Function-oriented synthesis of a didesmethyl triazacryptand analogue for fluorescent potassium ion sensing

Article abstract of DOI:10.1002/ejoc.201001450

Triazacryptand (TAC)-based fluorescent K⁺ sensors have broad biomedical utility, yet their advancement has been hindered because of their challenging synthesis. Herein, an efficient synthesis is reported that delivers a didesmethyl triazacrypta

Full text of DOI:10.1002/ejoc.201001450

FULL PAPER
DOI: 10.1002/ejoc.201001450
Function-Oriented Synthesis of a Didesmethyl Triazacryptand Analogue for
Fluorescent Potassium Ion Sensing
Richard D. Carpenter[a,b] and Alan S. Verkman*[a]
Dedicated to the memory of Prof. Aaron D. Mills
Keywords: Biosensors / Potassium / Ionophores / Cryptands / Dianionic oxidation
Triazacryptand (TAC)-based fluorescent K+ sensors have
broad biomedical utility, yet their advancement has been
hindered because of their challenging synthesis. Herein, an
efficient synthesis is reported that delivers a didesmethyl tri-
azacryptand (ddTAC) K+ sensor in twofold fewer steps and
ninefold higher overall yield than the original TAC synthesis.
Our synthesis utilizes a C–O dianionic oxidative macrocycli-
zation and reports new examples of aminoarylations and a
microwave route to xanthythilium chromophores. The K+
sensitivity and selectivity of the ddTAC-based sensor are
comparable to the TAC-based sensor.
Introduction
Fluorescent potassium (K+) ion sensors have great po-
tential utility in the fields of cell biology, neuroscience, drug
discovery, and clinical medicine. Figure 1a shows the ge-
neric structure of one type of ion sensor, which consists of
ionophore, linker, and chromophore domains. With suitable
linker and chromophore domains, ion binding to the iono-
phore region can increase fluorescence of the chromophore
by a charge-transfer mechanism.[1–6] The K+ sensor shown
in Figure 1b contains two macrocyclic rings and multiple
heteroatoms in its triazacryptand (TAC) ionophore and a
chromophore with its π system in conjugation with the
linker and ionophore regions.[7–9]
Figure 1. (a) Schematic of a generic ion sensor, where ion binding
produces an increase in chromophore fluorescence via a charge-
transfer mechanism; (b) chemical structure of a K+ sensor in a K+-
bound conformation, showing triazacryptand ionophore (blue),
linker (green), and xanthylium chromophore (red) domains.
He et al. reported the first TAC K+ sensor, yet their sen-
sor contained ionophore and chromophore moieties that
were not in π conjugation, and thus it was of limited use.[10]
The key precursor of He et al., TAC-CHO (2a in Figure 2),
was synthesized in 10 steps (longest linear sequence being
9 steps) and 2% overall yield. In addition to the low overall
yield of 2a, its synthesis was difficult to reproduce, as it
required two challenging 21-membered macrocyclization re-
actions. Nonetheless, on the basis of the route to 2a re-
ported by He et al., we synthesized a series of water-soluble
K+ sensors with different optical and physical properties[11]
and demonstrated their application to measurement of ex-
tracellular space K+ concentration in brain[12] and airway/
ocular surface liquids[13] and to K+ transporter drug discov-
ery.[14] Recently, we reported new ionophore 1 with excellent
K+ sensitivity and selectivity,[15] yet its synthesis utilizes an
ionophore bromide precursor that, compared to TAC-CHO
precursor 2a, limits further chromophore and bioconjugate
derivatization.
Our goal here was to develop an alternative, practical
synthetic route to an ionophore aldehyde precursor with
reduced number of steps and improved overall yield over
the original synthetic route to 2a. After studying the scheme
reported by He et al.[10] we speculated that the bistolyl moi-
eties likely served as blocking groups, thereby enabling re-
gioselective Vilsmeir–Haack formylation to afford 2a.[16,17]
[a] Departments of Medicine and Physiology, University of
California, San Francisco, Health Sciences East, Room 1246,
Box 0521,
513 Parnassus Ave., San Francisco, CA 94143-0521, USA
Fax: +1-415-665-3847
E-mail: Alan.Verkman@ucsf.edu
[b] Department of Biomedical Engineering, University of
California, Davis, Genome and Biomedical Sciences Facility,
451 Health Sciences Dr., Davis, CA 95616, USA
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201001450.
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Function-Oriented Synthesis of a Didesmethyl Triazacryptand Analogue
conversion of 79% determined by LC–MS and with com-
plete chemoselectivity. Using these conditions with the de-
sired diazacryptand starting material rapidly afforded bisar-
ylated amine 4 (76% yield; Scheme 1), which represents the
only example of a β-amino ether coupled with an iodo-
phenol. Of note, this transformation uses commercially
available 2-iodophenol in slight excess (2.8 equiv.). Prepara-
tion of TAC-CHO 2a by this route would require 2-iodo-5-
methylphenol, which, despite its structural simplicity, adds
one or two synthetic transformations from either an electro-
philic aromatic iodination of m-cresol[21] or an o-lithi-
ation[22] and subsequent demethylation of 3-methyl-
anisole.[23]
Figure 2. Structures of sensors 1, 2b–e, and 3b–c, with 2a and 3a
serving as key sensor precursors. In this report, sensors 2b/3b and
2c/3c are compared in their sensitivity and selectivity for K+ and
their insensitivity to pH.
Therefore, we recognized that removing these remote
methyl groups on the TAC ionophore would result in a sim-
plified structure without a predicted effect on K+ binding.
Herein, we report a streamlined synthesis of the function-
ally comparable didesmethyl triazacryptand (ddTAC) K+
sensors 3b and 3c by using a key template-directed di-
anionic oxidation reaction to close the alkyl phenyl ether
macrocycle. Our efficient and streamlined route to ddTAC
should enable the advancement of TAC-based K+ sensors
by using the ddTAC ionophore in place of the TAC iono-
phore.
Scheme 1. Initial attempts towards bismacrocyclic fragment 7.
This bisaminoarylation was then followed by a Finkel-
stein-modified Williamson ether synthesis of bisphenol 4
with tert-butyl bis(2-chloroethyl)carbamate to afford mono-
O-alkylated intermediate 5 with an adorning alkyl chloride
motif. Finkelstein-modified Williamson ether synthesis un-
der dilute conditions afforded Boc-protected amine 6; how-
ever, the yields were poor and variable (26Ϯ19%; n = 3)
for this two-step sequence.
Results and Discussion
This disappointing intramolecular displacement macro-
Our streamlined route maximizes step-economy based on cyclization employing a phenoxide anion is, nonetheless, an
function-oriented synthesis, convergence, and new reaction improvement over a similar transformation that was at-
methodology.[18,19] The synthesis of ddTAC-CHO 3a, the tempted but ultimately unsuccessful with the imidazole sys-
key ionophore aldehyde precursor that is subsequently con- tem.[15] To overcome this unsuccessful displacement reac-
jugated with various chromophores, began with a metal- tion with the imidazole system, we recently developed a new
mediated bisaminoarylation as outlined in Scheme 1. While template-directed organodipotassium oxidative macrocycli-
metal-mediated aminoarylations, so-called Buchwald–Hart- zation to install a C–N bond[15] that was inspired by Sar-
wig reactions,[20] have been key transformations in the syn- pong’s organodilithium oxidation.[24,25] During the course
thesis of anilines, there are no examples, to the best of our of this work, we discovered that our organodipotassium
knowledge, of the use of β-amino ethers, iodophenols, di- oxidation conditions could be used to close a variety of
amines, or any type of cyclic secondary amine. Given this, rings and make C–N or N–N bonds from either metal–
the sophistication of both reactants, and the expense of the halogen exchange or deprotonation of acidic C–H or N–
diazacryptand, optimization reactions were carried out by H.[15] Although unprecedented, we speculated that organ-
using morpholine and piperazine as proof-of-principle sub- odipotassium oxidation of 5 (structure shown in Scheme 1)
strates. During these optimization reactions, it was found would similarly make macrocycle 6; the phenol would be
that the use of DMA as a solvent, a predissolved 1 n solu- deprotonated and the alkyl chloride would undergo metal–
tion of NaOH, 2-iodophenol (2.8 equiv.), catalytic cop- halogen exchange to produce dianions that are held in close
per(I) iodide, and microwave heating to 105 °C for 12 min proximity through neighboring chelating groups prior to
afforded the desired N-arylated products with an average oxidation to install a C–O bond. Gratifyingly, alkyl chloride
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R. D. Carpenter, A. S. Verkman
FULL PAPER
5 was treated with Li0 sticks and KOtBu (Schlosser’s condi-
Crude diazonium aldehyde fragment 8 was then can-
tions) at cryogenic temperature, followed by treatment with nulated into a chilled DMSO solution (0 °C) of bismacro-
diphenyl diselenide and TFA to afford amine 7 (74% over cyclic fragment 7. This solution was then warmed to room
three steps) containing the bismacrocyclic entity of 3a temperature and, following purification by column
(Scheme 2).
chromatography, afforded didesmethylated TAC-CHO 3a in
83% yield over two steps. This metal-free aminoarylation
represents a rare example of a secondary amine reacting
with an aryldiazonium salt, as thiols, water, or iodide have
been the nucleophiles of choice.[27] In eight total steps, with
the longest linear sequence being five steps, ddTAC-CHO
3a was synthesized in 28% overall yield from commercially
available starting materials.
Having devised an efficient and high-yielding route to
ddTAC-CHO 3a, our attention turned towards the iono-
phore–chromophore union (Scheme 4). To investigate
whether the remote 4-methyl groups on the TAC ionophore
affect probe K+ sensitivity and selectivity, as well as pH
sensitivity, ddTAC-CHO 3a was conjugated to both xanthy-
lium and BODIPY chromophores to generate sensors 3b
and 3c (Figure 2). Briefly, aldehyde 3a was treated with 3-
dimethylaminophenol and a catalytic amount of acid fol-
lowed by oxidation with tetrachloro-1,4-benzoquinone to
afford ddTAC-Red (3b) in 62% yield from ddTAC-CHO
3a. Notably, key to the xanthylium chromophore synthesis
is the use of microwave heating, which rapidly afforded the
desired chromophores Ͼ100-fold faster and, remarkably, in
12-fold higher yield than the previously described route.[10]
Similarly, 3a was treated with 2,4-dimethylpyrrole and a
catalytic amount of TFA in a Friedel–Crafts reaction, fol-
lowed by oxidation with DDQ and treatment with
BF3·OEt2 to afford K+ ddTAC-BODIPY (3c) in 58% yield
over three steps.
Scheme 2. Improved route to bismacrocycle 7 featuring a dianionic
oxidation macrocyclization presumably via templation of one or
more K+ ions. The structures of 5–7 are shown in Scheme 1.
A series of control experiments were then performed to
ensure that this transformation likely goes through a dian-
ionic intermediate and not a nucleophilic displacement. No-
tably, combining any two of the three reagents (Li0, KOtBu,
and diphenyl diselenide) with 5 did not result in any ap-
preciable macrocyclization (i.e., 6). Therefore, a likely path-
way in this reaction involves elemental lithium reducing the
phenolic hydrogen and undergoing lithium–halogen ex-
change, the potassium alkoxide solution for transmetalation
and substrate templation, and diphenyl diselenide as an ap-
propriate oxidant in the presence of an organometallic spe-
cies.
With the construction of bismacrocycle 7 complete, benz-
aldehyde fragment 8 was synthesized (Scheme 3). The syn-
thesis of the aldehyde fragment began with O-alkylation of
electron-deficient 3-hydroxy-4-nitrobenzaldehyde under
Finkelstein conditions to obtain 9 in 68% yield. Tin(II)
chloride reduction of the nitro group[26] afforded aniline 10
(88%) followed by diazotization to presumably furnish dia-
zonium salt 8, which was not isolated.
Scheme 4. Synthesis of ddTAC-Red (3b) and ddTAC-Lime (3c).
Reagents and conditions: (a) 3-(dimethylamino)phenol, PTSA,
propionic acid, MW, 130 °C, 5 min; (b) tetrachloro-1,4-benzo-
quinone, MeOH/CHCl3, MW, 115 °C, 18 min; (c) 2,5-dimethyl-
pyrrole, TFA, CH2Cl2; (d) BF3·OEt2, DIEA, CH2Cl2; (e) DDQ,
CH2Cl2.
The cation sensitivity and selectivity and the pH sensiti-
vity of ddTAC-Red 3b and ddTAC-Lime 3c were deter-
mined and compared with those of TAC-based K+ sensors
2b/2c. Sensor selectivity was assessed via titration with sev-
eral alkali and alkali earth cations (Figure 3b). ddTAC-De-
rived sensors 3b/3c and TAC-derived sensors 2b/2c showed
comparably low sensitivity to Na+, Li+, Mg2+, and Ca2+
(data shown only for Na+) and comparable modest sensitiv-
ity to Rb+ and Cs+. Finally, pH sensitivity was determined
by titration with HCl at different concentrations of K+ (Fig-
ure 3c). Sensors 2b/2c and 3b/3c were comparably insensi-
tive to pH values greater than 6 and sensitive to pH values
Scheme 3. Synthesis of ddTAC-CHO 3a.
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Function-Oriented Synthesis of a Didesmethyl Triazacryptand Analogue
lower than 6. This is likely due to the presence of multiple fied precursor ddTAC-CHO 3a is synthesized in twofold
Lewis basic heteroatoms that are present in either iono- fewer total steps and with a ninefold increase in overall
phore.
yield compared to the original synthesis of 2a. Additionally,
this report introduces a new C–O bond-forming dianionic
oxidative macrocyclization, a much improved ionophore–
chromophore union, and new examples of metal-mediated
and metal-free aminoarylations. Future efforts will be fo-
cused on creating conjugates of 3 for various molecular,
cellular, translational, and clinical applications.
Experimental Section
Experiment Setup and Equipment Details: Oxygen- and moisture-
sensitive reactions were carried out in flame- or oven-dried glass-
ware sealed with a rubber septum under positive pressure of argon
from a balloon. Sensitive liquids and solutions were transferred by
syringe or cannula through septa under positive pressure of argon.
The term “concentration” refers to rotary evaporation (Büchi) with
a vacuum pump at a pressure of 3 Torr. Residual solvents were
removed from nonvolatile products on a vacuum line with a pres-
sure of 0.1–0.3 Torr. The temperature of microwave reactions was
maintained by using a Biotage microwave reactor, which heated
the sealed samples to the indicated temperature in 100–120 s and
maintained that temperature for the duration of the reactions.
Proof-of-principle reactions, carried out by using morpholine and
piperazine to optimize the synthesis of 4, were analyzed by TLC
and LC–MS and were neither purified nor characterized by ad-
ditional analytical methods.
Reagents, Solvents, and Buffers: Reagents and solvents were pur-
chased from commercial vendors and used without further purifi-
cation with the exception of the following: Ether and THF were
distilled from sodium–benzophenone ketyl under an atmosphere of
argon (CAUTION: do not distill to dryness). Dichloromethane was
distilled from calcium hydride under an atmosphere of argon. Ele-
mental lithium was weighed out in an oven-dried (120 °C) screw-
cap vial that was cooled in a desiccator by employing Drierite as
the desiccant. TAC-Red and TAC-Lime are described else-
where.[12,14] HEPES solution contained (in mm): 145 NaCl, 5 KCl,
1 MgCl2, 1 CaCl2, 10 d-glucose, and 10 HEPES (pH 7.4). The K+-
free solution was prepared by replacing 5 mm KCl with NaCl. The
200 mosm/LK+-free solution was prepared by removing 50 mm
NaCl and 5 mm KCl. The high-K+ solution was prepared by replac-
ing 100 mm NaCl with KCl. Other solvents and reagents were used
without purification unless otherwise noted.
Figure 3. (a) Fluorescence emission at 570 nm for xanthylium-de-
rived TAC-Red and ddTAC-Red (2b/3c) and at 533 nm for BOD-
IPY-derived TAC-Lime and ddTAC-Lime (2c/3c) at indicated [K+].
(b) Fluorescence of 2b/3b and 2c/3c in the presence of indicated
cations. (c) Fluorescence as a function of pH at indicated [K+].
Samples in (a–c) were excited at 480 nm for xanthylium-derived
sensors and 500 nm for BODIPY-derived sensors. Solutions in (a–
c) were balanced with NaCl or choline chloride to maintain a con-
stant ionic strength of 200 mm.
Chromatography and Purification: Thin-layer chromatography
(TLC) used glass plates coated with silica gel 60 F254, developed
in a chamber, and visualized under ultraviolet light (λex = 254 nm)
as well as by treatment with iodine or potassium permanganate
solution. Column chromatography used silica gel 60 packed in glass
columns with indicated eluents. Precipitation was carried out by
bubbling in hydrogen chloride (generated from the dropwise ad-
dition of concentrated sulfuric acid to solid sodium chloride) into
a dropping funnel containing anhydrous ether (ca. 3 h) followed by
the dropwise addition of this solution to a concentrated anhydrous
ether solution of the amine-containing compound. Following vac-
uum filtration, the amine–HCl salt was dissolved in sodium hydro-
gen carbonate and ether and extracted as indicated. Recrystalli-
zation was carried out by dissolving the compound with the indi-
cated boiling solvent followed by gradual cooling to ambient tem-
perature or treatment with the indicated nonpolar cosolvent at am-
Conclusions
In conclusion, the didesmethyl modification with two 4-
methyl groups removed results in a simplified sensor that
retains the fluorescence properties of TAC-based K+ sen-
sors, regardless of the chromophore. Functionally simpli- bient temperature.
Eur. J. Org. Chem. 2011, 1242–1248
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R. D. Carpenter, A. S. Verkman
FULL PAPER
Physical Properties and Spectroscopic Measurements: 1H and 13C
–78 °C. After cooling this solution for 10 min, a –78 °C chilled
NMR spectra were obtained by using Bruker Instruments (300 and
solution of crude alkyl chloride 5 (65 mg, 0.100 mmol) in THF
500 MHz for 1H; 75 and 125 MHz for 13C). 1H and 13C NMR (2 mL) was added via cannula over 5 min. After cooling for 20 min,
spectra were recorded in CDCl3 and [D6]DMSO with the solvent
potassium tert-butoxide (1.0 m in THF, 1.00 mL, 1.00 mmol) was
added, and the reaction mixture was stirred for an additional
1
signals used as a reference (CDCl3: 7.26 ppm for H, NMR 77.16
for 13C NMR; [D6]DMSO: 2.50 ppm for 1H, 39.52 ppm for 13C 20 min. To this solution was added diphenyl diselenide (312 mg,
NMR). 13C NMR spectra were recorded with complete hetero-
decoupling and with nOe. LC–MS was accomplished by using a
Waters Alliance instrument equipped with a Nova-Pak C18 column
(3.9ϫ150 mm). All yields reported refer to isolated material judged
to be homogeneous by TLC and NMR spectroscopy. HRMS was
performed at the University of California, Riverside. Fluorescence
data shown in Figure 3 was obtained by using a Spex fluorimeter
(λex = 480 nm for xanthylium chromophores; λex = 500 nm for
BODIPY chromophores) with all solutions balanced with either
sodium chloride or choline chloride in order to keep ionic strength
constant at 200 mm.
0.500 mmol) and the temperature was kept at –78 °C for 2 h, fol-
lowed by warming the solution to 0 °C (ca. 2.5 h). The reaction
was quenched with saturated aqueous ammonium chloride (10 mL)
and saturated aqueous sodium bisulfite (10 mL). The resulting
solution was extracted with diethyl ether (3ϫ20 mL), and the com-
bined organics were washed with brine (35 mL), dried (MgSO4),
vacuum filtered, and concentrated. This residue was crystallized
with CHCl3/petroleum ether and filtered to afford 6 as a light-
brown solid (46 mg, 74% from 4).
Method B: Crude alkyl chloride 5 (65 mg, 0.100 mmol) was dis-
solved in acetone (10 mL) and treated with a mixture of sodium
iodide (75 mg, 0.500 mmol) and sodium carbonate (53 mg,
0.500 mmol) portionwise over 1 h. Following this, the temperature
of the reaction was gradually raised to 50 °C over 2 h and kept at
this temperature for 8 h before dilution with water (15 mL) and
extraction with diethyl ether (2ϫ15 mL). The combined organics
were washed with brine (25 mL), dried (MgSO4), and concentrated.
The crude residue was crystallized with CHCl3/petroleum ether and
filtered to afford 6 as a light brown solid (16Ϯ12 mg, 26Ϯ19%; n
Sample Handling and Storage: Because derivatives of 2 and 3 bind
to K+ with high affinity, all prepared compounds described herein
that contain a cryptand ring should be handled with care. No ani-
mal toxicity data exist for these compounds. All sample vials were
wrapped in foil, sealed in a plastic bag, and stored at –20 °C.
2,2Ј-(1,4,10,13-Tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)di-
phenol (4): 1,4,10,13-Tetraoxa-7,16-diazacyclooctadecane (700 mg,
2.68 mmol), 2-iodophenol (847 μL, 7.50 mmol), copper(I) iodide
(51 mg, 0.268 mmol), and a predissolved solution of 1 n NaOH
(2 mL) were dissolved in DMA (8 mL) in a sealable microwave vial.
This mixture was flushed with argon, sealed, and microwave irradi-
ated at 105 °C for 12 min. The reaction mixture was then cooled
to ambient temperature, diluted with water (20 mL), and extracted
with diethyl ether (3ϫ15 mL). The combined organics were
washed with water (50 mL), brine (50 mL), dried (MgSO4), and
concentrated. The concentrate was precipitated by the dropwise ad-
dition of concentrated hydrogen chloride in anhydrous ether and
filtered. To remove the unwanted HCl salt, the precipitate was
taken up in diethyl ether (15 mL) and saturated NaHCO3 (15 mL).
Following separation, the organic layer was washed with brine
(15 mL), dried (MgSO4), and concentrated to afford 4 (1.81 g,
1
= 3). H NMR (300 MHz, CDCl3, 25 °C): δ = 6.74–6.58 (m, 8 H),
3.61–3.56 (m, 24 H), 2.80 (t, J = 4.8 Hz, 8 H), 1.46, (s, 9 H) ppm.
13C NMR (75 MHz, CDCl3, 25 °C): δ = 156.2, 145.1, 135.6, 119.9,
118.9, 115.8, 115.3, 80.0, 70.1, 70.0, 61.6, 52.5, 49.1, 28.4 ppm. MS
(ESI): m/z = 60.8 [M + H – tBu]+. HRMS (ESI): calcd. for
+
C33H50N3O8 616.3592; found 560.2963 (–tBu). Purity was found
to be 97% and 99% for methods A and B, respectively, on the basis
of HPLC analysis.
2,2Ј-[2,2Ј-(1,4,10,13-Tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)-
bis(2,1-phen-ylene)]bis(ethoxy)amine (7): Boc-Protected amine 6
was dissolved in TFA/CH2Cl2 (1:1, 50 mL), and the mixture was
stirred overnight at room temperature. The reaction mixture was
concentrated and treated with saturated aqueous sodium hydrogen
carbonate. The solid was filtered to afford 7 (428 mg, 74% from 4)
as a light brown solid. 1H NMR (300 MHz, CDCl3, 25 °C): δ =
6.73–6.58 (m, 8 H), 3.96 (br. s, 1 H), 3.68 (t, J = 5.0 Hz, 4 H),
3.61–3.57 (m, 20 H), 2.84 (t, J = 4.9 Hz, 8 H) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): δ = 145.2, 135.7, 119.9, 118.9, 115.8,
115.3, 70.3, 70.1, 61.3, 50.3, 49.7 ppm. MS (ESI): m/z = 516.8 [M
1
76%) as a light brown solid. H NMR (300 MHz, CDCl3, 25 °C):
δ = 6.74–6.58 (m, 8 H), 3.62–3.57 (m, 1 H), 2.81, (t, J = 4.8 Hz, 8
H) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ = 145.3, 135.8,
119.7, 118.9, 115.6, 115.3, 70.2, 49.2 ppm. MS (ESI): m/z = 47.3
+
[M + H]+. HRMS (ESI): calcd. for C24H35N2O6 [M + H]+
447.2490; found 447.2495. Purity was found to be 99% on the basis
of HPLC analysis.
+
+ H]+. HRMS (ESI): calcd. for C28H42N3O6 516.3068; found
tert-Butyl 2-Chloroethyl(2-{2-[16-(2-hydroxyphenyl)-1,4,10,13-tetra-
oxa-7,16-diazacycloocta-decan-7-yl]phenoxy}ethyl)carbamate (5):
Diphenol 4 (500 mg, 1.12 mmol), tert-butyl bis(2-chloroethyl) carb-
amate (271 mg, 1.12 mmol), sodium iodide (16 mg, 0.112 mmol),
and sodium carbonate (59 mg, 0.56 mmol) were dissolved in acet-
one (50 mL). The reaction mixture was stirred for 16 h before di-
lution with water (40 mL) and extraction with diethyl ether
(2ϫ30 mL). The combined organics were washed with brine
(50 mL), dried (MgSO4), and concentrated. This residue was crys-
tallized with CHCl3/petroleum ether and filtered to presumably af-
ford crude 5, which was quickly used without further purification
or characterization.
516.3061. Purity was found to be 100% on the basis of HPLC
analysis.
3-(2-Methoxyethoxy)-4-nitrobenzaldehyde (9): 3-Hydroxy-4-nitro-
benzaldehyde (1 g, 5.99 mmol), 1-chloro-2-ethoxyethane (684 μL,
7.19 mmol), sodium iodide (1.07 g, 7.19 mmol), and sodium carb-
onate (733 mg, 7.19 mmol) were dissolved in DMF (70 mL). The
reaction mixture was kept at room temperature for 1 h and then
heated to 75 °C for 16 h before dilution with water (40 mL) and
extraction with diethyl ether (2ϫ30 mL). The combined organics
were washed with brine (50 mL), dried (MgSO4), and concentrated.
This residue was purified by column chromatography (hexane/
1
EtOAc, 95:5) to afford 9 (795 mg, 68%) as a light yellow solid. H
tert-Butyl-2,2Ј-[2,2Ј-(1,4,10,13-tetraoxa-7,16-diazacyclo-octadecane-
7,16-diyl)-bis(2,1-phenylene)]bis(ethoxy)carbamate (6)
NMR (300 MHz, [D6]DMSO, 25 °C): δ = 10.0 (s, 1 H), 8.02 (d, J
= 8.3 Hz, 1 H), 7.58 (d, J = 1.6 Hz, 1 H), 7.48 (dd, J = 1.5, 8.3 Hz,
1 H), 3.50 (t, J = 6.5 Hz, 2 H), 3.34 (t, J = 5.3 Hz, 2 H), 3.24 (s, 3
H) ppm. 13C NMR (75 MHz, [D6]DMSO, 25 °C): δ = 192.2, 151.6,
140.9, 139.8, 125.8, 124.2, 119.7, 118.9, 73.8, 60.0, 57.9 ppm. MS
Method A: Lithium sticks (10 mm diameter; 7 mg, 1.00 mmol) were
quickly added to a flame-dried flask that was back-filled with ar-
gon. THF (5 mL) was added, and the suspension was chilled to
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Function-Oriented Synthesis of a Didesmethyl Triazacryptand Analogue
(ESI): m/z = 226.2 [M + H]+. HRMS (ESI): calcd. for C10H12NO5
966.4778; found 465.7542 [M + H – Cl–]2+. Purity was found to be
99% on the basis of HPLC analysis.
+
226.0710; found 226.0706. Purity was found to be 98% on the basis
of HPLC analysis.
ddTAC-Lime (3c): ddTAC-Lime 3c was synthesized in 58% yield
from ddTAC-CHO 3a by using the TAC-Lime protocol that is de-
scribed elsewhere.[8] 1H NMR (500 MHz, [D6]DMSO/D2O = 1:1,
25 °C): δ = 6.66–6.52 (m, 9 H), 6.42–6.39 (m, 8 H), 6.27 (s, 1 H),
5.52 (s, 2 H), 3.51–3.34 (m, 24 H), 3.23 (s, 3 H), 2.62–2.58 (m, 24
H), 2.10 (s, 6 H), 1.94 (s, 6 H) ppm. 13C NMR (125 MHz, [D6]-
DMSO/D2O = 1:1, 25 °C): δ = 145.6, 144.4, 136.6, 136.1, 126.9,
126.7, 119.8, 119.6, 118.9, 117.01, 116.96, 115.1, 114.9, 114.7,
113.8, 113.6, 107.21, 107.18, 74.1, 70.10, 70.07, 60.4, 60.3, 58.3,
51.6, 49.1, 13.0, 12.9, 12.2 ppm. MS (ESI): m/z = 912.7 [M + H]+.
4-Amino-3-(2-methoxyethoxy)benzaldehyde (10): Nitrobenzene 9
(500 mg, 2.22 mmol) was dissolved in methanol (300 mL) followed
by the addition of tin(II) chloride dihydrate (15.01 g, 66.7 mmol).
This reaction mixture was heated at reflux for 6 h and then cooled
to ambient temperature. The solution was concentrated and then
dissolved in ethyl acetate (200 mL). The solution was washed with
saturated aq. NaHCO3 (3ϫ100 mL), dried (MgSO4), and concen-
trated to afford 10 (795 mg, 68%) as a light yellow solid. 1H NMR
(300 MHz, [D6]DMSO, 25 °C): δ = 9.70 (s, 1 H), 7.29 (d, J = 2 Hz,
1 H), 7.27–7.23 (m, 1 H), 6.92 (d, J = 4 Hz, 1 H), 3.49 (t, J =
6.5 Hz, 2 H), 3.35–3.32 (m, 2 H), 3.24 (s, 3 H) ppm. 13C NMR
(75 MHz, [D6]DMSO, 25 °C): δ = 191.0, 152.1, 145.8, 128.8, 124.4,
115.5, 114.3, 73.8, 60.0, 58.0 ppm. MS (ESI): m/z = 196.1 [M +
+
HRMS (ESI): calcd. for C50H65BF2N5O8 912.4889; found
912.4894. Purity was found to be 100% on the basis of HPLC
analysis.
Supporting Information (see footnote on the first page of this arti-
cle): NMR spectra of all new compounds and tabulated fluores-
cence emission values for the graphs shown in Figure 3.
+
H]+. HRMS (ESI): calcd. for C10H14NO3 196.0968; found
196.0970. Purity was found to be 97% on the basis of HPLC analy-
sis.
ddTAC-CHO (3a): A solution of aniline 10 (50 mg, 0.256 mmol) in
concentrated HCl (2 mL) was cooled to 0 °C. A solution of NaNO2
(17 mg, 0.256 mmol) in H2O (3 mL) was added, and the mixture
was stirred at 0 °C for 1 h. This mixture, presumably containing
diazonium salt 8, was added dropwise over 30 min to a solution of
7 (131 mg, 0.256 mmol) in CH3CN (10 mL) at 0 °C. The reaction
mixture was warmed to 25 °C, and the solution was stirred at this
temperature for 12 h. The mixture was slowly diluted with satu-
rated aqueous sodium hydrogen carbonate (75 mL) and extracted
with diethyl ether (3ϫ100 mL). The combined extracts were
washed with brine (100 mL), dried (MgSO4), and concentrated.
This residue was purified by column chromatography (hexane/
EtOAc, 90:10) to afford 3a (147 mg, 83%) as a light-brown solid:
1H NMR (300 MHz, CDCl3, 25 °C): δ = 9.69 (s, 1 H), 7.48–7.08
(m, 3 H), 6.79–6.58 (m, 8 H), 3.73–3.48 (m, 24 H), 3.39 (s, 3 H),
2.82–2.74 (m, 12 H) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ =
191.0, 155.5, 148.0, 147.5, 145.0, 136.9, 135.5, 128.2, 125.6, 120.1,
119.0, 116.0, 115.8, 115.3, 113.7, 69.4, 68.9, 60.8, 50.8, 49.2 ppm.
MS (ESI): m/z = 694.5 [M + H]+. HRMS (ESI): calcd. for
Acknowledgments
This work was supported by the National Institutes of Health
(NIH) (grants EB00415, DK86125, DK35124, HL73856, EY13574,
and DK72517), the Cystic Fibrosis Foundation, and the Guthy–
Jackson Charitable Foundation.
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+
Eur. J. Org. Chem. 2011, 1242–1248
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1247

R. D. Carpenter, A. S. Verkman
FULL PAPER
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Published Online: January 17, 2011
1248
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 1242–1248