Synthesis and properties of molecular probes for the rescue site on mutant cystic fibrosis transmembrane conductance regulator

Article abstract of DOI:10.1021/jm201335c

Cystic fibrosis is a genetic disease caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In vitro experiments have demonstrated that 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol (VRT-532, 1) is able t

Full text of DOI:10.1021/jm201335c

Article
pubs.acs.org/jmc
Synthesis and Properties of Molecular Probes for the Rescue Site on
Mutant Cystic Fibrosis Transmembrane Conductance Regulator
Bashar Alkhouri,† Robert A. Denning,† Patrick Kim Chiaw,‡ Paul D. W. Eckford,‡ Wilson Yu,‡ Canhui Li,‡
Jovanka J. Bogojeski,† Christine E. Bear,‡ and Russell D. Viirre*,†
†Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, Ontario M5B 2K3, Canada
‡Programme in Molecular Structure and Function in the Research Institute, Hospital for Sick Children, 555 University Avenue,
Toronto, Ontario M5G 1X8, Canada
ABSTRACT: Cystic fibrosis is a genetic disease caused by
mutations in the gene for the cystic fibrosis transmembrane
conductance regulator (CFTR) protein. In vitro experiments have
demonstrated that 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol
(VRT-532, 1) is able to partially restore the function of mutant
CFTR proteins. To help elucidate the nature of the interactions
between 1 and mutant CFTR, molecular probes based on the
structure of 1 have been prepared. These include a photoreactive aryl azide derivative 11 and a fluorescent dansyl sulfonamide
15. Additionally, a method for hydrogen isotope exchange on 1 has been developed, which could be used for the incorporation of
radioactive tritium. Using iodide efflux assays, the probe molecules have been demonstrated to modulate the activity of mutant
CFTR in the same manner as 1. These probe molecules enable a number of biochemical experiments aimed at understanding
how 1 rescues the function of mutant CFTR. This understanding can in turn aid in the design and development of more
efficacious compounds which may serve as therapeutic agents in the treatment of cystic fibrosis.
systems, this trafficking defect can be partially overcome by
incubating cells at 27 °C,6 however the protein’s ability to
function as a chloride channel at the cell surface remains
impaired relative to that of the wild-type CFTR.7 Another less
common mutation: G551D-CFTR undergoes normal bio-
genesis and trafficking to the cell surface5 yet it exhibits
defective channel activation at that location.8 Thus, two classes
of small molecules have been identified, which may prove to be
useful in the pharmacological treatment of CF.9,10 “Correctors”
are those molecules which, through either direct interactions
with mutant CFTR or perhaps any of the various chaperone
proteins, rescue the cell’s ability to correctly traffic the protein
to the cell membrane. “Potentiators” are those molecules which
result in a restoration of normal channel activity in the mutant
protein once it appears in the cell membrane.
In 2006, a group from Vertex Pharmaceuticals reported that
4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol (VRT-532, 1)
was identified in a high-throughput screen for the ability to
potentiate channel activity in mouse NIH 3T3 cells expressing
wild-type CFTR or F508del-CFTR after low-temperature
correction of the trafficking defect.11 Subsequent reports have
shown that 1 also partially promotes proper protein trafficking
of F508del-CFTR from the endoplasmic reticulum (ER) to the
cell membrane.12 Thus, 1 is both a potentiator and a corrector.
It has been suggested on the basis of limited proteolysis studies
that this molecule may modify the conformation of F508del-
CFTR such that it more closely resembles that of the wild-type
INTRODUCTION
■
Cystic fibrosis (CF) is one of the most common genetic
diseases among Caucasians. The illness is best known for
affecting the respiratory system, where the lungs become
obstructed with thick sticky mucus, resulting in difficulty
breathing and increased susceptibility to bacterial infections.
The disease also affects the pancreas, liver, intestines, and male
reproductive system.
CF is caused by mutations in the gene for the cystic fibrosis
transmembrane conductance regulator protein (CFTR). In
healthy individuals, CFTR acts as a phosphorylation and
nucleotide regulated channel which mediates the flux of
chloride across the apical membrane of epithelial cells.1 In
CF patients, mutant CFTR fails to correctly mediate chloride
flux, and as a consequence, transepithelial chloride, salt, and
water transport is impaired. This transport defect leads to
dehydration of the airway surface fluid, mucus desiccation, and
obstruction with recurrent episodes of inflammation and
infection. Currently, CF is treated through careful control of
diet, physiotherapy, antibiotics and, when lung function is
severely degraded, lung transplantation.2 The average life
expectancy for a CF patient is 47 years.3 There are currently
no pharmacological agents approved for use in the clinic that
address the basic molecular defect underlying the disease.
The most common mutation in CFTR, occurring on at least
one allele in 70% of CF patients, is the deletion of a
phenylalanine residue at position 508 in the amino acid
sequence (F508del-CFTR).4 This defect results in protein
misfolding, retention in the endoplasmic reticulum, and its
failure to reach the cell membrane.5 In cell culture expression
Received: October 6, 2011
Published: November 10, 2011
© 2011 American Chemical Society
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protein, and that this interaction is sufficient to partially rescue
both protein biogenesis and channel activity.13 Understanding
the molecular basis for this interaction will provide insight into
the mechanism of action of 1 as a modulator of CFTR and
provide a template for development of therapeutically effica-
cious compounds.
Our synthetic efforts to prepare derivatives of 1 including the
photoaffinity label, aryl azide 11 are illustrated in Scheme 1.
Reaction of 2′-hydroxy-5′-methylacetophenone 2 with 4-
iodobenzoyl chloride 3 afforded the expected ester 6 in 86%
yield. The ester was subjected to the Baker−Venkataraman
rearrangement by heating with t-BuOK in THF, intending to
isolate diketone 8.15 The crude product of this reaction was a
1
yellow solid which by H NMR spectroscopy suggested the
presence of numerous species. Repeated attempts at recrystal-
lization failed to yield any material with apparent improved
purity. Reasoning that perhaps the additional signals arise from
a mixture of keto−enol tautomers, which ought to be
competent in the subsequent pyrazole formation, it was
decided to carry the crude yellow solid through the next step.
Treatment with hydrazine in acetic acid afforded the desired
pyrazole 9 in a serviceable 68% yield (from ester 6). Exchange
of the iodide for azide using a Cu/proline catalyzed
methodology reported by Ma16 was hampered by the fact
that the desired azide 11 and starting material iodide 9 have
identical chromatographic properties on silica gel. It was
therefore impossible to gauge reaction progress by TLC and
impossible to isolate 11 from the reaction mixture by column
To help elucidate the nature of the interaction between 1 and
mutant CFTR proteins, we have designed and prepared
derivatives of 1, modified with functional groups useful in
biochemical studies. These derivatives include a photoaffinity
label and a fluorescent probe. We have also developed a
protocol for hydrogen isotope exchange (deuteration by D2O,
as a model for tritiation). We show in functional assays of the
channel activity of the mutant CFTR proteins that these
derivatives retain the potentiator activity of the parent
compound and, hence, have the potential to define the
molecular basis for this activity.
1
chromatography. In fact, it was only by H NMR analysis of
what appeared by TLC analysis to be clean recovered 9 that we
were able to observe that the material was actually a nearly 1:1
mixture of 9 and another compound with the same NMR
splitting pattern. After this discovery, TLC analysis of this
material using a variety of common eluents failed to resolve the
two compounds. Treatment of a portion of the mixture with
triphenylphosphine in wet THF (Staudinger reaction con-
ditions)17 led to the formation of the amine compound 12,
confirming that the second component of the 1:1 mixture was
in fact the azide 11, formed in only about 50% conversion in
the iodide-for-azide exchange reaction. Given this poor
conversion, as well as the inability to separate 11 from 9 by
any practical means, we opted instead to pursue a different
route to the azide. Nitro compound 10 was prepared by the
RESULTS AND DISCUSSION
■
Synthesis of 3,5-Diarylpyrazoles and a Photoaffinity
Label. A photoaffinity label is a molecule that contains a
recognition element for specific interactions with a site on a
target protein as well as a photoactivatible functional group that
reacts to form a covalent bond to the protein upon
photoirradiation. The labeled site can then be identified using
any of a number of biochemical techniques, for instance limited
proteolysis followed by gel electrophoresis and/or mass
spectrometry to suggest the location of the binding site for
the unlabeled ligand.14
a
Scheme 1
a
Reagents and conditions: (i) pyridine, 0 °C, 1 h; (ii) KOt-Bu, THF, 50 °C, 30 min; (iii) H2N−NH2·xH2O, AcOH, 65 °C, 16 h; (iv) NaN3, CuI, L-
proline, NaOH, DMSO, 100 °C, 16 h; (v) PPh3, H2O, THF; (vi) SnCl2·2H2O, EtOH, 78 °C, 16 h; (vii) t-BuONO, TMS-N3, MeCN, 0 °C to rt, 1 h.
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a
Scheme 2
a
(a) Typical photoreaction of aryl azide photoaffinity labels. Upon irradiation, the azide decomposes to the reactive nitrene, which rearranges to the
cyclic ketenimine, which is ultimately quenched by a nucleophilic functional group from the protein target. (b) Photoreaction of 11, with trapping by
diethylamine.
same sequence as described above. Stannous chloride reduction
of 10 provided amine 12 in 71% yield. Diazotization of 12 by
treatment with t-butyl nitrite, followed by treatment with
azidotrimethylsilane,18 provided the desired azide 11 in
excellent 94% yield.
fluorophore. The N-propargyl dansyl amide 14 was prepared21
and found to react cleanly with iodide 9 under Sonogashira
reaction22 conditions to afford 15 in 47% yield (Scheme 3).
a
Scheme 3
Subsequent to these experiments, we discovered that the
entire synthesis of the pyrazole core from acetophenone 2 and
benzoyl chloride 5 could be accomplished in one pot, using
pyridine as a solvent, at 50 °C. With monitoring by TLC,
additional 5 was added until 2 was completely converted. At
this point, t-BuOK was added, again using TLC to gauge
complete consumption of the intermediate ester. Finally,
aqueous hydrazine was added, followed by acetic acid. Thus,
in less than one day’s reaction time, in one pot, and with only a
single, final chromatographic purification step, 1 was isolated in
65% yield from 2. Using the same one-pot sequence, 10 has
also been prepared from 2 and 4 in 24% yield.
Under UV irradiation, aryl azides extrude a molecule of N2,
transiently forming a reactive nitrene intermediate, which
rearranges to an electrophilic cyclic ketenimine.14 In photo-
affinity labeling experiments, this occurs within a protein
binding site, and the ketenimine is quenched by a nucleophilic
functional group from the protein, thus forming a covalent
linkage between the label and the protein (Scheme 2a). To
demonstrate that azide 11 is capable of undergoing this
chemistry, a benzene solution of 11, containing an excess of
diethylamine, was irradiated with an unfiltered (full spectrum)
mercury lamp (Scheme 2b). After workup and chromato-
graphic purification, diethylamine adduct 13 was isolated in
40% yield. Thus, 11 exhibits the necessary photoreactivity for
photoaffinity labeling experiments.
Synthesis and Properties of a Fluorescent Conju-
gate. The amino group on compound 12 was envisaged to be
a handle for further functionalization, for instance by reaction
with fluorescent electrophiles like 4-chloro-7-nitrobenzofurazan
(NBD chloride)19 or 5-(dimethylamino)naphthalene-1-sulfonyl
chloride (dansyl chloride),20 however no reaction was observed
on treatment of 12 with either of these reagents under their
prescribed reaction conditions. Rather than attempt to optimize
reaction with these costly reagents, we reasoned that
incorporation of a glycine amide might provide a more reactive
aliphatic amino group for further derivatization. Unfortunately,
attempts to acylate 12 with Boc-glycine using a variety of
standard peptide coupling agents also failed. Ultimately, we
decided to abandon this approach, instead choosing to focus on
palladium-catalyzed cross-coupling chemistry to install a
a
Reagents and conditions: (i) PdCl2·dppf·CH2Cl2, CuI, NEt3, THF,
45 °C, 15 h.
Given the success of this reaction, Boc-propargylamine 16 was
also prepared23 and reacted with 9, affording 17 in 69% yield.
Upon Boc-deprotection, this compound will afford a
nucleophilic primary aliphatic amine, which can be readily
derivatized with electrophiles, should the need arise to prepare
new derivatives in the future.
Dansyl sulfonamides typically display fluorescence that is
sensitive to their local environments. Figure 1 shows the
emission spectrum of 15, recorded in solvents of different
polarity. When dissolved in chloroform, fluorescence emission
is maximal and centered at about 504 nm. In the most polar of
the media tested, an aqueous buffer solution, the emission
maximum was red-shifted by 37 nm to 541 nm, with an
intensity 32% that of the emission in chloroform. In the organic
solvents dimethyl sulfoxide and methanol, which are increas-
ingly more polar than chloroform, but less so than water,
fluorescent emissions maxima were intermediate (529 nm in
both) and intensity decreased with increasing polarity. Probe
15 contains the recognition element of 1, conjugated to a
fluorescent dansyl group through a propargylamine linker and,
as such, is expected to be a useful probe for the binding
interaction between 1 and CFTR. The direct binding of probe
15 to CFTR, and/or the conformational changes which
mediate protein folding and function, may be detected in
fluorescence anisotropy measurements.24 The probe’s fluo-
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comparison of the binding sites for various CFTR modulators
as they are identified.
While it would be possible to incorporate a radioactive
electrophile onto the propargylamino group accessible from 17,
a method that would be much less disturbing to the original
structure of 1 would be to replace a “non-exchangeable”
hydrogen atom for a tritium atom. As a model for this, a study
was conducted on the incorporation of less expensive and more
safely handled nonradioactive deuterium. A small sample of 1
was dissolved in 1 M NaOH in D2O and heated at 90 °C.
Under these conditions, in addition to the classically
exchangeable phenolic O−H and pyrazole N−H, the pyrazole
C4−H also gradually exchanged for deuterium (Figure 2a). The
reaction progress was readily monitored by 1H NMR
spectroscopy. The singlet at 6.7 ppm, arising from the pyrazole
C4−H, was observed to gradually decrease in integration,
reaching ∼50% deuterium incorporation over 24 h, decreasing
to ∼20% (i.e., ∼80% deuterium incorporation) after 48 h. Parts
Figure 1. Fluorescence emission spectra of 20 μM solution of 15 in
CHCl3 (black solid line), DMSO (black dotted line), MeOH (gray
solid line), and aqueous buffer containing 20 mM MOPS, 75 mM KI,
1 mM n-dodecyl-β-D-maltoside (gray dotted line).
rescence intensity or emission maximum may change upon
binding to a hydrophobic pocket of CFTR, which would be
monitored by fluorescence experiments. Alternatively, as the
emission for the tryptophan residues in CFTR overlaps with
the excitation of this dansyl derivative, there may be measurable
resonance energy transfer upon binding to the protein.25 Lastly,
we anticipate that competition experiments with unlabeled
drugs will identify other small molecules that bind directly to
CFTR and permit quantification of their relative binding
constants.
Hydrogen Isotope Exchange. A radiolabeled version of 1
would also be useful for quantitative studies of its binding
properties to CFTR. Radioligands have shown to be invaluable
tools for the understanding of the mechanism of action of
membrane proteins, receptors, transporters, and channels.26 To
date, binding of CFTR modulators has been detected using
functional assays which report the consequences of binding
rather than binding per se.11,27,28 Hence, the development of
radiolabeled 1 would enable direct assay of the relative affinities
for binding to CFTR and mutant CFTR proteins, thereby
providing insight into potential genotype-specific structural
differences in the binding site. Further, this tool would enable
1
b and c of Figure 2 show the aromatic region of the H NMR
spectrum of 1 at the beginning (t = 0) and end (t = 48 h) of the
experiment, respectively. After workup (including aqueous acid
to re-exchange the phenolic O−H and the pyrazole N−H for
protons) and TLC purification, 1 was reisolated in 88% yield
and determined to have 80% deuterium incorporation at
pyrazole C4. If these conditions were to be repeated using
commercially available tritiated water (radioactivity level 185
GBq/g),29 they would be expected to provide [3H]-1 at a level
of 1.33 × 1012 Bq/mol,30 without the need for multiple
synthetic steps or purification of radioactive materials.
Biological Activity. For these molecular probes to be of
use in biochemical experiments, it must be demonstrated that
the substitutions, in every case at the 4-position of the phenyl
ring of the parent structure 1, do not interfere with the
compound’s ability to interact with the protein. To determine
this, the effects of 9−12 and 15 on CFTR channel function
were assessed using patch clamp electrophysiology.11,31,32 In
our version of this assay,27,28 baby hamster kidney (BHK) cells
expressing F508del-CFTR at the cell membrane (after
Figure 2. (a) Experimental conditions for hydrogen isotope exchange. (b) Aromatic region of the 1H NMR spectrum of 1 in 1 M NaOH/D2O, prior
to any heating. The signal from the proton on pyrazole C4 is indicated by the arrow. (c) The same region of the spectrum after heating at 90 °C for
48 h, wherein ∼80% of the pyrazole C4 protons have been exchanged for deuterium.
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biosynthetic rescue by low temperature culture conditions)6 are
loaded with NaI, stimulated using forskolin (10 μM), and then
treated acutely with the compounds at a final concentration of
10 μM at approximately the 500 s mark. The potentiation of
channel function is observed as an increased rate of iodide
efflux from the cells, measured using an iodide-sensitive
electrode positioned in the cell bath. After sufficient data is
collected (around 800 s), cells are lysed, releasing all remaining
iodide. Figure 3a depicts the measured level of iodide in the cell
The reduced activity in the iodide efflux assay of dansyl
derivative 15 may be attributable to the compound’s reduced
ability to permeate the cell membrane rather than some
hindered interaction with CFTR. To test this possibility, we
examined the effect of this molecule in a less complex
reconstitution system, which allows the direct interactions of
small molecules with isolated CFTR to be studied. G551D-
CFTR, a clinically relevant mutant which exhibits more severely
defective channel activity than F508del-CFTR, was reconsti-
tuted into liposomes of egg phosphatidylcholine. In this
reconstituted system, a population of protein molecules are
oriented inside out within the liposome (i.e., domains that are
normally intracellular in live cells face out of the liposome, and
vice versa). Therefore a compound does not need to cross the
membrane in order to interact with the intracellular domains of
the protein. If 15 retains the ability to interact with CFTR, it
should potentiate channel activity in this population of protein
molecules.
The liposomes were loaded with potassium iodide and
suspended in a 10 μM solution of the compound to be tested in
buffer lacking iodide. The concentration of iodide outside the
liposomes is continuously measured using an iodide sensitive
probe, and CFTR activity is observed after addition of protein
kinase A and ATP to activate CFTR, and valinomycin to
prevent charge build-up across the membrane due to CFTR-
mediated iodide efflux which would slow and eventually stop
further efflux. Figure 4a shows the traces of iodide
Figure 3. (a) Typical iodide efflux traces for 1 (black line), 11 (dark-
gray), and DMSO control (light-gray). Cells were activated with
forskolin at 120 s (arrow i), treated with compound at 500 s (arrow ii),
and completely lysed at 800 s (arrow iii). (b) Potentiating effects of all
compounds tested (measured as the maximum slope of the iodide
efflux trace prior to addition of the compound, subtracted from the
maximum slope post addition).
bath over time, after acute treatment with either 1 or 11 at a
final concentration of 10 μM or DMSO control. The
experiment was also repeated with addition of 9, 10, 12, and
15 (traces not shown). The channel potentiating effects of the
compounds (measured as the maximum slope of the line prior
to addition subtracted from the maximum slope after addition
of the compound) are depicted in figure 3b. The DMSO
control showed negligible change in halide efflux post forskolin
stimulation (1.4 ± 0.4 nM/s, n = 4) whereas acute addition of 1
displayed a marked increase in efflux post forskolin stimulation
(9.9 ± 2.3 nM/s, n = 7). Analogues 9−12 were found to be
biologically active, with efflux rates ranging from 5.1 to 7.5 nM/
s. In this assay, fluorescent analogue 15 proved to be inactive
(1.7 ± 1.2 nM/s, n = 6). The effects of 1 and 9−12 were all
determined to be significantly different from DMSO control, as
assessed using analysis of variance (ANOVA) statistics.
Importantly, the rates of efflux promoted by 9−12 were not
significantly different than the rate observed for the parent
compound 1, thus suggesting that substitution of a small
functional group at the 4-position of the phenyl ring is not
detrimental to the function of the molecule as a potentiator of
CFTR channel function.
Figure 4. (a) Iodide efflux assay on purified G551D-CFTR,
reconstituted in proteoliposomes for compound 1 (black line),
fluorescent derivative 15 (dark-gray) and DMSO control (light-
gray). (b) The effect of 1 and 15 on iodide efflux in this system,
measured as the maximum slope of the iodide efflux trace after
valinomycin activation.
concentration vs time for compound 1 (black line) and 15
(dark-gray) as well as a DMSO control (light-gray). Figure 4b
shows the effect of each compound, measured as the maximum
slope of the iodide efflux trace following valinomycin activation.
Under these conditions, compound 15 proved to be an
effective potentiator of channel activity, with an iodide efflux
rate 11.18 ± 2.08 nM/s, n = 3, although with slightly
diminished activity compared to 1 (16.85 ± 1.26 nM/s, n = 5).
These data suggest that 15 maintains the ability of the parent
compound 1 to interact with CFTR and modulate its activity
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which the gradient was 100% H2O to 50% MeCN over the course of 1
h.
but has a diminished ability to cross the cell membrane.
Therefore 15 should be a useful fluorescent probe in systems of
purified and reconstituted protein and other in vitro experi-
ments not involving whole live cells.
2′-(4-Iodobenzoyloxy)-5′-methylacetophenone (6). Solid 2′-
hydroxy-5′-methylacetophenone (2, 3.948 g, 26.3 mmol) was added to
an ice-bath cooled suspension of 4-iodobenzoyl chloride (3, 7.008 g,
26.3 mmol) in pyridine (140 mL). The resultant clear yellow solution
was stirred over an ice-bath for one hour, after which time it was
quenched by pouring into an ice/HCl mixture (2M, 600 mL). This
aqueous phase was extracted three times with CH2Cl2 (200 mL each
time). The combined organic extracts were washed aqueous HCl (1M,
200 mL), then NaHCO3 (saturated, 200 mL), and finally NaCl
(saturated, 200 mL), before drying over Na2SO4 and evaporating to
afford the title ester 6 as a yellow solid, which was used without further
purification. Yield: 86% (8.566 g). 1H NMR (400 MHz, (CD3)2SO): δ
7.99−7.95 (m, 2H), 7.84−7.81 (m, 2H), 7.76 (d, J = 1.5 Hz, 1H), 7.45
(ddd, J = 8.5 Hz, J = 2.0 Hz, J = 0.5 Hz, 1H), 7.22 (d, J = 8.5 Hz, 1H),
2.46 (s, 3H), 2.38 (s, 3H). 13C NMR (100 MHz, (CD3)2SO) δ 196.87,
163.89, 145.74, 137.41, 135.39, 133.53, 130.92, 130.27, 129.58, 127.97,
123.19, 102.14, 28.83, 19.81.
CONCLUSION
■
The interaction between 1 and mutants of CFTR imbues the
protein with properties more like the wild-type, including
improved protein maturation and halide channel function (the
two key deficiencies which lead to the symptoms of cystic
fibrosis). However, little is known about the exact nature of this
interaction. As tools to help elucidate these details, useful
molecular probes based on the structure of 1 have been
synthesized and characterized. Compound 11 bears an azide
group substituted at the 4-position of the parent compound’s
phenyl ring. The compound (and other derivatives bearing
small substitutions at the same position) maintains the ability to
potentiate channel function in live cells expressing the major
mutant F508del-CFTR and also undergoes a photoreaction
generating a strong electrophile. These properties combined
suggest that 11 will be useful in photoaffinity labeling
experiments. Sonogashira coupling between iodide 9 and
propargylamine derivatives provided further useful compounds,
including the fluorescent dansyl sulfonamide 15. This
compound is active as a potentiator in a purified proteolipo-
some assay, however exhibits almost no activity in live cells,
suggesting that 15 maintains the ability to interact with CFTR
mutants but that the larger substituent abrogates the ability to
cross the cell membrane. The environment-dependent
fluorescence properties of 15 will make it a useful probe
molecule for experiments in purified protein and/or cell lysates.
Finally, it has been demonstrated that the pyrazole C4−H in 1
undergoes gradual exchange in hot alkaline water, and a
protocol to exploit this property to incorporate radioactive
tritium has been developed. These molecular probes enable an
array of biochemical experiments to determine how and where
1 interacts with mutant CFTR, which will in turn enable the
design of new, more potent and specific compounds as
potential treatments for cystic fibrosis. These experiments are
currently underway, and their results will be reported in due
course.
2′-(4-Nitrobenzoyloxy)-5′-methylacetophenone (7). Pre-
pared as described above for 6, but using 2 (2.508 g, 16.7 mmol)
and 4-nitrobenzoyl chloride (4, 4.324 g, 23.3 mmol). As above for 6,
this compound was used after extractive workup without further
1
purification. Yield 98% (4.898 g); mp 274−276 °C. H NMR (400
MHz, CDCl3): δ 8.38−8.36 (m, 4H), 7.68 (d, J = 2 Hz, 1H), 7.42
(ddd, J = 12 Hz, J = 2 Hz, J = 1 Hz, 1H), 7.13 (d, J = 8 Hz, 1H), 2.54
(s, 3H), 2.45 (s, 3H). 13C NMR (100 MHz, (CD3)2CO) δ 196.79,
163.47, 150.91, 146.75, 136.45, 135.19, 134.02, 131.32, 131.03, 130.22,
123.66, 123.55, 28.65, 19.97.
4-Methyl-2-(5-(4-iodophenyl)-1H-pyrazol-3-yl)phenol (9).
Potassium tert-butoxide (613 mg, 5.46 mmol) was dissolved in THF
(25 mL). The solution was warmed to 50 °C, and ester 6 (2.000 g,
5.26 mmol) was added, upon which a yellow precipitate was observed
to form. The mixture was stirred at 50 °C for 30 min, then cooled in
an ice-bath and quenched by addition of aqueous acetic acid (10% v/v,
20 mL). The aqueous mixture was extracted three times with CH2Cl2
(50 mL each time). The combined organic extracts were dried over
Na2SO4 and evaporated to afford a yellow solid. This solid was taken
up in glacial acetic acid (30 mL) and cooled in an ice-bath. Hydrazine
hydrate (“50−60%”, 5.7 mL, ∼100 mmol) was added dropwise to the
mixture. The ice bath was removed, and the mixture was warmed to 65
°C for 16 h. The mixture was then cooled to room temperature and
quenched with water (30 mL) and then extracted three times with
CH2Cl2 (50 mL each time). The combined organic extracts were dried
over Na2SO4 and evaporated. The crude product was purified by silica
gel column chromatography, eluting with a gradient of 1:10 to 1:1
ethyl acetate/hexanes, to afford the title compound 9 as a yellow solid.
Yield 68% (1.346 g); mp 211−213 °C. 1H NMR (400 MHz,
(CD3)2CO) δ 13.03 (bs, 1H), 10.64 (bs, 1H), 7.86 (d, J = 8 Hz, 2H),
7.68 (d, J = 8 Hz, 2H), 7.58 (s, 1H), 7.28 (s, 1H), 7.01 (d, J = 8 Hz,
1H), 6.84 (d, J = 8 Hz, 1 H), 2.29 (s, 3H). 13C NMR (100 MHz,
(CD3)2CO) δ 177.09, 138.16, 135.28, 135.11, 129.74, 127.91, 127.44,
127.03, 124.44, 123.56, 118.14, 116.49, 116.23, 19.77. ESI-TOF MS:
m/z = 377.0140 (M + H) calculated for C16H14N2OI: 377.0145.
HPLC rt = 25.2 min.
4-Methyl-2-(5-(4-nitrophenyl)-1H-pyrazol-3-yl)phenol (10).
Prepared as described above for 9 but beginning with ester 7 (3.500 g,
11.7 mmol) and driving the rearrangement step with 17.6 mmol of
sodium tert-butoxide. The rest of the procedure was scaled up directly.
The crude product was purified by silica gel column chromatography,
eluting with a 3:17 ethyl acetate/hexanes, to afford the title compound
10 as an orange solid. Yield 67% (2.313 g); mp 105−106 °C. 1H NMR
(400 MHz, (CD3)2CO) δ 12.94 (bs, 1H), 10.81 (bs, 1H), 8.35 (d, J =
1.5 Hz, 2H), 8.17 (d, J = 9 Hz. 2H), 7.62 (d, J = 1.5 Hz, 1H), 7.46 (s,
1H), 7.04 (dd, J = 8.5 Hz, J = 1.5 Hz, 1H), 6.89 (d, J = 6 Hz, 1H), 2.30
(s, 3H). 13C NMR (100 MHz, (CD3)2CO) δ 161.24, 148.25, 136.27,
130.83, 129.35, 128.17, 127.12, 125.08, 124.46, 119.15, 117.37, 116.78,
102.01, 20.55. ESI-TOF MS: m/z = 296.1024 (M + H) calculated for
C16H14N3O3: 296.1029. HPLC rt = 31.2 min.
EXPERIMENTAL SECTION
■
General (Chemistry). All reagents and solvents were used as
received from commercial suppliers, except for tetrahydrofuran, which
was dispensed under nitrogen from an Mbraun solvent purification
system immediately prior to use as a reaction solvent. Propargylamine
derivatives 1421 and 1623 were prepared according to literature
methods. TLC was carried out on silica gel 60 F254 aluminum backed
plates, supplied by EMD chemicals, eluting with the solvent system
indicated, and visualizing with UV light. Column chromatography was
carried out on Silicycle SiliaFlash P60, 40−63 μm silica gel, eluting
with the solvent system indicated below for each compound. Nuclear
magnetic resonance (1H NMR, 400 MHz, and 13C NMR, 100 MHz)
spectroscopy was carried out on a Bruker Avance 400 instrument in
the deuterated solvents indicated below for each compound, and
spectra were referenced to the solvent residual proton and 13C peaks.
High-resolution mass spectrometry was performed using ESI-TOF or
DART-TOF as indicated. UV/Visible spectroscopy was performed
using a Perkin-Elmer Lambda 20 instrument. Melting points were
determined in open air and are uncorrected. The purity of all
compounds subjected to biological assays was determined to be ≥95%
by HPLC analysis, using an Agilent Zorbax Rx-C8 4.6 mm × 150 mm
column, eluting at 0.6 mL/min with a binary gradient of 100% H2O to
100% MeCN over the course of 1 h, except for compound 15, for
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4-Methyl-2-(5-(4-aminophenyl)-1H-pyrazol-3-yl)phenol
(12). Stannous chloride dihydrate (3.850 g, 17 mmol) was added to a
solution of 10 (1 g, 3.4 mmol) in 95% ethanol (30 mL). The mixture
was heated to reflux for 16 h. The mixture was cooled to room
temperature and poured into aqueous NaOH (1 M, 150 mL). The
mixture was extracted twice with ethyl acetate (75 mL each time). The
combined organic extracts were washed with aqueous NaCl (saturated,
75 mL), then dried over Na2SO4 and evaporated. The crude product
was purified by silica gel column chromatography, eluting with a 1:4
ethyl acetate/hexanes, to afford the title compound to afford the title
compound 12 as a pale-yellow solid. Yield 71% (638 mg); mp: 200−
251.1175 (M + H) calculated for C16H15N2O: 251.1184. HPLC rt =
34.3 min. The synthesis of 1 has been previously reported,11,33
however no characterization data aside from melting point have been
published.
4-Methyl-2-(5-(4-nitrophenyl)-1H-pyrazol-3-yl)phenol (10)
(One-Pot Method). Prepared by a method analogous to 1 above,
beginning with 1 g (6.7 mmol) of 2 and employing a total of 1.781 g
(9.6 mmol) of 4-nitrobenzoyl chloride 4. Yield: 24% (475 mg).
Characterization data were the same as reported above for this
compound.
Photoreaction of 11. A solution of 11 (40 mg, 0.14 mmol) and
diethylamine (0.4 mL) in C6D6 (1.5 mL) in a quartz NMR tube was
irradiated with a 140 W Hanovia Utility ultraviolet quartz lamp. The
tube was held ∼10 cm from the light bulb, and a stream of air was
blown over the tube to maintain the contents at room temperature.
After 5 h of exposure, the mixture was evaporated under reduced
pressure, and the crude product was purified by silica gel column
chromatography, eluting with 3:7 ethyl acetate/hexanes to afford the
1
202 °C. H NMR (400 MHz, (CD3)2CO) δ 13.2 (s, 1H), 10.9 (s,
1H), 7.54 (s, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.03 (s, 1H), 6.97 (d, J = 8
Hz, 1H), 6.79 (d, J = 8 Hz, 1H), 6.63 (d, J = 8 Hz, 2H), 5.42 (s, 2H),
2.27 (s, 3H). 13C NMR (100 MHz, (CD3)2CO) δ 154.17, 149.38,
129.54, 129.34, 127.66, 126.77, 126.69, 126.32, 120.08, 116.72, 116.35,
114.36, 97.06, 19.70. ESI-TOF MS: m/z = 266.1288 (M + H)
calculated for C16H16N3O: 266.1287. HPLC rt = 29.0 min.
1
4-Methyl-2-(5-(4-azidophenyl)-1H-pyrazol-3-yl)phenol
(11). Conversion of the amine to the azide was conducted using the
method reported by Moses.18 Amine 12 (265 mg, 1 mmol) was
dissolved in acetonitrile (10 mL) and cooled in an ice-bath. To this
was added tert-butyl nitrite (178 μL, 154 mg, 1.5 mmol), followed by
azidotrimethylsilane (158 μL, 138 mg, 1.2 mmol). The mixture was
warmed to room temperature and stirred for one hour, after which the
solvent was evaporated and the crude product was purified by silica gel
column chromatography, eluting with a gradient of 1:10 to 1:1 ethyl
acetate/hexanes, to afford the title compound 11 as a yellow solid.
Yield 94% (273 mg); mp 153−154 °C. 1H NMR (400 MHz,
(CD3)2CO) δ 12.82 (bs, 1H), 10.70 (bs, 1H), 7.93 (d, J = 8.5 Hz,
2H), 7.59 (d, J = 2 Hz, 1 H), 7.28−7.22 (m, 3H), 7.02 (dd, J = 8.5 Hz,
J = 2 Hz, 1H), 6.83 (d, J = 8 Hz, 1H), 2.30 (s, 3H). 13C NMR (100
MHz, (CD3)2CO) δ 153.89, 143.72, 140.18, 129.68, 127.99, 127.14,
126.97, 126.38, 125.50, 119.63, 116.49, 116.37, 99.14, 19.76. ESI-TOF
MS: m/z = 291.1196 (M + H) calculated for C16H14N5O: 292.1192.
HPLC rt = 33.8 min.
4-Methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol (1) (One-Pot
Method). Benzoyl chloride (5, 510 μL, 617 mg, 4.4 mmol) was
added to a solution of 2 (601 mg, 4 mmol) in pyridine (10 mL), and
the mixture was warmed to 50 °C. At 15 min intervals, more 5 (50 μL,
61 mg, 0.4 mmol) was added, until TLC analysis (1:3, ethyl acetate/
hexanes) indicated complete conversion of 2 (Rf = 0.8) into the ester
(Rf = 0.7) (three aliquots were added over 45 min, for a total of 660
μL, 800 mg, 5.7 mmol benzoyl chloride in the reaction). While stirring
at 50 °C, solid potassium tert-butoxide (987 mg, 8.8 mmol) was
gradually added to the reaction mixture. After 15 min, TLC analysis
(1:3, ethyl acetate/hexanes) indicated substantial but incomplete
conversion of the ester (Rf = 0.7) to the rearranged “dione” (under
these conditions, the TLC of this material was a streak extending from
the baseline to a major spot with Rf = 0.6). More potassium tert-
butoxide (258 mg, 2.3 mmol) was added, and after an additional 30
min of stirring, TLC analysis indicated complete consumption of the
ester. Hydrazine hydrate (“50−60%”, 2.5 mL, ∼44 mmol) was added
over the course of one minute, followed by glacial acetic acid (2.0 mL,
2.1 g, 35 mmol). After 1.5 h, TLC analysis indicated completed
conversion to the desired product (1:3, ethyl acetate/hexanes, Rf =
0.5). The reaction mixture was poured into an ice/HCl mixture (1M,
250 mL), which was then extracted three times with ethyl acetate (100
mL each time). The combined organic extracts were washed with
NaHCO3 (saturated, 100 mL) and NaCl (saturated, 100 mL) and then
dried over MgSO4 and evaporated. The crude product was purified by
silica gel column chromatography, eluting with a 1:9 ethyl acetate/
hexanes, to afford the title compound 1 as a yellow solid. Yield 65%
(648 mg); mp 160−161 °C (lit.33 156−157 °C). 1H NMR (400 MHz,
(CD3)2CO) δ 12.78 (bs, 1H), 10.65 (bs, 1H), 7.908−7.876 (m, 2H),
7.606 (d, J = 2 Hz, 1H), 7.542−7.488 (m, 2H), 7.454−7.397 (m, 1H),
7.274 (s, 1H), 7.022 (ddd, J = 8.5 Hz, J = 2 Hz, J = 0.5, 1H), 6.838 (d,
J = 7.5 Hz, 1H), 2.303 (s, 3H). 13C NMR (100 MHz, (CD3)2CO) δ
154.82, 152.95, 145.16, 130.50, 130.28, 129.92, 129.57, 128.79, 127.79,
126.48, 117.33, 117.28, 100.04, 20.59. DART-TOF MS: m/z =
diethylamine adduct 13. Yield 40% (17.3 mg). H NMR (400 MHz,
(CD3)2CO) δ 10.75 (bs, 1H), 7.54 (s, 1H), 7.21 (d, J = 8.0 Hz, 1H),
6.98 (dd, J = 8.5 Hz, J = 1.5 Hz, 1H), 6.92 (s, 1H), 6.80 (d, J = 8.0 Hz,
1H), 6.04 (d, J = 8.0 Hz, 1H), 5.73 (t, J = 7.5 Hz, 1H), 3.46 (q, J = 7.0
Hz, 4H), 2.28 (s, 3H), 1.29 (bs, 2H), 1.26−1.01(m, 6H). 13C NMR
(100 MHz, (CD3)2CO) δ 154.05, 152.09, 146.01, 144.21, 142.88,
129.44, 129.40, 127.74, 126.79, 116.56, 116.35, 109.33, 106.77, 99.40,
42.95, 30.97, 19.66, 13.46.
5-Dimethylamino-N-(3-(4-(3-(2-hydroxy-5-methylphenyl)-
1H-pyrazol-5-yl)phenyl)-2-propynyl)-1-naphthalenesulfona-
mide (15). A 10 mL Schlenck tube was fitted with a magnetic stirring
bar and a glass stopper and flushed with argon gas. The tube was then
charged with 9 (203 mg, 0.54 mmol), 1421 (312 mg, 1.08 mmol), and
PdCl2·dppf·CH2Cl2 (22 mg, 0.027 mmol). The tube was evacuated
and backfilled with argon. Under a stream of argon, THF (4 mL),
triethylamine (0.12 mL, 0.8 mmol), and finally CuI (4 mg, 0.02 mmol)
were added. The tube was sealed, and the mixture was heated to 45 °C
for 15 h and then cooled back to room temperature. The mixture was
diluted with ethyl acetate (25 mL) and extracted with 0.1 M HCl (10
mL), then brine (10 mL), then dried over anhydrous sodium sulfate
and evaporated under reduced pressure. The crude material was
purified by silica gel column chromatography, eluting with 1:4 ethyl
acetate/hexanes, to afford the title compound 15 as a yellow solid.
Yield 47% (135 mg); mp 216−218 °C. 1H NMR (400 MHz,
(CD3)2CO) δ 12.82 (bs, 1H), 10.62 (bs, 1H), 8.51 (d, J = 8.5 Hz,
1H), 8.42 (d, J = 8.5, 1H), 8.33 (dd, J = 7.5 Hz, J = 1.0 Hz, 1H), 7.72
(d, J = 8 Hz, 2H), 7.66 − 7.57 (m, 3H), 7.29 − 7.23 (m, 3H), 7.02
(dd, J = 8 Hz, J = 2 Hz, 1H), 6.94 (d, J = 8 Hz, 2H), 6.84 (d, J = 8 Hz,
1 H), 4.12 (d, J = 5 Hz, 2H), 2.81 (s, 6H), 2.30 (s, 3H). 13C NMR
(100 MHz, (CD3)2CO) δ 152.85, 137.24, 137.21, 132.72, 130.98,
130.90, 130.75, 130.59, 130.29, 130.27, 128.93, 128.73, 127.86, 126.08,
124.27, 123.27, 120.40, 117.36, 117.28, 117.12, 115.97, 100.47, 86.47,
83.50, 45.59, 33.77, 20.57 (one 13C signal was not observed after
15000 scans). DART-TOF MS: m/z = 537.1958 (M + H) calculated
for C31H29N4O3S: 537.1960. HPLC rt = 25.2 min.
O-t-Butyl-N-(3-(4-(3-(2-hydroxy-5-methylphenyl)-1H-pyra-
zol-5-yl)phenyl)-2-propynyl)carbamate (17). Prepared by a
method analogous to 15 above, but using 9 (610 mg, 1.62 mmol),
1623 (500 mg, 3.24 mmol), PdCl2·dppf·CH2Cl2 (66 mg, 0.084 mmol),
THF (12 mL), triethylamine (0.35 mL, 2.5 mmol), and CuI (11 mg,
0.06 mmol). Title compound 17 was isolated as an orange/brown
1
solid. Yield 69% (445 mg); mp 185−187 °C. H NMR (400 MHz,
(CD3)2CO) δ 12.86 (bs, 1H), 10.66 (bs, 1H), 7.87 (d, J = 8 Hz, 2H),
7.60 (d. J = 1.5 Hz, 1H), 7.54 (d, J = 7 Hz, 2H), 7.31 (s, 1H), 7.03 (dd,
J = 8.5 Hz, J = 2 Hz, 1H), 6.87−6.82 (m, 1H), 6.45 (bs, 1H), 4.13 (d, J
= 6 Hz, 2H), 2.30 (s, 3H), 1.45 (s, 9H). 13C NMR (100 MHz,
(CD3)2CO) δ 156.32, 154.70, 136.16, 135.98, 133.02, 130.59, 128.90,
127.87, 126.46, 124.12, 119.04, 117.36, 117.16, 100.53, 89.87, 82.18,
79.38, 31.30, 28.59, 20.58. DART-TOF MS: m/z = 404.1966 (M + H)
calculated for C24H26N3O3: 404.1974. HPLC rt = 19.8 min.
Hydrogen Isotope Exchange on 1. To a standard NMR tube
containing 1 mL of 1 M NaOH in D2O was added 1 (25 mg, 0.1
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mmol). The tube was capped and heated in a sand bath at 90 °C.
Reaction progress was monitored by periodically collecting a 1H NMR
spectrum and observing the relative integration of the pyrazole C4−H
signal at δ 6.7 ppm. After 48 h, the reaction was cooled to room
temperature and diluted with 10 mL of H2O. The mixture was cooled
in an ice bath and acidified to pH ∼1 by dropwise addition of 1 M
HCl. The mixture was extracted with 3 × 10 mL Et2O, concentrated
under reduced pressure, and purified by preparative TLC eluting with
1:3 EtOAc/hexanes to afford 22 mg (88%) of 1C41‑D with ∼80%
approximately 1:300 (w/w) by incubation in the presence of 20 mM
MOPS, 75 mM KI, 1 mM DDM, and lipid for 30 min, followed by
passage through an Extracti-Gel D detergent-binding column (Pierce
Corp. Rockford, IL).
Iodide Efflux Measurements for Purified and Reconstituted
G551D-CFTR. The external iodide in the reconstituted protein
sample was exchanged for 75 mM K-glutamate by Sephadex G50
gel filtration resin equilibrated with 20 mM MOPS, 75 mM K-
glutamate, yielding G551D-CFTR liposomes containing 75 mM KI on
the vesicle interior and 75 mM K-glutamate in the bulk solution. The
increase in external iodide concentrations was monitored continuously
using an iodide-selective electrode (Lazar Research Laboratories, Los
Angeles, CA) interfaced to the Digidata 1320A data acquisition system
and controlled by Clampex 8 software (Axon Instruments, Sunnyvale,
CA), as described above for the cell-based assay. Protein kinase A
(catalytic subunit; Promega Corp. Madison, WI) and Mg-ATP (200
nM and 1 mM, respectively) were added to the external solution to
initiate iodide flux, and 20 nM valinomycin was added to prevent
charge-buildup across the membrane that would limit iodide flux.
Control liposomes were prepared and treated identically but in the
absence of G551D-CFTR. Verification that sufficient iodide was
trapped in the proteoliposomes was done by lysing the liposomes with
0.5% Triton X-100 at late time points. Stock solutions of 5 mM 1 or
15 were prepared in DMSO. A working solution of 20 μM was
prepared from the stock in 20 mM MOPS and 75 mM K-glutamate
buffer and added to the vesicle solution at a ratio of 1:1 to yield a final
concentration of 10 μM, 5 min before initiation of flux by valinomycin.
1
deuteration as determined by H NMR integration. H NMR (400
MHz, (CD3)2CO) δ 12.78 (bs, 1H), 10.65 (bs, 1H), 7.91−7.88 (m,
2H), 7.61 (d, J = 2 Hz, 1H), 7.53−7.49 (m, 2H), 7.45−7.41 (m, 1H),
7.27 (s, 0.2H), 7.02 (dd, J = 8.5 Hz, J = 1.5 Hz, 1H), 6.84 (d, J = 8.5
Hz, 1H), 2.30 (s, 3H). 13C NMR (100 MHz, (CD3)2CO) δ 155.94,
154.82, 142.80, 130.49, 129.97, 129.56, 128.80, 127.82, 126.48, 117.33,
117.30, 100.06, 20.58 (one 13C signal not observed after 13686 scans).
Fluorescence Emission Spectra Determination of 15. A stock
solution of 15 (5 mM in DMSO) was diluted to 20 μM in 100%
chloroform, 100% methanol, 100% DMSO or buffer containing 20
mM MOPS, 75 mM KI, and 1 mM n-dodecyl-β-D-maltoside (DDM).
Emission spectra were obtained with an excitation wavelength of 360
nm, and 4 nm excitation and emission bandpass, at 22 °C in a 0.5 cm
micro quartz cuvette on a PTI (Photon Technology International,
London, ON, Canada) Quantamaster QM/80 steady state spectro-
fluorimeter. Curves are an average of two measurements at 0.5 nm
increments with a 1 s integration time and were corrected using a
solvent blank measured under identical conditions.
Continuous Recording Cell-Based Iodide Efflux Assay. Using
BHK cells stably expressing F508del-CFTR, continuous recording cell-
based iodide efflux assays were performed as previously described.27 In
brief, cells were grown to approximately 90−100% confluency. Cells
were loaded with NaI loading buffer (3 mM KNO3, 2 mM Ca(NO3)2,
11 mM glucose, 20 mM HEPES, and 136 mM NaI) at 37 °C for 1 h.
NaI loading buffer was aspirated, and cells were washed four times in
iodide-free efflux buffer (3 mM KNO3, 2 mM Ca(NO3)2, 11 mM
glucose, 20 mM HEPES, and 136 mM NaNO3). Cells were scraped in
1 mL of iodide-free efflux buffer and collected by centrifugation (350 g
for 5 min at 25 °C). Iodide-free efflux buffer was removed, and the cell
pellet was resuspended in 400 μL of iodide-free efflux buffer. Iodide
efflux was measured at room temperature using an iodide-sensitive
electrode (Lazar Research Laboratories, Los Angeles, CA). F508del-
CFTR at the cell surface was stimulated with 10 μM forskolin,
followed by addition of 10 μM of compound (1, 9, 10, 11, 12, or 15).
Five minutes after addition of each compound, Triton X-100 (Sigma)
was used to lyse the cells to ensure iodide was properly loaded. The
maximal iodide efflux rate was quantified over a 1 min interval
associated with the largest positive slope during the 4- to 5-min time
period after the addition of the activation cocktail. Traces were
recorded using the Digidata 1320A data acquisition system with
Clampex 8 software (Molecular Devices, Sunnyvale, CA).
G551D-CFTR Purification and Reconstitution. Mutant
G551D-CFTR was purified from Sf9 cells expressing the channel
with a C-terminal His10 tag from 0.5 L of culture. Cells were
homogenized in the presence of protease inhibitors using an
Emulsiflex C3 high-pressure homogenizer (Avenstin, Ottawa, ON.
Canada), and plasma membranes were isolated on a 35% sucrose
cushion by ultracentrifugation. One-fifth of the membrane pellet was
solubilized by the presence of 2% fos-choline 14 (Anatrace, Maumee,
OH) for 1 h with resuspension by 30 G needle, and insoluble material
was removed by ultracentrifugation. The sample was bound to Ni-
NTA (Qiagen Inc. Mississauga, ON, Canada), and the beads were
washed with 10−50 mM imidazole buffer containing 1 mM
dodecylmaltoside (DDM). G551D-CFTR was eluted with 600 mM
imidazole buffer containing 1 mM DDM, and lower molecular weight
contaminants and imidazole were removed by centrifugation with an
Amicon Ultra centrifugal filter device (Milipore Corp. Billerica, MA),
yielding up to ∼0.25 mg of pure G551D-CFTR protein per liter of
culture used, in 20 mM MOPS, 75 mM KI, and 1 mM DDM, pH 7.4.
G551D-CFTR was reconstituted into 5 mg of egg phosphatidylcholine
(Avanti Polar Lipids, Alabaster, AB) at a protein:lipid ratio of
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (416) 979-5000 ext 4951. E-mail: rviirre@ryerson.ca.
ACKNOWLEDGMENTS
■
These studies were supported by the Canadian Institutes of
Health Research (CIHR) through Operating Grant MOP-
97954 and Emerging Team Grant GPG-102171, both awarded
to R.D.V. and C.E.B. P.D.W.E. was supported by postdoctoral
fellowships from Cystic Fibrosis Canada and the Canadian
Institutes of Health Research.
ABBREVIATIONS USED
■
ANOVA, analysis of variance; BHK, baby hamster kidney; CF,
cystic fibrosis; CFTR, cystic fibrosis transmembrane con-
ductance regulator; DANSYL, 5-(dimethylamino)naphthalene-
1-sulfonyl; DDM, n-dodecyl-β-D-maltoside; DPPF, 1,1′-bis-
(diphenylphosphino)ferrocene; ER, endoplasmic reticulum;
F508del, deletion of phenylalanine at position 508; G551D,
glycine to aspartic acid missense mutation at position 551;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
MOPS, 3-(N-morpholino)propanesulfonic acid; NBD chloride,
4-chloro-7-nitrobenzofurazan; NIH 3T3, mouse embryonic
fibroblast cell line; NTA, nitrilotriacetic acid; Sf9, Spodoptera
frugiperda pupal ovarian cell line
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