Improved synthesis of the triazacryptand (TAC) and its application in the construction of a fluorescent TAC-BODIPY conjugate for K+ sensing in live cells

Article abstract of DOI:10.1002/ejoc.201403600

In this report, a vastly improved method for the synthesis of [2.2.3]-triazacryptand (TAC), an excellent K⁺-selective cryptand, is disclosed, which provides a shorter synthetic route with > fivefold higher overall yield. On the basis of the imp

Full text of DOI:10.1002/ejoc.201403600

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SHORT COMMUNICATION
DOI: 10.1002/ejoc.201403600
Improved Synthesis of the Triazacryptand (TAC) and its Application in the
Construction of a Fluorescent TAC-BODIPY Conjugate for K+ Sensing in
Live Cells
Binglin Sui,[a] Xiling Yue,[a] Michael G. Tichy,[a] Taihong Liu,[a,b,c] and
Kevin D. Belfield*[a,b,c]
Keywords: Potassium / Sensors / Cryptands / Fluorescence / Cell imaging
In this report, a vastly improved method for the synthesis of
[2.2.3]-triazacryptand (TAC), an excellent K+-selective
cryptand, is disclosed, which provides a shorter synthetic
route with Ͼ fivefold higher overall yield. On the basis of the
oped for K+ sensing. TAC-BODIPY is a fluorescence turn-on
K+ sensor exhibiting high selectivity toward K+ over other
biologically relevant metal cations. Cell imaging studies are
presented and demonstrate that TAC-BODIPY is suitable for
improved synthesis, a TAC-BODIPY conjugate was devel- dynamic K+ sensing in live cells.
As the most abundant intracellular metal cation, a [2.2.3]-triazacryptand (TAC) platform as a K+-selective
potassium plays indispensable roles in a wide range of bio- receptor unit, which exhibits very high selectivity for
logical processes, such as nerve transmission, cardiac excit- detecting K+ over the interfering Na+.[5] Subsequently,
ability, epithelial fluid transport, muscle contraction, cell fluorescent potassium sensors employing TAC as the K+-
proliferation, and cellular ionic homeostasis.[1] On the other recognition moiety have been synthesized and applied to
hand, abnormal potassium levels are early symptoms for biological imaging.[6]
certain diseases, including hypertension, heart disease, sei-
zures, stroke, bulimia, anorexia, diabetes, alcoholism, renal
disease, myasthenia, cancer, and AIDS.[2] Therefore, a deli-
cate balance of potassium is essential for human health.
Due to the biological significance of potassium, continuous
efforts have been devoted to intracellular and extracellular
potassium detection. Relative to other techniques, fluores-
cence approaches have attracted more and more interest be-
cause of their distinct advantages including non-invas-
iveness, high sensitivity, and convenience. Among various
proposed fluorescent potassium sensors, PBFI (potassium-
binding benzofuran isophthalate), which incorporates a di-
aza-18-crown-6 ether as the recognition unit and a benzo-
furan derivative as the fluorophore, is the most popular and
currently the only one commercially available.[3] However,
PBFI suffers insufficient potassium binding strength and
poor selectivity for K+ over Na+.[4] To overcome the
problem of poor K+/Na+ selectivity, He et al. developed
However, the synthesis of TAC involves multistep reac-
tions, which indirectly limits its wide application. Herein,
we describe an optimized synthesis of TAC with a shorter
synthetic route and a higher overall yield (Scheme 1) and
its application in the construction of TAC-BODIPY, a
BODIPY (4,4-difluoro-4-bora-3a,4a-diza-s-indacene) based
fluorescent K+-selective sensor.
The synthetic precursor compound 3 was obtained from
compounds 1 and 2 (see Supporting Information). In the
literature, this reaction was performed in refluxing MeCN
with KI as catalyst and K2CO3 as base. However, after re-
fluxing for 16 h, only a monosubstituted aniline derivative
was produced as the major product and the desired bis-
substituted aniline product was formed in trace amount.
Repeated addition of 1 every 20 h (three times) resulted in
a modest yield (ca. 50%) of compound 3. DMF was also
employed as the reaction medium rather than MeCN, and
the reaction temperature was raised to 120 °C, but similar
results were obtained. Finally, a totally different reaction
system was adopted to perform this reaction, by using a
mixture of 1,4-dioxane and water (2:3) as the reaction me-
dium and CaCO3 as the base (reaction details in Supporting
Information). As a result, 3 was obtained in significantly
higher yield (91%).
[a] Department of Chemistry, University of Central Florida,
4111 Libra Drive, Orlando, FL 32816, USA
E-mail: belfield@njit.edu
[b] College of Science and Liberal Arts, New Jersey Institute of
Technology,
323 Dr. Martin Luther King Jr. Blvd., Newark, NJ 07102, USA
http://csla.njit.edu/people/belfield.php
In the next step, a hydrogenation reaction was used as in
literature methods for the reduction of the nitro groups of
compound 3 to amino groups in a DMF solution of 3 at
2.2 atm, with Pd/C (palladium on activated carbon) as cata-
[c] School of Chemistry and Chemical Engineering, Shaanxi
Normal University,
Xi’an 710062, China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201403600.
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B. Sui, X. Yue, M. G. Tichy, T. Liu, K. D. Belfield
SHORT COMMUNICATION
Scheme 1. Synthetic route of TAC-BODIPY.
lyst. In this way, amine
yield. To avoid the use of hydrogen gas, hydrazine mono- eliminates the synthesis of compounds 6 and 7.
hydrate was employed as the reducing reagent. A compar- Enlightened by the results obtained in the synthesis of 3,
4 was prepared in 97% 4 and TAC directly from 5, as displayed in Scheme 1, which
able yield (97%) was obtained by this method relative to in which the MeCN/K2CO3 system worked well in prepar-
that under Pd/C catalytic conditions. Furthermore, a more ing mono-substituted anilines but poorly in producing bis-
economic catalyst FeCl3/C (a mixture of FeCl3 and acti- substituted anilines, we employed those conditions to pre-
vated carbon) also provided compound 4 in a similarly high pare macrocycle 5. Different conditions were evaluated to
yield (96%).
achieve the optimized reaction conditions, which included
The following two 21-membered macrocyclization reac- reactants, bases, and solvents. As shown in Table S1, the A/
tions are the two key steps for the synthesis of TAC. In the K2CO3/MeCN system (entry 1) turned out to be the most
literature methods, both macrocycles were achieved by an efficient one among the systems examined. Macrocyle 5 was
amide synthesis (steps Sd and Sf in Scheme S1). Two amide generated by reaction of 4 and 1,2-bis(2-iodoethoxy)-
groups were synchronously formed through reactions be- ethane with K2CO3 as base in MeCN under reflux. The
tween the amines (compound 4 for step Sd and compound yield (40%) of this reaction was moderate but comparable
5 for step Sf) and 3,6-dioxaoctanedioic acid dichloride to the overall yield (42%) of the literature two-step meth-
(DODC), which was obtained from reaction of 3,6-dioxa- ods. Similarly, TAC was produced in 69% yield, much
octanedioic acid with oxalyl chloride. Following each of higher than the overall yield (32%) of the literature two-
these two steps, a reduction reaction takes place, which re- step method by refluxing a mixture of 5, 1,2-bis(2-iodo-
duces the newly formed amides (compounds 6 and 7 in ethoxy)ethane, and CaCO3 in 1,4-dioxane/water (2:1).
Scheme S1) to amines (compound 5 and TAC), respectively.
TAC-CHO was obtained by following a literature pro-
Here we attempted to synthesize macrocycle 5 directly from cedure.[5] Subsequently, a TAC-BODIPY conjugate was
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Fluorescent TAC-BODIPY Conjugate for K+ Sensing in Live Cells
synthesized for K+ sensing through a trifluoroacetic acid
catalyzed condensation reaction of TAC-CHO with 2,4-di-
methylpyrrole. The fluorescence response of TAC-BODIPY
to K+ ions was investigated in 5 mm HEPES buffer (pH
7.2)/MeCN (4:1, v/v). Figure 1 shows the fluorescence emis-
sion spectra of TAC-BODIPY in the presence of K+ and
other biologically relevant metal cations at their physiologi-
cal concentrations. As expected, TAC-BODIPY exhibits
very high selectivity for K+ over the other metal cations. In
the absence of cations, free TAC-BODIPY emits a very
weak fluorescence (quantum yield: 0.03) at 512 nm. Upon
addition of K+ ions, the fluorescence intensity
dramatically increases while the addition of Na+ and other
biologically important metal cations induces no obvious
change to the fluorescence spectrum of TAC-BODIPY.
Figure 2. Enhancements in fluorescence emission spectra of TAC-
BODIPY (10 μm) in 5 mm HEPES buffer (pH 7.2)/MeCN (4:1,
v/v) upon continuous addition of 150 mm K+ ions. Ionic strength
was maintained at 150 mm by addition of NaCl.
Encouraged by these results, TAC-BODIPY was used to
detect changes of intracellular potassium levels. Figure 3
shows images of cells taken at different times (0, 30, 60, 90,
120 min), and the average fluorescence emission intensity
of each image is shown in Figure S2. U87MG cells were
incubated with a 5 μm solution of TAC-BODIPY in com-
plete MEM medium for 10 min. As expected, the cells were
highly fluorescent as shown in Figure 3A and F, which is
consistent with the forgoing observed fact that the intra-
cellular K+ level is high enough to trigger strong fluores-
cence of TAC-BODIPY. To examine the ability of TAC-
BODIPY to respond to changes in K+ levels in living cells,
a mixture of nigericin (5 μm), ouabain octahydrate (10 μm),
and bumetanide (10 μm) in complete MEM medium was
pumped into the cell chambers and images of the living cells
were taken at different times. It has been shown that the
combination of nigericin, ouabain octahydrate, and bumet-
anide can effectively bring about K+ efflux of cells.[6e,8]
With the depletion of K+ induced by nigericin, ouabain
octahydrate, and bumetanide, the fluorescence intensity of
the cells gradually decreases (from Figure 3F–J). For the
control experiment, after the same time, the cells still
appeared highly fluorescent (Figure 3B–E), which exhibit
little visible change relative to their original state (Fig-
ure 3A). These results indicate that TAC-BODIPY is a
Figure 1. Fluorescence emission spectra of TAC-BODIPY (10 μm)
in 5 mm HEPES buffer (pH 7.2)/MeCN (4:1, v/v) upon addition
of various biologically relevant metal cations at their physiological
concentrations: Na+ (150 mm), K+ (150 mm), Ca2+ (0.5 mm), Mg2+
(2.5 mm), Fe3+ (18 mm), Zn2+ (45 mm), Mn2+ (0.9 mm), Cu2+
(16 mm).
To further examine the efficacy of TAC-BODIPY
towards sensing K+ ions, fluorescence emission titration ex-
periments were carried out in 5 mm HEPES buffer (pH 7.2)/
MeCN (4:1, v/v). Solutions were balanced with NaCl to
maintain a constant ionic strength of 150 mm. As illustrated
in Figure 2, a gradual enhancement of the fluorescence in-
tensity of TAC-BODIPY was observed upon progressive
addition of K+ ions. The dissociation constant (Kd) of the
sensor was determined to be 65 mm (see Supporting Infor-
mation), according to a literature method.[6e] The linear
fitting of the plot (Figure S1) evidenced a 1:1 binding ratio
between TAC-BODIPY and K+.[6e] The fluorescence inten-
sity increased 14-fold in the presence of 150 mm K+ (quan-
tum yield: 0.43). Higher concentrations of K+ ions were not
able to further increase the fluorescence intensity of TAC-
BODIPY. Moreover, TAC-BODIPY was capable of de-
tecting 1 mm K+ with the co-existence of a high concentra-
tion of Na+. The detection limit of TAC-BODIPY for K+
was determined to be 1.5ϫ10–6 m. Since the extra-
cellular and intracellular concentrations of potassium are
of the order of 5 mm and 150 mm,[7] respectively, the TAC-
BODIPY probe is potentially suitable for determining
physiological K+ levels.
Figure 3. Images of live U87MG cells in MEM medium (A–E),
and in an MEM medium solution of nigericin (5 μm), ouabain oc-
tahydrate (10 μm), and bumetanide (10 μm) (F–J), at 0 min (A, F),
30 min (B, G), 60 min (C, H), 90 min (D, I), and 120 min (E, J)
after incubation with TAC-BODIPY (5 μm).
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B. Sui, X. Yue, M. G. Tichy, T. Liu, K. D. Belfield
SHORT COMMUNICATION
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In summary, the synthesis of TAC was improved signifi-
cantly. The synthetic route was shortened and the overall
yield was increased fivefold from 4% to nearly 20%. A
fluorescent K+-selective sensor, TAC-BODIPY, was then
synthesized and characterized. TAC-BODIPY exhibited ex-
cellent selectivity toward K+ over other biologically
important metal cations and high sensitivity for K+ sensing.
Fluorescence microscopy experiments demonstrated that
the TAC-BODIPY probe is capable of sensing K+ in living
cells.
Supporting Information (see footnote on the first page of this arti-
cle): Information on the procedures for the improved synthesis of
compounds 1, 3–5, and TAC, synthesis of TAC-BODIPY, cell cul-
ture for imaging, fluorescence intensity of cell images, and copies
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Acknowledgments
We wish to acknowledge support from the National Science Foun-
dation (CBET-1517273 and CHE-0832622) and the U. S. National
Academy of Sciences (PGA-P210877).
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Received: December 9, 2014
Published Online: ᭿
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Fluorescent TAC-BODIPY Conjugate for K+ Sensing in Live Cells
Fluorescent Sensors
The dye, TAC-BODIPY, emits very weak
fluorescence at 512 nm. Upon binding with
K+, it becomes highly fluorescent, emitting
very strong fluorescence. TAC-BODIPY
exhibits excellent selectivity toward K+
over Na+ and desirable sensitivity for K+
sensing. This fluorescent K+ sensor is
capable of efficiently sensing the K+ levels
in living cells through cell imaging with
fluorescence microscopy.
B. Sui, X. Yue, M. G. Tichy, T. Liu,
K. D. Belfield* .................................. 1–5
Improved Synthesis of the Triazacryptand
(TAC) and its Application in the Construc-
tion of a Fluorescent TAC-BODIPY Con-
jugate for K+ Sensing in Live Cells
Keywords: Potassium / Sensors / Cryp-
tands / Fluorescence / Cell imaging
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