Fluorescent bis-cyclen tweezer receptors for inositol (1,4,5)-trisphosphate

Article abstract of DOI:10.1016/j.tet.2011.03.092

Herein, we report the development of two fluorescent sensors for inositol 1,4,5-trisphosphate (InsP₃), containing either two free cyclen or zinc(II)/cyclen groups preorganized into a binding cleft. Preorganization was achieved using a rigid acr

Full text of DOI:10.1016/j.tet.2011.03.092

Tetrahedron 67 (2011) 3803e3808
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Tetrahedron
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Fluorescent bis-cyclen tweezer receptors for inositol (1,4,5)-trisphosphate
*
Chi-Linh Do-Thanh, Meng M. Rowland, Michael D. Best
Department of Chemistry, The University of Tennessee, 1420 Circle Drive, Knoxville, TN 37996, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report the development of two fluorescent sensors for inositol 1,4,5-trisphosphate (InsP3),
containing either two free cyclen or zinc(II)/cyclen groups preorganized into a binding cleft. Pre-
organization was achieved using a rigid acridine backbone, which was also exploited for fluorescence
signal transduction. In addition, click chemistry was used to facilitate receptor synthesis and produce
triazole moieties that add to structural rigidity and recognition. In binding studies, both receptors were
Received 1 February 2011
Received in revised form 24 March 2011
Accepted 28 March 2011
Available online 2 April 2011
found to bind InsP3 with high affinity (Ka¼0.5e1.0ꢀ106
M
ꢁ1) in competitive media (1:1 methanol/water).
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
the quintessential biologically active inositol phosphate19e21 due to
its prominent role as a second messenger in calcium release, origi-
nally reported in 1983.22 InsP3 is produced through the hydrolysis of
phosphatidylinositol (4,5)-bisphosphate by phospholipase C.23e25
This soluble product then binds to its cognate receptors, the inosi-
tol trisphosphate receptors (InsP3Rs),26e29 which leads to the
opening of these gated ion channels, directly resulting in calcium
release by gradient diffusion into the cytoplasm. InsP3-mediated
calcium release is critical due to the numerous physiological
processes that are mediated by this ion, which includes cell pro-
liferation, differentiation, secretion, muscular contraction, embry-
onic development, immune responses, and many others.26,29
Due to the significance of InsP3, this compound has been a target
for sensor design. Anslyn and co-workers initially reported a hexa-
guanidinium receptor with a binding cavity engineered via the
1,3,5-triethylbenzene scaffold.30 To achieve optical detection, a dye
displacement assay was implemented, and strong binding
(Ka¼2.2ꢀ104 Mꢁ1) was observed in aqueous solution. More re-
cently, Kimura and co-workers took non-fluorescent receptors
containing zinc/cyclen chelating groups and incorporated them
into a ruthenium tris-pyridyl system to achieve fluorescence sig-
naling.31 In addition, Ahn and co-workers developed a tris zinc/
dipicolylamine receptor, and again employed a dye displacement to
achieve detection.32 Matile and co-workers used a synthetic mul-
tifunctional pore based on rigid p-octiphenyl rods organized into
The development of molecular sensors that are effective for
detecting important biomolecular targets in competitive media
remains a significant challenge in the field of supramolecular
chemistry.1,2 This process involves the design of a macromolecular
host containing binding groups that are preorganized to instill
geometric complementarity with a target guest and thus increase
the affinity and selectivity of binding. In addition, a signal trans-
duction element is installed for detection purposes, of which
fluorescence is advantageous due to its high sensitivity.3e5 Anion
sensing is critical as biological systems are highly reliant upon the
manipulation of anionic functional groups as a regulatory means,
particularly through the control of biomolecule phosphorylation.
As a result, sensors for phosphorylated molecules are particularly of
interest.6,7
Within the field of supramolecular chemistry, anion recognition
has proven problematic due to inherent challenges including the
large and varying shapes of anions, their multiple pH-dependent
charge states, and the difficulty in synthetically incorporating an-
ion-chelating moieties into receptors.5,8e10 Despite these chal-
lenges, a number of receptor motifs have been developed for anion
recognition, including polyammonium macrocycles,11e13 porphy-
rins,14 the triethylbenzene system,15 dimetallic hosts,16 calixar-
enes,17 and steroidal constructs.18 However, the generation of
sensors capable of detecting anionic targets for real world appli-
cations remains a significant barrier due to the difficulty of
achieving strong binding in competitive media.
a b-barrel for fluorimetric detection of InsP3 binding transduced by
pore opening and closing.33 Approaches other than molecular re-
ceptors have been successful for InsP3 detection, particularly
through the use of labeled or engineered protein receptors.34e36
Finally, receptors for phytate (InsP6), another inositol phosphate
isomer, have been reported as well.37,38
An important target for molecular sensing is the signaling
molecule inositol (1,4,5)-trisphosphate (InsP3, 2, Scheme 1). This is
While synthetic sensors capable of coordinating InsP3 have been
successfully designed, we set out to design a receptor with specific
attributes. First, we targeted a system with a built-in fluorescent
* Corresponding author. Fax: þ1 865 974 9332; e-mail address: mdbest@utk.edu
(M.D. Best).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.03.092

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C.-L. Do-Thanh et al. / Tetrahedron 67 (2011) 3803e3808
Polyammonium
binding groups
Linker
N
H
N
O
O
H
Cl
Cl
NH
H2N
NH
NH
N
OPO3-2
OH
H2N
N
-2 O3PO
HO
N
H
H
N
N
H
N
N
N
H
N
N
-2
-2
O3PO
OPO3
-2 O3PO
OH
OH
HO
N
N
2
-2
OH
O3PO
NH
N
N
N
H
N
H
H
N
H
N
Cl
N
N
Cl
N
H2N
N
H2N
H
N
H
N
O
O
i
Acrid ne:
Rigid scaffold /
Fluorescent signaling
1
3
Scheme 1. Design of receptor 1 and hypothetical binding complex with target guest InsP3 (2).
signaling group for direct detection of binding.39 In addition, we
sought to implement a rigid scaffold to preorganize binding groups
in the creation of a binding cavity. Finally, we looked to exploit the
strong affinities of polyammoniums and their zinc complexes for
anions40e44 including phosphate to create a receptor containing
two of these groups preorganized into a tweezer structure.
was obtained from acridine (10) by bis-bromomethylation to 11,63
followed by azide substitution. Here, the acridine unit was also
beneficial as a result of the convenient derivatization of this pre-
cursor at the desired 4- and 5-positions. For binding studies, InsP3
(2) was synthesized as described in the Supplementary data.
Following the completion of this synthetic route, receptor 1 was
employed for titration binding studies to evaluate the binding of
InsP3 (2) with concomitant fluorescence signal transduction. First,
UV scans of the host were performed, with each exhibiting an ab-
sorption peak centered around 355 nm, with excitation at this
wavelength leading to an emission signal located around 440 nm.
Next, 2 was titrated into host 1 in such a way as to keep the total
host concentration constant. Here, dose-dependent decreases in
the emission signal of host 1 were observed upon guest addition.
This was performed in a range of solvents varying from 0 to 100%
methanol in water buffered at pH 7.4. From these studies, a solvent
mixture of 50% methanol/water was identified as a competitive
solvent in which strong binding was observed. A representative
plot indicating the decrease in emission properties of host 1 upon
addition of 2 in this solvent mixture is shown in Fig. 1. While the
changes in emission properties were somewhat modest, dropping
by only 10e15%, these signals could nevertheless be reproducibly
correlated to a binding curve for 2, as shown in Fig. 2. Here, the
change in fluorescence intensities upon addition of 2 are shown as
averaged values over several runs, with error bars included
depicting the standard error. From these results, an association
constant of 1.0ꢀ106 Mꢁ1 (Table 1) was calculated for the complex,
indicating high-affinity binding in competitive media.
2. Results and discussion
To develop a fluorescent sensor for InsP3, we initially designed
host 1 (Scheme 1), which includes a number of features. First, two
polyammonium groups were installed to take advantage of the
strong anion-binding properties of these moieties. Ditopic poly-
ammonium ligands have previously been investigated, with re-
ceptor designs consisting of polyammonium moieties separated by
bridging linkers.11,12,45e57 However, these scaffolds have often been
designed around relatively flexible scaffolds, which may be too
floppy to maximize preorganization. In order to preorganize these
two groups into an effective binding cavity in our receptor, an
acridine backbone substituted at the 4- and 5-positions was in-
troduced as a rigid unit for steric gearing. The acridine was also
beneficial due to its photophysical properties so as to achieve direct
fluorescence signal transduction of guest binding.
In the host design, triazole-based linkers were incorporated
between the acridine scaffold and the polyammonium binding
groups, which were also envisaged to be advantageous for multiple
reasons. First, this would allow for a convergent receptor synthesis
in which these groups could be conveniently merged using click
chemistry.58,59 In addition, the resulting triazole moieties were
expected to be beneficial by introducing rigidity to the tether, fur-
ther assisting in the preorganization of binding groups. Finally,
triazoles have also been shown to be effective for forming hydrogen
bonding interactions with anionic guests,60 and thus could con-
tribute to the binding of phosphorylated analytes. In this way, re-
ceptor 1 was designed to complement multiply phosphorylated
biomolecules, such as InsP3, by forming complexes exemplified by
hypothetical model 3 in Scheme 1.
Next, a series of related phosphorylated compounds were
employed for binding studies to further investigate the recogni-
tion properties of host 1. These included D-fructose 1,6-bisphos-
phate trisodium salt (FBP, 12, Fig. 3), cyanoethyl phosphate barium
salt (13), and sodium dihydrogen phosphate (14), which were
selected due to their variation in the number of phosphate and
alkyl groups. The binding curves for these guests are shown
alongside those corresponding to 2 in Fig. 2, and the resulting
association constants are indicated in Table 1. In general, each of
these guests exhibited fairly strong binding, indicating that host 1
is generally effective at complexing a range of phosphorylated
guest structures in competitive solvents. Nevertheless, slight var-
iations in binding affinity were observed in the order of
2>12>13>14, indicating very modest selectivity for InsP3, as well
as for multiply phosphorylated guests (2 and 12) over singly
phosphorylated guests (13 and 14) in the series.
With this receptor design in place, a convergent synthetic route
to receptor 1 was devised, as shown in Scheme 2. Here, the key step
employed click chemistry to link alkynyl-cyclen intermediate 4 and
bis-(azidomethyl)acridine structure 5 to form 6, which was then
deprotected. Ion exchange was next performed to access host 1
with chloride counteranions, and it is estimated that four chlorides
are associated with the cationic host structure. In precursor syn-
thesis, cyclen (7) was used to access 4 via tris-Boc protection to 8,61
followed by coupling with alkyne 9.62 Bis-(azidomethyl)acridine 5
A benefit of employing polyamine moieties for anion binding is
the modular nature of these groups. This is because these receptors

C.-L. Do-Thanh et al. / Tetrahedron 67 (2011) 3803e3808
3805
O
Br
N
Boc
N
Boc
N
H
N
H
Boc2O
Et3N
CHCl3
80%
9
O
K2CO3
HN
NH
BocN
NH
BocN
N
N
H
MeCN
92%
N
H
N
N
Boc
Boc
7
8
4
Br
Br
N3
N3
N
NaN3
MeO
Br
N
N
H2SO4
25%
DMF
87%
10
11
5
Boc
N
O
N
N
N
Boc
N
N
H
N
N
Boc
CuSO4•5H2O
Na ascorbate
1. TFA, CH2Cl2
N
4
5
1
THF, H2O
55%
2. Ion exchange
98%
Boc
N
N
H
N
N
N
N
Boc
N
O
N
Boc
6
Scheme 2. Synthetic route to bis-cyclen tweezer host 1.
600
500
400
300
200
100
0
370
420
470
520
570
620
670
Wavelength (nm)
Fig. 1. Plots exhibiting the decrease in the emission properties of host 1 upon addition
of guest 2.
Fig. 2. Binding curves for guests studied in titrations, including inositol 1,4,5-tris-
phosphate (2), fructose 1,6-bisphosphate (12), cyanoethyl phosphate (13), and sodium
dihydrogen phosphate (14).
effectively bind anions in both their unchelated polyammonium
form, as with receptor 1, and also when chelated to a metal ion, in
which the metal center then acts as the electrophilic binding site.
As a result, we next decided to take advantage of this modularity by
exploring the binding properties of the bis-zinc complex of this
host (1-Zn2, Scheme 3) to compare binding properties. This meta-
lated receptor was synthesized through reaction with zinc acetate.
We next performed titration binding experiments similar to
those previously described, this time using host 1-Zn2 in order to
compare recognition properties. Once again, titration with 2 yiel-
ded a decrease in emission for 1-Zn2, for which a representative
Table 1
Association constants calculated for the binding of host 1 to phosphorylated guests
Guest
Ka (Mꢁ1
)
Inositol (1,4,5)-triphosphate (2)
Fructose 1,6-bisphosphate (12)
Cyanoethyl phosphate (13)
1.0ꢀ106
9.5ꢀ105
8.9ꢀ105
7.8ꢀ105
Sodium dihydrogen phosphate (14)

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C.-L. Do-Thanh et al. / Tetrahedron 67 (2011) 3803e3808
OH
OH
HO
HO
O
O
P
O
P
O
P
O
P
-2
O3PO
O3PO
-2
-2
-2
NC
O3PO
O
O
OH
O
O
OPO3
O
HO
O
-2
O
O
O
OH
O3PO
HO
2
12
13
14
15
Fig. 3. Structures of guests used for binding studies with receptor 1 and 1-Zn2.
H
N
H
N
O
O
Zn2+ NH
N
Zn2+
N
NH
N
N
N
H
N
H
N
N
N
H
N
N
N
H
-2
-2
OPO3
O3PO
2
Zn(OAc)2
OH
OH
HO
1
N
N
CH3OH
100%
-2
O3PO
H
N
H
N
N
N
H
N
H
N
N
N
N
N
Zn2+
N
N
Zn2+
NH
NH
O
O
N
H
N
H
1-Zn2
3-Zn2
Scheme 3. Synthesis of bis-zinc receptor 1-Zn2 and hypothetical binding complex with target guest InsP3 (2).
plot is shown in Fig. 4. While still modest, the emission changes of
1-Zn2 upon addition of 2 (20e25%) were greater than those of re-
ceptor 1. Once again, receptor 1-Zn2 exhibited strong binding of 2,
with an association constant of 5.1ꢀ105 Mꢁ1 (Table 2). Despite the
enhanced fluctuation in emission properties upon guest addition,
this binding affinity was slightly weaker than that of receptor 1. It is
possible that host 1 exhibits slightly stronger binding to multiply
phosphorylated guests because the positive charge is spread onto
multiple ammonium groups, rather than being localized on the two
zinc atoms of 1-Zn2, which may better complement the charge
distribution in these guests.
Table 2
Association constants calculated for the binding of host 1-Zn2 to phosphorylated
guests
Guest
Ka (Mꢁ1
)
Inositol (1,4,5)-trisphosphate (2)
Fructose 1,6-bisphosphate (12)
Cyanoethyl phosphate (13)
Sodium dihydrogen phosphate (14)
Sodium pyrophosphate (15)
5.1ꢀ105
4.9ꢀ105
9.4ꢀ105
4.3ꢀ105
4.7ꢀ105
association constants are indicated in Table 2. Once again, each
guest exhibited strong binding, and only slight variations in affinity
were observed. In this case, cyanoethyl phosphate surprisingly
yielded the strongest binding, and an ordering of 13>2>12>15>14
was observed. However, in these studies multiply phosphorylated
guests (2, 12, and 15) all produced higher changes in fluorescence
than those analytes containing a single phosphate group (13 and
14). This provided evidence for variation in binding activity
Fig. 4. Plots exhibiting the decrease in the emission properties of host 1-Zn2 upon
addition of guest 2.
Next, the same series of phosphorylated guests were analyzed
for binding with 1-Zn2, along with the addition of sodium pyro-
phosphate (15). The binding curves for these guests are shown
alongside those corresponding to 2 in Fig. 5, and the resulting
Fig. 5. Binding curves for guests studied in titrations, including inositol 1,4,5-tris-
phosphate (2), fructose 1,6-bisphosphate (12), cyanoethyl phosphate (13), sodium
dihydrogen phosphate (14), and sodium pyrophosphate (15).

C.-L. Do-Thanh et al. / Tetrahedron 67 (2011) 3803e3808
3807
resulting from the presence of more than one phosphate in the
guest structure.
4.1.2. 2-Bromo-N-(propargyl)acetamide
(9). Propargylamine
(0.36 mL, 5.61 mmol) and triethylamine (0.78 mL, 5.61 mmol) were
added to a solution of bromoacetyl bromide (0.50 mL, 5.61 mmol) in
dichloromethane (25 mL) at 0 ꢂC. After stirring at 0 ꢂC for 1 h, the re-
action mixture was concentrated through rotary evaporation, resus-
pended in ethyl acetate (50 mL), and then filtered through a pad of
silica gel. The filtrate was then concentrated to provide the product as
a dark orange solid (0.96 g, 98%). Characterizations matched previous
reports.62
3. Conclusions
This article describes the development of fluorescent sensors for
InsP3 based upon bis-cyclen tweezer structures either containing
free polyammonium or zinc chelated groups. In the design of the
host, an acridine backbone was installed as both a fluorescent
moiety for signal transduction as well as a rigid scaffold for pre-
organization. Furthermore, triazole units were implemented for
convenient host synthesis via click chemistry, preorganization of
cyclen groups due to the rigidity of the triazoles, and since these
groups have previously been shown to bind anions. The resulting
sensors, 1 and 1-Zn2, were both found to bind InsP3 with high af-
finity in competitive media (methanol/water mix), with detection
of binding observed through changes in the host emission spectra
upon binding. While receptor 1 exhibited a slightly enhanced
binding affinity for InsP3 compared to other phosphorylated guests,
this difference was very modest. This is a common obstacle in anion
binding as enhancing selectivity remains a significant challenge.
Additionally, receptor 1-Zn2 generally benefited from greater
emission changes than 1 upon guest addition. Furthermore, the en-
hanced emission changes of 1-Zn2 upon addition of multiply phos-
phorylated guests as opposed to monophosphate analytes points to
the benefits of preorganizing two phosphate binding groups into
acavityforcomplexationofmultiplyphosphorylatedtargets. Itisclear
that polyammonium macrocycles are highly effective for binding
anions, particularly when they are incorporated within preorganized
macromolecular structures. However, to fully harness these proper-
ties and achieve the ultimate goal of selective recognition, the preor-
ganized scaffold must be carefully designed to maximize interactions
withthe targetguest. Tofurtherthisgoal, wearecurrentlyconsidering
alternate templates for enhancing anion-binding specificity.
4.1.3. N-(Propargyl)-1,4,7-tris(tert-butyloxycarbonyl)-1,4,7,10-tet-
raazacyclododecan-10-yl-acetamide (4). A solution of 9 (0.19 g,
1.06 mmol) in acetonitrile (5 mL) was added to a solution of 8
(0.34 g, 0.71 mmol) in acetonitrile (20 mL) at rt. Anhydrous potas-
sium carbonate (0.15 g,1.06 mmol) was then added, and the reaction
mixture heated to 80 ꢂC, at which it was stirred for 2 days. The
mixture was then filtered, followed by concentration of the filtrate by
rotary evaporation. Column chromatography over silica gel with
gradient elution from 50 to 100% ethyl acetate/hexanes then pro-
vided the product as a white solid (0.37 g, 92%). 1H NMR (300 MHz,
CDCl3)
d 7.16 (s, 1H), 4.05e3.98 (m, 2H), 3.71e3.11 (m, 14H),
2.92e2.45 (m, 4H), 2.18 (s,1H),1.51e1.43 (m, 27H).13C NMR (75 MHz,
CDCl3) d 170.72, 155.52, 79.88, 79.18, 71.18, 58.19, 56.22, 50.95, 49.78,
28.50. DARTeHRMS [MþH]þ calcd: 568.3710, found: 568.3605.
4.1.4. 4,5-Bis(azidomethyl)acridine (5). Sodium azide (0.51 g,
7.82 mmol) was added to a solution of 4,5-bis(bromomethyl)acri-
dine63 (0.48 g, 1.30 mmol) in N,N-dimethylformamide (20 mL) at rt.
The reaction mixture was then heated to 80 ꢂC, and allowed to stir
overnight. The reaction crude was then filtered, and the filtrate was
concentrated by rotary evaporation. Column chromatography over
silica gel with gradient elution from 20 to 100% dichloromethane/
hexanes gave the product as a light yellow solid (0.33 g, 87%).1H NMR
(300 MHz, CDCl3)
d
8.72 (s, 1H), 7.94 (d, J¼8.5 Hz, 2H), 7.78 (d,
J¼6.8 Hz, 2H), 7.52 (dd, J¼8.5, 6.8 Hz, 2H), 5.20 (s, 4H). 13C NMR
4. Experimental section
4.1. General experimental
(75 MHz, CDCl3) d 146.43, 136.59, 134.41, 129.77, 128.61, 126.60,
125.67, 51.49. DARTeHRMS[MþH]þ calcd:290.1154, found:290.1074.
4.1.5. Hexa-tert-butyl
10,100-((((1,10-(acridine-4,5-diylbis(-
Generally, reagents were purchased from Acros, Aldrich or AK
Scientific and used as received. Dry solvents were obtained from
a Pure Solv solvent delivery system purchased from Innovative
Technology, Inc. Column chromatography was performed using
230e400 mesh silica gel purchased from Sorbent Technologies.
Chloride counteranion ion exchange, as well as C18 reverse phase
solid phase extraction (SPE) syringe columns were obtained from
Silicycle. NMR spectra were obtained using a Varian Mercury 300
methylene))bis(1H-1,2,3-triazole-4,1-diyl))bis(methylene))bis(azane-
diyl))bis(2-oxoethane-2,1-diyl))bis(1,4,7,10-tetraazacyclododecane-
1,4,7-tricarboxylate) (6). A solution of alkyne 4 (0.084 g, 0.15 mmol)
in tetrahydrofuran (1 mL) was added to a solution of bis-azide 12
(0.021 g, 0.074 mmol) in tetrahydrofuran (1 mL) at rt. Copper sulfate
pentahydrate (0.0092 g, 0.037 mmol), sodium ascorbate (0.037 g,
0.19 mmol), and water (2 mL) were then added, and the reaction
mixture was stirred at rt overnight. After the addition of water
(20 mL), the mixture was extracted with dichloromethane
(2ꢀ20 mL). The combined organic layers were then dried over mag-
nesium sulfate, filtered, and concentrated. Column chromatography
over silica gel with gradient elution from 3 to 10% methanol/
dichloromethaneprovided theproductasa paleyellowsolid (0.058 g,
spectrometer. Mass spectra were obtained with
a
JEOL
DARTeAccuTOF spectrometer and an ABI Voyager DE Pro MALDI
spectrometer with high resolution capabilities. UVevis absorption
spectra were obtained with a Thermo Evolution 600 UVevis spec-
trophotometer, and fluorescence spectra were obtained with a Perkin
Elmer LS 55 luminescence spectrometer. InsP3 (2) used for binding
studies was synthesized from protected inositol intermediates that
we previously reported,64,65 as described in the Supplementary data.
55%). 1H NMR (300 MHz, CDCl3)
d
8.82 (s, 1H), 8.03 (d, J¼8.5 Hz, 2H),
7.82 (s, 2H), 7.71 (d, J¼6.2 Hz, 2H), 7.58e7.49 (m, 2H), 7.37 (s, 2H), 6.30
(s, 4H), 4.45 (d, J¼5.0 Hz, 4H), 3.52e3.00 (m, 28H), 2.62 (s, 8H),
1.52e1.30 (m, 54H). 13C NMR (75 MHz, CDCl3)
d 170.98,167.27,146.06,
4.1.1. 1,4,7-Tris(tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclodode-
cane (8). A solution of di-tert-butyl dicarbonate (1.77 g, 8.12 mmol)
in chloroform (10 mL) was added slowly via addition funnel to a so-
lution of cyclen (7, 0.51 g, 2.90 mmol) and triethylamine (1.25 mL,
8.99 mmol) in chloroform (40 mL) at rt. The reaction mixture was
stirred at rt overnight and then concentrated through rotary evapo-
ration. The resulting residuewaspurifiedbycolumnchromatography
over silica gel with gradient elution from ethyl acetate to 5% metha-
nol/dichloromethane to yield the product as a white solid (1.02 g,
80%). Characterizations matched previous reports.61
137.11, 129.30, 126.60, 125.78, 79.80, 50.45, 47.88, 34.97, 28.43. MAL-
DIeHRMS [MþNa]þ calcd: 1446.8238, found: 1446.8245.
4.1.6. N,N0-((1,10-(Acridine-4,5-diylbis(methylene))bis(1H-1,2,3-tri-
azole-4,1-diyl))bis(methylene))bis(2-(1,4,7,10-tetraazacyclododecan-
1-yl)acetamide) (1). Trifluoroacetic acid (3 mL) was added
dropwise to a solution of 6 (0.024 g, 0.017 mmol) in dichloro-
methane (3 mL) at rt. The reaction mixture was stirred at rt for 3 h,
followed by concentration via rotary evaporation. The crude
product was next dissolved in deionized water, loaded onto

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a chloride counteranion exchange column (0.5 g, Si-TMA chloride),
and eluted with deionized water. Purification on a reversed-phase
column (2 g, C18) with gradient elution from 0 to 50% methanol/
water gave the product as a yellow solid (0.016 g, 98%). 1H NMR
(300 MHz, CD3OD)
7.78 (d, J¼6.6 Hz, 2H), 7.67e7.59 (m, 2H), 6.36 (s, 4H), 4.50 (s, 4H),
3.39 (s, 4H), 3.24e2.70 (m, 32H).13C NMR (75 MHz, CD3OD)
174.06,
d
9.09 (s, 1H), 8.19 (d, J¼8.3 Hz, 2H), 8.04 (s, 2H),
d
147.03, 145.90, 138.48, 134.30, 132.21, 130.69, 127.88, 126.84, 124.95,
56.87, 51.73, 51.43, 47.85, 45.93, 44.25, 44.01, 35.76. MALDIeHRMS
[MþHClþH2OþH]þ calcd: 878.5145, found: 878.5143.
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raacetate complex as a light yellow solid (0.026 g, quant.). 1H NMR
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(300 MHz, CD3OD)
d
9.11 (s, 1H), 8.21 (d, J¼8.8 Hz, 2H), 8.06e7.90
(m, 2H), 7.90e7.76 (m, 2H), 7.69e7.60 (m, 2H), 6.34 (s, 4H), 4.49
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formed through excitation at 355 nm at rt. In these studies, a 10
buffered solution of host 1-Zn2 (2 mL) was titrated stepwise with 25
aliquots of a buffered solution consisting of 10 M host 1-Zn2 and
30 M guest. After each addition, the fluorescence spectra were
mM
mL
m
m
recorded. The changes in fluorescence intensities at the emission lmax
(440 nm) were then plotted against the concentration of added guest.
Binding constants were next determined in two ways. First, the data
were analyzed by non-linear regression analysis using saturation curve
fitting in SigmaPlot.66 Apparent Kd values were also evaluated using
the half-maximal point of the binding curves, and both methods
resulted in similar values. It should be noted that binding curves ex-
hibit some sigmoidal, or s-shaped, character, and thus are depicted
using a four-parameter sigmoidal curve fit in Fig. 2. Sigmoidal prop-
erties typically indicate the presence of more than one equilibria or
cooperativity.
Acknowledgements
M. D. B. acknowledges research support from the National Sci-
ence Foundation (CHE-0954297 and DMR-0954297).
52. Develay, S.; Tripier, R.; Le Baccon, M.; Patinec, E.; Serratrice, G.; Handel, H.
Dalton Trans. 2005, 3016.
53. Bernier, N.; Allali, M.; Tripier, R.; Conan, F.; Patinec, V.; Develay, S.; Le Baccon,
M.; Handel, H. New J. Chem. 2006, 30, 435.
Supplementary data
54. Costa, J.; Balogh, E.; Turcry, V.; Tripier, R.; Le Baccon, M.; Chuburu, F.; Handel,
H.; Helm, L.; Toth, E.; Merbach, A. E. Chem.dEur. J. 2006, 12, 6841.
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Supplementary data associated with this article can be found in
online version at doi:10.1016/j.tet.2011.03.092.
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