use this chemical tool to investigate
intracellular levels of labile copper in cultured cells. The preparation of CS1 is anticipated to take 4–5 d, and imaging assays can
be performed in 1–2 d with cultured cells.
INTRODUCTION
Copper is an essential nutrient for living organisms1,2, and cells
tightly regulate this redox cofactor for proteins that control aerobic
respiration, iron transport, oxidative stress protection, hormone
production, neurotransmitter processing and angiogenesis3–7. On
the other hand, mismanagement of cellular copper stores is impli-
cated in severe diseases such as cancer8,9 and cardiovascular dis-
orders10, as well as neurogenerative diseases including Alzheimer’s
disease11–16, Menkes and Wilson diseases17,18, familial amyotrophic
lateral sclerosis19–22, and prion diseases23,24. Despite the importance
of copper homeostasis to situations of health and disease, mechan-
isms of copper accumulation, trafficking, and function remain
incompletely characterized. Fluorescence imaging of labile,
subcellular copper pools with selective copper-responsive dyes offers
a potentially powerful technique for elucidating many of these
pathways with spatial and temporal resolution; analogous small-
molecule reagents have greatly aided researchers of calcium
biology25, and a pyrazoline dye has been applied successfully for
detecting exchangeable copper in fixed cells with ultraviolet excita-
tion26. The present protocol describes a recipe-style preparation of
CS1 (Fig. 1), a BODIPY-based fluorophore for selective and
sensitive detection of copper(I) ions in aqueous solution, as well
as its application for imaging ionic copper pools in living cells27.
CS1 is composed of a BODIPY chromophore possessing visible-
wavelength excitation and emission profiles to minimize cellular
autofluorescence and photodamage coupled to a thioether-rich
receptor to achieve selective and stable binding of the Cu+ ion in
water over abundant cellular cations, including divalent magnesium,
calcium, and zinc ions. Molecular recognition of Cu+ by CS1 triggers
up to a 10-fold increase in emission intensity for the probe. The
sensor is membrane-permeable and can be used to report changes in
labile copper levels within living cells using standard fluorescence
microscopy. Control experiments with a chelator must be performed
to establish that any observed fluorescence signals are due to copper
binding. In addition, cell experiments are repeated at least in triplicate
on separate days to verify results. Potential advantages of this CS1
fluorescence method over traditional atomic absorption and radio-
activity assays include the ability to resolve labile copper pools with
spatial resolution and redox selectivity, whereas potential limitations
include the inability to assess total copper content and the current
availability of only one emission color and Cu+ binding affinity.
MATERIALS
REAGENTS
.3,6,12,15-Tetrathia-9-monoazaheptadecane 2 prepared as described
elsewhere28
.Copper chelator N-ethyl-3,6,12,15-tetrathia-9-monoazaheptadecane
4 prepared as described elsewhere27
.2,4-Dimethyl-3-ethylpyrrole (kryptopyrrole; Sigma-Aldrich)
.Chloroacetyl chloride (Acros)
.Triethylamine (EM Science)
.Boron trifluoride diethyl etherate (Sigma-Aldrich)
.Potassium carbonate
.Potassium iodide
.Acetonitrile (distilled from CaH2).Sodium sulfate
.Dichloromethane (Fisher)
.Toluene (Fisher)
.Hexanes (Fisher)
.Silica gel 60
.DMSO HPLC grade (EMD)
.Millipore-purified water
.CuCl2 (anhydrous; Sigma-Aldrich).DMEM (Invitrogen)
.Glutamine (Sigma-Aldrich)
.Poly-L-lysine (Sigma-Aldrich)
EQUIPMENT
.Petri dishes (35 mm)
.Hotplate magnetic stirrer with contact thermometer
.Oil bath
.Dual nitrogen-vacuum manifold with vacuum pump
.Rotary evaporator
.Three-neck round-bottom flask (1,000 ml)
.Two-neck round-bottom flask (50 ml)
.Rubber septa
.Water-cooled condensers
.Air-inlet adapters
.Graduated cylinders
.Disposable glass Pasteur pipettes
.Pipette bulbs
.Single-neck round-bottom flasks (200–300 ml)
.Separatory funnels (250–1,000 ml)
.Powder funnels
.Fluted filter paper
.Columns for chromatography
.Fluorescence microscope
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REAGENT SETUP
Cells for imaging Cells for imaging are grown on glass coverslips in medium.
Cells typically used include mammalian cell lines HEK293T, COS-7 and
HeLa. A representative example is given here. Briefly, HEK293T cells are
cultured in DMEM supplemented with 10% (vol/vol) FBS (Invitrogen) and
glutamine (2 mM). One day before imaging, cells are passaged and plated on
18-mm glass coverslips coated with poly-L-lysine (50 mg ml–1). Adherent
cells for imaging are grown to 50–80% confluency.
EQUIPMENT SETUP
Petri dishes Petri dishes were used in our laboratory in conjunction with
water-immersion microscope objectives; this protocol can be readily adapted
to coverslip holders and inverted microscopes with oil-based objectives.
PROCEDURE
Synthesis of 8-chloromethyl-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (compound 1)
1| Dry the three-neck round-bottom flask, a magnetic stirbar, condenser, and air-inlet adapter overnight in an electric oven
at 130 1C.
2| After cooling the glassware to room temperature (20–25 1C), assemble the apparatus used for the reaction consisting of
the 1,000-ml three-neck round-bottom flask and stirbar, condenser, septum, and air-inlet adapter.
3| To the flask add 200 ml of dry dichloromethane and then 7.38 g of 2,4-dimethyl-3-ethylpyrrole.
4| Next add 3.39 g of chloroacetyl chloride dropwise to the reaction.
5| Heat the resulting solution under a nitrogen atmosphere to 50 1C for 1 h.
6| Cool the reaction to room temperature, and remove the solvent under vacuum.
7| Add 400 ml of toluene, 20 ml of dichloromethane and 17 ml of triethylamine, and stir at room temperature for 15 min.
8| Add 19 ml of boron trifluoride diethyl etherate, and heat the resulting mixture to 50 1C for 1 h.
9| Cool the reaction to room temperature, and remove the solvent under vacuum.
10| Redissolve the residue in 200 ml of dichloromethane, pour the solution into a separatory funnel and wash the organic
extract three times with water (100 ml each wash).
11| Separate the organic layer, dry over sodium sulfate, gravity-filter through fluted filter paper and remove the solvent by
rotary evaporation.
12| Flash column chromatography on silica gel (2 � 24-inch column) by using 1:1 dichloromethane to hexanes as an eluant
gives 5.8 g of compound 1 as an orange solid. Yield: 55% from chloroacetyl chloride. Rf ¼ 0.7 (1:1 dichloromethane to
hexanes). The identity and purity of the compound can be established by proton nuclear magnetic resonance (1H NMR) and
mass spectrometry (MS). 1H NMR (CDCl3, 300 MHz): d 4.82 (2H, s), 2.50 (6H, s), 2.45 (6H, s), 2.40 (4H, q, J ¼ 7.5 Hz),
1.05 (6H, t, J ¼ 7.5 Hz). FAB-MS: calculated for [M+] 352, found 352.
Synthesis of CS1 (3)
13| Dry the 50-ml two-neck round-bottom flask, stirbar, condenser, and air-inlet adapter overnight in an electric oven at 130 1C.
14| After cooling the glassware to room temperature, assemble the apparatus used for the reaction consisting of the 50-ml
two-neck round-bottom flask and stirbar, condenser, septum, and air-inlet adapter.
15| To the flask, add 20 ml of dry acetonitrile, and then 71 mg of compound 1 from step 12, 125 mg of 3,6,12,15-tetrathia-9-
monoazaheptadecane (2), 73 mg of potassium iodide and 61 mg potassium carbonate.
16| Heat the stirred dark-red solution at reflux under a nitrogen atmosphere for 18 h.
17| Let the reaction cool to room temperature, and evaporate to dryness using a rotary evaporator.
18| Redissolve the residue in 100 ml dichloromethane, and transfer to an Erlenmeyer flask.
19| Pour the solution into a separatory funnel, and wash the organic extract three times with water (50 ml each wash).
2
N
H
+
O
Cl Cl
Cl NH
S
S
S
S
N N
N
S
S
S
S
F F
B
N N
B
F F 1
2
3 (CS1)
BF3OEt2
Figure 1 | Synthesis of CS1 (compound 3).
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20| Separate the organic layer, dry over sodium sulfate, gravity-filter through fluted
filter paper and remove the solvent by rotary evaporation.
21| Flash column chromatography on silica gel (1 � 12-inch column) using
dichloromethane as an eluant gives 26 mg of CS1 (3) as a red solid. Yield: 22% from
compound 1. Rf ¼ 0.6 (dichloromethane). The identity and purity of the compound
can be established by 1H NMR and MS. 1H NMR (CDCl3, 400 MHz): d 4.02 (2H, s), 2.87
(4H, t, J ¼ 7.6 Hz), 2.52–2.65 (16H, m), 2.50 (s, 6H), 2.40 (s, 6H), 2.38 (4H, q, J ¼ 7.6 Hz), 1.24 (6H, t, J ¼ 7.6 Hz) 1.05 (6H,
t, J ¼ 7.6 Hz). FAB-MS: calculated for [MH+] 630, found 630.
Preparation of reagent stock solutions for imaging experiments
22| Prepare a 1 mM stock solution of CS1 (MW 630 g/mol) in DMSO by dissolving 0.63 mg of solid CS1 per milliliter of DMSO solvent.
’ PAUSE POINT Stock solutions of CS1 can be stored for months at room temperature in the dark. Solutions can also be stored
frozen and thawed when needed, but repeated freeze/thaw cycles may lead to decomposition.
23| Prepare a 10 mM stock solution of CuCl2 (MW 134 g/mol) by dissolving 1.34 mg of solid CuCl2 per milliliter of Millipore
water solvent.
24| Prepare a 100 mM stock solution of N-ethyl-3,6,12,15-tetrathia-9-monoazaheptadecane (4, MW 342 g/mol, Fig. 2) in DMSO
by dissolving 34.2 mg of the liquid chelator per milliliter of DMSO solvent.
’ PAUSE POINT Stock solutions of the chelator can be stored for months at room temperature in the dark under a nitrogen
atmosphere.
CS1 labeling of live cells
25| Remove cells from incubator, and transfer one coverslip into a 35-mm Petri dish containing 3 ml PBS buffer.
Should you wish to supplement the cells with copper, add an aliquot of the CuCl2 stock solution to the growth medium and
return the cells to the incubator for 1–8 h before continuing to Step 26. To add a competing chelator to copper-supplemented
cells, remove cells from the incubator and add 15 ml of a 100 mM compound 4 stock solution to give a final chelator
concentration of 500 mM. Mix thoroughly, and incubate in the dark for 5 min at 25 1C, before continuing to Step 26.
m CRITICAL STEP In the case of, for example, HEK293T cells growing in 1 ml of medium, add 10 ml of the 10 mM CuCl2 stock
solution to give a final copper concentration of 100 mM. A 5- to 10-fold excess of chelator over copper supplementation gives
optimal results.
26| To the Petri dish, add 15 ml of a 1 mM CS1 stock solution to give a final dye concentration of 5 mM. Mix thoroughly.
m CRITICAL STEP Higher concentrations of dye may result in high levels of background fluorescence.
27| Incubate for 5–20 min in the dark at 251 or 37 1C.
Imaging the CS1-labeled cells
28| CS1 can be imaged using any type of fluorescence microscope, including epifluorescence, confocal and multiphoton.
For standard confocal experiments, best results were obtained with 543 nm excitation to match the absorption maximum of
the apo and Cu+-bound probe.
� TIMING
With appropriate precursors in hand, the synthesis and purification of BODIPY (1) and CS1 (3) is anticipated to require 4–5 d.
Imaging experiments with cells will require 1–2 d with cells in culture.
ANTICIPATED RESULTS
The approaches described here have
been found useful for detecting labile
pools of copper in a variety of cell
types, including mammalian cell
line and primary culture sources. For
example, the ability of CS1 to detect
various levels of intracellular copper
within living cells is demonstrated
here using live HEK293T cells
(Fig. 3).
Figure 2 | Structure
of the copper chelator
N-ethyl-3,6,12,15-
tetrathia-9-
monoazaheptadecane
(compound 4).
S
S
N
S
S
4
a b c
Figure 3 | Live-cell copper imaging with CS1. (a) HEK293T labeled with 5 mM CS1 for 5 min at 25 1C.
(b) HEK293T grown with 100 mM CuCl2 supplement for 6 h at 37 1C and stained with CS1 using the same
conditions as in a. (c) Copper-supplemented HEK293T treated with 500 mM of the competing Cu+ chelator
4 for 5 min at 25 1C and stained with CS1 using the same conditions as in a and b. Scale bar ¼ 38 mm.
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COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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