Synthesis of a sensitive and selective potassium-sensing fluoroionophore

Article abstract of DOI:10.1021/ol902836c

"Chemical Equation Presented" An efficient synthesis is reported that delivers in 5 steps and 52% overall yield a new structurally simplified fluorescent K⁺ sensor with improved K⁺ sensitivity and selectivity over existing K⁺ sensors. The synthesis procedure utilizes a new template-directed oxidative C-N bond-forming macrocyclization reaction and reports new approaches to Pd(O), Sandmeyer-like and metal-free aminoarylations, as well as organotitanium additions to vinylogous sulfonates.

Full text of DOI:10.1021/ol902836c

ORGANIC
LETTERS
2010
Vol. 12, No. 6
1160-1163
Synthesis of a Sensitive and Selective
Potassium-Sensing Fluoroionophore
Richard D. Carpenter† and A. S. Verkman*
Departments of Medicine and Physiology, CardioVascular Research Institute,
UniVersity of California, San Francisco, California
alan.Verkman@ucsf.edu
Received December 8, 2009
ABSTRACT
An efficient synthesis is reported that delivers in 5 steps and 52% overall yield a new structurally simplified fluorescent K+ sensor with
improved K+ sensitivity and selectivity over existing K+ sensors. The synthesis procedure utilizes a new template-directed oxidative C-N
bond-forming macrocyclization reaction and reports new approaches to Pd(0), Sandmeyer-like and metal-free aminoarylations, as well as
organotitanium additions to vinylogous sulfonates.
Efficient syntheses of complex, biomedically relevant com-
pounds provide the impetus for novel reaction methodolo-
gies.1 In the field of ion sensing, of great importance are
fluorescent potassium (K+) indicators that function in aque-
ous media and have good optical properties, high K+
sensitivity and selectivity, pH insensitivity, and perhaps most
importantly, a feasible synthetic route.2 K+ is a major analyte
that plays a vital role in normal cell function and various
diseases.3 Our laboratory and others have made advances in
K+ biosensing, including the development of an aqueous-
compatible sensor,4a an ionophore that is insensitive to pH
in the range of 5-8,4b and a conjugated ionophore-chro-
mophore system that by photoinduced electron transfer
produced a 14-fold increase in fluorescence with increasing
K+.4c,d The reported synthetic methods for these sensors
involve 12-16 steps (longest linear sequence 11-13 steps),
which include two 21-membered macrocyclizations and a
poorly yielding two- to three-step ionophore-chromophore
union late in the synthesis.4 Limited by these laborious routes,
fine-tuning the K+ sensitivity and selectivity of these sensors
has not been done, nor have these sensors been widely
available to biomedical scientists.
Herein is described a concise synthesis of structurally
simplified and functionally superior K+ sensor 1 via three
interesting C-N bond-forming reactions and an efficient
ionophore-chromophore C-C bond-forming reaction as
shown in Figure 1. Highlights of this novel approach include
a rapid microwave-mediated aminoarylation using a pal-
ladium-QuinaPhos catalyst, a template-directed macrocy-
† Presently at the Department of Biomedical Engineering, University of
California, Davis, California.
(1) Wender, P. A.; Verma, V. A.; Paxton, T. J.; Pillow, T. H. Acc. Chem.
Res. 2008, 41, 40–49.
(2) (a) Callan, J. F.; de Silva, A. P.; Magri, D. C. Tetrahedron 2005,
61, 8551–8588. (b) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson,
T.; Huxley, A. J. M.; McCoy, P.; Rademacher, J. T.; Rice, T. E. Chem.
ReV. 1997, 97, 1515–1566.
(3) (a) Sack, J. T.; Shamotienko, O.; Dolly, J. O. J. Gen. Physiol. 2008,
131, 415–420. (b) Dudev, T.; Lim, C. J. Am. Chem. Soc. 2009, 131, 8092–
8101. (c) Frolov, R. V.; Brelim, I. G.; Saptal, S. J. Biol. Chem. 2008, 283,
1518–1524. (d) Schroeder, B. C.; Bjorn, C.; Kubisch, C.; Stein, V.; Jentsch,
T. J. Nature 1998, 396, 687–690. (e) Alper, S. L. Annu. ReV. Physiol. 2002,
64, 899–923. (f) Rosenhouse-Dantsker, A.; Sui, J. L.; Zhao, Q.; Rusinova,
R.; Rodriguez-Menchaca, A. A.; Zhang, Z.; Logothetis, D. E. Nat. Chem.
Biol. 2008, 4, 624–631.
(4) (a) Namkung, W.; Padmawar, P.; Mills, A. D.; Verkman, A. S. J. Am.
Chem. Soc. 2008, 130, 7794–7795. (b) He, H.; Mortellaro, M. A.; Leiner,
M. J. P.; Fratz, R. J.; Tusa, J. K. J. Am. Chem. Soc. 2003, 125, 1468–1469.
(c) Padmawar, P.; Yao, X.; Bloch, O.; Manley, G. T.; Verkman, A. S. Nat.
Methods 2005, 2, 825–827. (d) Magzoub, M.; Padmawar, P.; Dix, J. A.;
Verkman, A. S. J. Phys. Chem. B 2006, 110, 21216–21221.
10.1021/ol902836c  2010 American Chemical Society
Published on Web 02/11/2010

Figure 1. Structures and key disconnections of K+ sensor 1 and
ionophore bromide precursor 2.
clization representing a conformationally unrestricted oxi-
dative C-N bond-forming reaction, the first example of
employing a secondary amine in a Sandmeyer-like reaction,
a metal-free aminoarylation, and a one-step organotitanium-
mediated ionophore-chromophore forming reaction. Each of
these transformations not only is applied to the synthesis of
1 but also has been extended to demonstrate additional
examples of their scope and utility. The efficient synthesis
route developed here should enable the development and
application of K+ sensors for important biomedical applica-
tions, such as high-throughput screening/drug discovery,5a
in ViVo K+ imaging,5b artificial receptors,5c fiberoptic K+
sensing,5d and organellar K+ measurement.
Figure 2. (a) Conditions shown in entry 14 used QuinaPhos and
microwave irradiation (20 min) to afford 3 (93%). (b) Utility of
this aminoarylation reaction is demonstrated in the syntheses of
4-6. (c) Finkelstein alkylation of 3 to afford 7 (87%). Footnotes:
aDetermined by LCMS. The conversion % value in entry 14 also
represents the isolated yield. bConventionally heated reactions
occurred in an oil bath at reflux for 18 h. Microwave reactions
Our step-economical synthesis focused on simplifying and
optimizing the K+ binding motif as well as improving the
ionophore-chromophore union. Previous ionophores have
shown >30-fold selectivity for K+ over biological ions such
as Ca2+, Mg2+, and to a lesser extent, Na+.4 However, these
sensors lack selectivity for the larger ions Cs+ and Rb+. We
postulated that improved K+ binding affinity and selectivity
could be achieved by decreasing the size of the ionophore
cage by four atoms and using Lewis basic bisimidazoles in
lieu of tolyl moieties. Additionally, we envisioned that
replacing the methoxyethoxyphenyl motif with pyrimidine
would likely maintain or improve affinity and that ionophore
bromide 2 as well as subsequent ionophore-chromophore
conjugates would be more synthetically accessible than
sensors derived from an ionophore aldehyde, the precursor
used in prior syntheses.4
c
were heated to 95 °C for 20 min. Conditions used from ref 6a,
e
f
dref 6b, ref 6c. Desired T could not be achieved.
in high yield without requiring protection of the imidazole
NH. Entry 14 highlights the conditions found that feature
2-chloroimidizole as the aryl halide, QuinaPhos as a new
ligand, and microwave irradiation as a heat source to afford
3 in 93% yield. Additionally, these aminoarylation conditions
afforded tertiary arylamines 4-6 in excellent yields in 20
min using a variety of aryl chlorides with acyclic and cyclic
secondary amines. This aminoarylation was then followed
by a Finkelstein-mediated monoalkylation of diimidazole 3
with tert-butyl bis(2-chloroethyl)carbamate to provide alkyl
chloride 7 (87%) as shown in Figure 2c.
With an efficient two-step route to 7, effort was then
focused on the synthesis of ionophore bromide 2 as shown
in Figure 3. Unfortunately, various intramolecular N-alky-
lation attempts produced intermolecular or elimination
products as shown in Chart 1 of Supporting Information.
Inspired by recent dianionic oxidations on conformationally
constrained systems,7 we envisioned that deprotonating the
N-H of 7 coupled with a metal-halogen exchange would
The function-oriented synthesis of 1 began with attempts
toward a transition-metal-mediated bisaminoarylation as
shown in Figure 2a. Unfortunately, established Cu(I)6a and
Pd(0)6b,c methods were unsuccessful in our hands, as depicted
in entries 2, 10, and 11. The halogen of the aryl halide, Pd(0)
source and ligand, base, solvent, and heating element were
all found to be critical components to obtain diimidazole 3
(5) (a) Dittrich, P. S.; Man, A. Nat. ReV. Drug DiscoVery 2006, 5, 210–
218. (b) Trachtenberg, J. T.; Chen, B. E.; Knott, G. W.; Feng, G.; Sanes,
J. R.; Welker, E.; Svoboda, K. Nature 2002, 420, 788–794. (c) Xia, W. S.;
Schmehl, R. H.; Li, C. J. Am. Chem. Soc. 1999, 121, 2319–2320. (d) Xia,
W. S.; Schmehl, R. H.; Li, C. J. Am. Chem. Soc. 1999, 121, 2319–2320.
(6) (a) Job, G. E.; Buchwald, S. L. Org. Lett. 2002, 4, 3703–3706. (b)
Shen, Q.; Ogata, T.; Hartwig, J. F. J. Am. Chem. Soc. 2008, 130, 6586–
6596. (c) Fors, B. P.; Watson, D. A.; Biscoe, M. R.; Buchwald, S. L. J. Am.
Chem. Soc. 2008, 130, 13552–13554.
(7) (a) Bisai, A.; West, S. P.; Sarpong, R. J. Am. Chem. Soc. 2008,
130, 7222–7223. (b) West, S. P.; Bisai, A.; Lim, A. D.; Narayan, R. R.;
Sarpong, R. J. Am. Chem. Soc. 2009, 131, 11187–11194.
Org. Lett., Vol. 12, No. 6, 2010
1161

oxidative C-N bond formation from a conformationally
unrestricted system.
To investigate the scope of this reaction, a small collection
of cyclic secondary amines was synthesized as shown in
Figure 3b. These conditions afforded 6- to 15-membered
cyclic alkoxyamines 9-11 and 13 as well as alkoxyhydrazine
12 in very high yields. Interestingly, the anion source could
be generated from either a lithium-halogen exchange or a
deprotonation of an o-tolyl C-H or an aniline N-H.
Attempts to prepare a cyclic secondary amine from Boc-
protected 12-chlorododecylamine were unsuccessful, sug-
gesting that an in-chain “RCH2K”-chelating motif is an
essential component.
Returning to the synthesis of ionophore 2, Boc-protected
amine 8 was treated with TFA followed by precipitation
with aqueous sodium carbonate to quantitatively afford
free amine 14. A solution containing diazonium salt 15
(prepared from the oxidation of 5-bromo-2-aminopyrimi-
dine) was then cannulated into a THF solution of amine
14 with catalytic CuOAc and warmed to reflux temperature
to afford 2 (78%). When catalytic CuOAc is not used,8
the ionophore bromide 2 is obtained in 86% yield. These
represent, to our knowledge, the first examples of a
secondary amine participating in a Sandmeyer-like or a
metal-free aminoarylation from aryldiazonium salts.9 To
investigate the scope of this aminoarylation reaction with
and without catalytic copper(I), amines 16-22 were
prepared in 58-89% yield (Figure 3d). Interestingly,
electron-deficient heteroaryl diazonium salts afforded
higher % yield than electron-efficient systems.
Having devised an efficient route to ionophore bromide 2
(see Figure 1), our attention turned toward the ionophore-
chromophore union as shown in Figure 4a. In prior reports,
this union required several steps to afford a K+ sensor
because of the need to construct the chromophore around
the ionophore aldehyde carbon.4 In order to accomplish this
union in one transformation, we envisioned an organometallic
addition to an activated xanthylium such as the vinylogous
sulfonate 23. Indeed, treatment of 2 with t-BuLi, followed
by ClTi(Oi-Pr)3, delivered the corresponding organotitanium
reagent. Quenching this anion with a chilled solution of
xanthylium triflate 23 (prepared from the corresponding
xanthone)10 gave 1 in 82% yield. Since there are no
examples, to our knowledge, of organotitanium additions to
vinylogous systems in the literature, the general utility of
this transformation was further demonstrated as shown in
Figure 3. (a) Conditions shown in entry 10 used Li sticks/KOt-Bu
and (PhSe)2 to afford 8 in an isolated yield of 91% yield. (b) Utility
of this oxidative cyclization is demonstrated in the syntheses of
cyclic secondary amines 9-13 (P ) Boc). (c) Synthesis of
ionophore 2 featuring an oxidative C-N bond-forming cyclization
and either a Sandmeyer-like reaction (+CuOAc) or a metal-free
aminoarylation (-CuOAc) with a secondary amine. (d) Utility of
these aminoarylation conditions used in Figure 2c is demonstrated
a
in the synthesis of tertiary arylamines 16-22. Footnotes: Deter-
mined by LCMS. Isolated yield shown in parentheses. bMajor
product was a dimer. cStarting material recovered. dMajor product
was an alkene. eMajor product was an alkane. fFirst/second values
rerpresent the % yield obtained with/without CuOAc (cat.).
afford a dimetalated intermediate that, in the presence of
several chelating neighboring groups, would bring both
dimetalated moieties in close proximity, thereby enabling a
template-directed oxidative macrocyclization. As depicted
in Figure 3a, several metals and oxidants were assessed;
however, many of these were unsuccessful because of
decomposition of the starting material, protonation of the
chloride-derived anion, and/or dimerization. Dilithiation with
Li sticks, followed by trans-metalation with KOt-Bu presum-
ably generated an organodipotassium intermediate that was
subsequently oxidized with diphenyl diselenide to afford 8
(91%) containing the complete macrocyclic backbone of 2.
We believe that this template-directed macrocyclization,
which presumably occurs via polar or SET mechanisms in
accord with Sarpong’s findings,7b is a new example of an
(8) CuOAc was chosen over CuCN, as the undesired benzonitrile was
observed as a minor product and poisonous HCN is a likely byproduct.
When stoichiometric CuCN was used, the benzonitrile was obtained in 60%
yield. Beletskaya’s conditions afforded the benzonitrile in 89% yield:
Beletskaya, I. P.; Sigeev, A. S.; Peregudov, A. S.; Petrovskii, P. V. J.
Organomet. Chem. 2004, 689, 3810–3812.
(9) The Sandmeyer reaction implies a radical process involving Cu(I)
or other metals. Presumably, this happens with this Cu(I) version. However,
metal-free aminoarylations occur with aryldiazonium salts using thiols,
water, or iodide. It is not known, to our knowledge, whether these metal-
free versions occur via a radical, ipso substitution, or dissociative mecha-
nism. For metal-free versions, see: Filimonov, V. D.; Trusova, M.;
Postnikov, P.; Krasnokutskaya, E. A.; Lee, Y. M.; Hwang, H. Y.; Kim, H.;
Chi, K.-W. Org. Lett. 2008, 10, 3961–3964.
(10) Kenmoku, S.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem.
Soc. 2007, 129, 7313–7318.
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Org. Lett., Vol. 12, No. 6, 2010

Figure 4. (a) One-step synthesis of sensor 1 from ionophore
bromide 2 via an organotitanium intermediate to vinylogous
sulfonate 23. (b) Cyclohexenones 24-27 were prepared with an
average yield of 81% with complete chemo- and regioselectivity,
thereby demonstrating the utility of this organotitanium addition
to a model vinylogous sulfonate.
Figure 4b. Organotitanium reagents (1.0 equiv) derived from
aryl or alkyl bromides were added to a model vinylgous
sulfonate and afforded 3-substituted cyclohexenones 24-27
in an average yield of 81% with expected control of chemo-
and regioselectivity.
The K+ sensitivity and specificity of sensor 1 were
compared to reference sensor TAC-Red4c as shown in Figure
5. TAC-Red and 1 (each at 7 µM) were dissolved in pH 7
HEPES buffer balanced with KCl/NaCl to maintain constant
ionic strength at 200 mM. The structurally simplified 1 was
slightly more sensitive to K+ than TAC-Red as shown in
Figure 5a. Figure 5b shows that 1 was remarkably more
selective for K+ versus Cs+ or Rb+ than TAC-Red and
had comparable low sensitivity to Na+, Li+, Mg2+, and
Ca2+ (the latter three ions not shown in Figure 5b). Finally,
1 had comparable pH insensitivity to TAC-Red at pH
greater than 6 as shown in Figure 5c. These results are
likely attributed to 1 having a smaller ionophore cage that
excludes the larger cations like Cs+ and Rb+ yet brings
Lewis basic nitrogen and oxygen atoms in a favorable
position for K+ binding.
Figure 5. (a) Fluorescence emission at 570 nm of 1 (blue) and
TAC-Red4c (red) at various [K+]. (b) Fluorescence at 570 nm of 1
(blue line) and TAC-Red (red line) in the presence of indicated
cations. (c) Fluorescence of 1 as a function of pH at indicated [K+].
Samples in a-c were excited at 480 nm, and solutions were
balanced with NaCl or choline chloride to maintain a constant ionic
strength of 200 mM.
from sensor 1 to measure [K+] in major molecular, cellular,
translational, and clinical applications will be reported in due
course.
Acknowledgment. This research is dedicated in remem-
brance of Prof. Aaron D. Mills (University of Idaho). Helpful
discussions with Profs. Mark J. Kurth and Dean J. Tantillo
(University of California, Davis) are gratefully acknow-
eldged. This work was supported by NIH grants EB00415,
DK86125, DK35124, HL73856, EY13574, and DK72517
and grants from the Cystic Fibrosis Foundation and Guthy-
Jackson Charitable Foundation.
In summary, the streamlined synthesis reported here, which
represents over a 2-fold decrease in steps compared to TAC-
Red, affords the functionally superior yet structurally simpli-
fied K+ sensor 1 in 52% overall yield with the longest linear
sequence being 4-5 steps. Additionally, this report highlights
new ligand and microwave conditions for aminoarylations,
a C-N bond-forming oxidative cyclization reaction to
generate cyclic secondary alkoxyamines, the first example
of secondary amines in Sandmeyer-like reactions, new
examples of metal-free aminoarylations, and an efficient route
to chromophore conjugates via an organotitanium addition
to vinylogous sulfonates. Creating bioconjugates derived
Supporting Information Available: Experimental pro-
cedures and analytical data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL902836C
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