Switchable cucurbituril-bipyridine beacons

Article abstract of DOI:10.1002/chem.200903067

4-Aminobipyridine derivatives form strong inclusion complexes with cucurbit[6]uril, exhibiting remarkably large enhancements in fluorescence intensity and quantum yields. The remarkable complexation-induced pK_a shift (ΔpK_a =3.3) highlights the strong charge-dipole interaction upon binding. The reversible binding phenomenon can be used for the design of switchable beacons that can be incorporated into cascades of binding networks. This concept is demonstrated herein by three different applications: 1) a switchable fluorescent beacon for chemical sensing of transition metals and other ligands; 2) direct measurement of binding constants between cucurbit[6]uril and various nonfluorescent guest molecules; and 3) quantitative monitoring of biocatalytic reactions and determination of their kinetic parameters. The latter application is illustrated by the hydrolysis of an amide catalyzed by penicillin G acylase and by the elimination reaction of a b-cabamoyloxy ketone catalyzed by aldolase antibody 38C2.

Full text of DOI:10.1002/chem.200903067

DOI: 10.1002/chem.200903067
Switchable Cucurbituril–Bipyridine Beacons
Mantosh K. Sinha,[a] Ofer Reany,[a] Galit Parvari,[a] Ananta Karmakar,[a] and
Ehud Keinan*[a, b]
Abstract: 4-Aminobipyridine deriva-
tives form strong inclusion complexes
with cucurbit[6]uril, exhibiting remark-
ably large enhancements in fluores-
cence intensity and quantum yields.
The remarkable complexation-induced
pKa shift (DpKa =3.3) highlights the
strong charge–dipole interaction upon
binding. The reversible binding phe-
nomenon can be used for the design of
switchable beacons that can be incor-
porated into cascades of binding net-
works. This concept is demonstrated
herein by three different applications:
1) a switchable fluorescent beacon for
chemical sensing of transition metals
and other ligands; 2) direct measure-
ment of binding constants between cu-
curbit[6]uril and various nonfluorescent
guest molecules; and 3) quantitative
monitoring of biocatalytic reactions
and determination of their kinetic pa-
rameters. The latter application is illus-
trated by the hydrolysis of an amide
catalyzed by penicillin G acylase and
by the elimination reaction of a b-caba-
moyloxy ketone catalyzed by aldolase
antibody 38C2.
Keywords: biocatalysis · cucurbitur-
ils · fluorescence · inclusion com-
pounds · sensors
Introduction
ils[2] as preferred host molecules because their unique bind-
ing properties and rigid structure,[3] which includes two
highly polar portals and a hydrophobic interior, could be
harnessed for altering the photophysical properties of vari-
ous guest molecules.[4] It has been demonstrated that cucur-
bit[7]uril (CB[7]) can encapsulate various fluorescent dye
molecules and thereby modify their UV/Vis absorption
spectra, fluorescence intensities, and pKa values.[5]
While searching for guest molecules that could participate
in other binding events, we were attracted by the 2,2’-bipyri-
dine derivatives due to their fluorescence properties and in-
dependent ability to form stable complexes with essentially
any transition metal. It has been recently reported that 5,5’-
dimethyl-2,2’-bipyridine binds to either tetramethyl- or dicy-
clohexyl-CB[6] (CB[6]=cucurbit[6]uril), but that binding
event did not lead to fluorescence enhancement.[6] On the
contrary, the formation of 1:1 host–guest complexes between
those molecules resulted in fluorescence quenching. This
phenomenon probably reflects a binding mode in which
only one of the two pyridine rings is included within the cu-
curbituril cavity whereas the second ring remains exposed to
Biological systems are characterized by complex, intercon-
nected networks of noncovalent binding equilibria in which
one binding event triggers a cascade of other binding phe-
nomena. These equilibria cascades can finally give rise to a
specific functional output, such as the folding of proteins
and RNA.[1] This concept can be applied also to nonbiologi-
cal systems for the construction of specific chemical sensors.
Of particular interest are sensors that are based on host–
guest chemistry because they can be designed to be specific,
selective, and highly sensitive.
Aiming at the construction of a network of interconnected
binding events, we focused our attention on the cucurbitur-
[a] M. K. Sinha, Dr. O. Reany, G. Parvari, Dr. A. Karmakar,
Prof. E. Keinan
Schulich Faculty of Chemistry
Technion—Israel Institute of Technology
Technion City, Haifa 32000 (Israel)
Fax : (+972)48293913
E-mail: keinan@technion.ac.il
À
the solvent and rotates freely around the C C single bond,
[b] Prof. E. Keinan
Department of Molecular Biology and
the Skaggs Institute for Chemical Biology
The Scripps Research Institute
10550 North Torrey Pines Road
La Jolla, California 92037 (USA)
thus populating many stable conformations. This binding
mode, particularly with 5,5’-dimethyl-2,2’-bipyridine and tet-
ramethyl-CB[6], was predicted by quantum mechanical cal-
culations and confirmed by single-crystal X-ray crystallogra-
phy.[6a]
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200903067.
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FULL PAPER
We assumed that this situation could be drastically
changed if the bipyridine guest were forced to reside com-
pletely within the cavity of CB[6] (1). We anticipated that
this goal could be achieved by substituting position 4 of the
bipyridine molecule with an amine group to allow for a
double interaction of the guest molecule with both portals
of the CB host. We also expected that 1, being the smallest
practical host in the cucurbituril family, could induce signifi-
cant changes in the photophysical properties of the guest
molecule by imposing maximal restriction on its conforma-
tional freedom. In this respect, host 1 seems to have advan-
tages over CB[7], which has been extensively used by Nau
for substrate-selective supramolecular assays.[1b,7]
that the beacon fluorescence intensity can be harnessed for
chemical sensing, for direct measurement of binding con-
stants between 1 and various nonfluorescent guest molecules
(6, 7, and 8), and for quantitative monitoring of biocatalytic
reactions and determination of their kinetic parameters.
Herein we report 4-aminobipyridine derivatives, such as
2, 3, 4, and 5, which exhibit remarkably large enhancements
in fluorescence intensity and quantum yields upon formation
of inclusion complexes with 1. We also report that this en-
hanced fluorescence phenomenon can be utilized for the
design of switchable beacons that can be incorporated
within cascades of binding networks. In addition, we show
Abstract in Hebrew:
Results and Discussion
Compounds 2, 3, and 5 were prepared from 2-bromo-5-ami-
nopyridine (9), and compound 4 was prepared from 4-iodoa-
niline (10; Scheme 1). Thus, protection of the amine group
in 9 in the form of a 1,5-dimethylpyrrole derivative (11) was
followed by a Stille cross coupling with 2-(tributyltin)pyri-
dine and a palladium(0) catalyst to give protected bipyridine
13. Deprotection of the amine group by using aqueous hy-
droxylamine gave 4-amino-2,2’-bipyridine 2 in 67% overall
yield. By starting with 10 and using the same sequence of re-
actions, 4-(2-pyridyl)aniline (4) was obtained in a similarly
high overall yield. Compound 5 was obtained from 9 by first
converting it to the bis-Boc derivative (15) and then per-
forming a Suzuki coupling with phenylboronic acid by using
Fuꢁs palladium catalyst.[8] Finally, deprotection of the amine
group gave 5 in 60% overall yield. Monoprotection of 9
with only one Boc group to give 17 followed by double al-
ACHUTNGRENkNUG ylACHUTNGTRENaNNGU tion with 1,4-bis(bromomethyl)benzene under basic con-
ditions gave 18. The latter underwent the above-mentioned
Stille coupling to give 19, which, upon deprotection under
acidic conditions, afforded 3.
The absorption spectra of 2 and 2@1 were found to be
quite similar, with the latter being redshifted by 15 to 18 nm
(Figure 1 and Table 1, and Figure S1 in the Supporting Infor-
mation). These spectra are dominated by an intense p–p*
transition of the protonated (low-energy band) and the neu-
tral (high-energy band) bipyridine ligands.[9] Protonation of
2 is associated with a nine-fold intensity enhancement in the
low-energy band and a two-fold intensity decrease in the
high-energy band. The analogous effects in the spectrum in-
tensities of 2@1 are a five-fold enhancement and a two-fold
decrease, respectively. The spectral changes of 2 exhibit two
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E. Keinan et al.
isosbestic points at l=323 and
254 nm, which allow for deter-
mination of the two pKa values
(Scheme 2). The first pKa value
of 4.77 was determined by mon-
itoring the intensity changes at
l=302 and 353 nm, which char-
acterize the bipyridine part of
the molecule (Scheme 2 and
Figure 2, right),[9] whereas the
second pKa value of 5.04, which
is associated with the amine
group, was determined by mon-
itoring the changes at l=245
and 260 nm (Figure 2, left). A
second protonation of the bi-
Scheme 1. Synthesis of guest molecules 2–5. Reagents and conditions: a) hexane-2,5-dione, p-TsOH (cat.), tol-
uene, reflux, 4 h; b) 2-(tributyltin)pyridine, [Pd(PPh3)4], toluene, 1308C, 24 h; c) NH2OH·HCl, Et3N, EtOH/
H2O, 1008C, 24 h; d) (Boc)2O, Et3N, 4-dimethylaminopyridine (DMAP), CH2C2, RT, 48 h; e) PhB(OH)2,
CsCO3, dioxane, Pd(tBu3P)2 (5 mol%), 808C, 16 h; f) 4n HCl, EtOH, RT, 16 h; g) (Boc)2O, Et3N, CH2Cl2, RT,
12 h; h) 1,4-bis(bromomethyl)benzene, NaH, N,N-dimethylformamide (DMF), RT, 24 h; f) 4n HCl, EtOH,
RT, 16 h.
pyridine
fragment
usually
occurs at pKa =2.5,[10] however,
we have not observed it under
our experimental conditions.
Double protonation of bipyri-
dine results in repulsive interac-
tion between the two positive
charges and leads to a torsion
angle of at least 408, which is
unlikely to occur within the
cavity of 1.
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
In contrast, the absorption
spectrum of 2@1 exhibits only
one isosbestic point at l=
332 nm (Figure S1 in the Sup-
porting Information) that re-
lates to the bipyridine part,
with a remarkably high pKa
value of 8.1. The absence of a
second isosbestic point is not
surprising because deprotona-
tion of both the bipyridinium
and the ammonium group is ex-
pected to result in dissociation
of the complex. It has already
been reported that the pKa
values of bis-ammonium guest
Figure 1. Changes in the UV/Vis spectrum of 2 as a function of pH (3–11) at 298 K.
Table 1. Spectral properties of bipyridine ligands 2 and 3 and their com-
plexes with 1 (298 K, pH 3).
Absorption
emax [mÀ1 cmÀ1
Emission
lmax [nm]
lmax [nm]
]
Scheme 2.
2
287
353
302
371
254
302
377
305
371
12000
17000
4000
9000
8900
11000
15000
6400
417
2@1
3
455
molecules increase upon formation of host–guest complexes
with CB[n], as expected for a binding mode that is largely
composed of charge–dipole interactions.[5c]
The emission spectrum of 2 (lex =360 nm) is characterized
by an intense band at l=417 nm with tailing up to l=
453
513
3@1
455
8000
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Cucurbituril–Bipyridine Beacons
FULL PAPER
Consequently, 2@1 can be con-
veniently used as a fluorescent
sensor within a broad range of
over 3 pKa units in which the
complex is doubly protonated
and highly fluorescent whereas
the free ligand is neutral and
essentially nonfluorescent.
Compound 3 and its complex
3@1 exhibit essentially the
same UV/Vis absorption spec-
trum with no redshift of the
latter (Figure S3 in the Support-
ing Information and Figure 5).
The pH dependence of these
spectra is much more complex
than those of 2 and 2@1 be-
cause 3 contains four protona-
tion sites and is conformational-
Figure 2. Determination of the pKa values of 2 by monitoring the changes in its UV/Vis absorbance at l=245
and 260 nm (left) and at l=302 and 353 nm (right). The absorbance intensity changes were recorded for pH
values of 3–11.
455 nm, which can be ascribed to locally excited and charge
transfer excited states, respectively (Figure S2 in the Sup-
porting Information).[11] The emission of doubly protonated
2 at pH 3 is 300 times more intense than that of the neutral
molecule at pH 10. This phenomenon is expected because
the nonbonding electrons of the amine group in the neutral
molecule can efficiently quench the excited state. Accord-
ingly, monitoring the emission intensity at l=417 nm as a
function of pH between 3 and 9 allowed us to determine the
pKa of the amine group as 5.65Æ0.05. The second pKa
(ꢀ4.35) could also be determined by a similar analysis of
another very small variation in the fluorescence intensity as
a function of pH (Figure S2, upper inset, in the Supporting
Information).
In contrast, the emission spectrum of 2@1 (lex =360 nm)
is characterized by an intense band at l=455 nm with essen-
tially no tailing, which probably arises from a dominant in-
tramolecular charge transfer excited state (Figure 3). The
emission of doubly protonated 2@1 at pH 3 is about 200
times more intense than that of free 2 at pH 10. Monitoring
the emission intensity at l=455 nm as a function of pH al-
lowed for determination of the pKa of the bipyridine group
(7.45Æ0.05). It has been suggested that when molecules in
solution are continuously excited, their acid–base equilibri-
um is strongly dependant on the reversible formation of an
internal charge transfer excited state (S1ICT).[11] Thus, the ob-
served increased acidity of the bipyridine unit in the excited
state of 2@1 (pKa =7.45) in comparison with that of the
ground state (pKa =8.1) can be explained by an internal
charge transfer. A similar behavior was observed with 2, in
which the bipyridine unit was more acidic in the excited
state (pKa =4.35) than in the ground state (pKa =4.77).
The remarkable pKa shift between the free and bound
ligand, 2 versus 2@1, in either the ground state (DpKa =3.3,
Figure 4) or the excited state (DpKa =3.1) highlights the
strong charge–dipole interaction upon binding (Table 2).
Figure 3. Effect of pH on the fluorescence intensity of 2@1 (lex
=
360 nm). Inset: Evaluation of the pKa value by using a logarithmic linear
regression plot: pKa =pHÀlog[(IobsÀImin)/(ImaxÀIobs)]; y=À0.7387x+
5.5064, R2 =0.9849.
Figure 4. A comparison of the UV/Vis absorption of 2 (lmax =362 nm,
normalized) and 2@1 as a function of pH. The dashed lines represent
curve fittings by using a simple iterative least-squares analysis: Iobs
=
[Imax(10ACHUTNGRTEG(NNUN pHÀpKa))+Imin]/(1+10ACHTUGNRTGENN(U pHÀpKa)).
AHCTUNGTRENNUNG
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E. Keinan et al.
emission at l=510 nm is quite unusual.[12] It can be ex-
plained by excimer formation, which is known for many aro-
matic hydrocarbons, such as 1,4-bis(4’-pyridylethynyl)ben-
zene[13] and 2,2’-bipyridine in silica gel glass,[14] which exhibit
excimer fluorescence in solution at room temperature. Intra-
molecular excimer fluorescence is known in molecules that
have two or more identical aromatic groups (segmers)
linked by a flexible tether.[15] If an excited-state segmer can
move into the vicinity of a ground-state segmer during the
lifetime of the excited state, it
Table 2. pKa values of free 2 and 3 and bound 2@1 and 3@1 at 298 K.
Ground state pKa
Excited state pKa
2
4.77
5.04
ꢀ4.35
5.65
2@1
3
8.10
4.38
7.45
5.20
4.8, 5.24
7.53
6.05
3@1
6.80[a]
[a] Average value (see text).
may form an intramolecular ex-
cimer (1D*) that is character-
ized by structureless fluores-
cence spectrum at lower energy
than the monomeric emission.
The observed excimer fluores-
cence of 3 seems to reflect an
effective p–p stacking between
two segmers, one of which is in
the excited state. The intensity
of this high-energy band is
slightly dependent on pH
within the range of 6–10, and
reveals a pKa value of 5.20Æ
0.05
(lex =360 nm,
lem =
455 nm; Table 2) associated
with protonation of the bipyri-
dine unit. The lower transition
Figure 5. UV/Vis absorption spectra of 3@1 as a function of pH at 298 K exhibiting an isosbestic point at l=
341 nm. Arrows indicate intensity changes as the pH was increased from 3 to 12. Inset: Absorbance changes at
l=308 and 371 nm reveal a pKa value of 7.53.
ly more flexible. The spectrum of 3@1 exhibits the same
pH-dependence trend seen with 2 and 2@1 (increased inten-
sity of the high energy p–p* absorption and decreased in-
tensity of a low-energy band along with one isosbestic
point), whereas the spectrum of 3 exhibits three bands
(lmax =254, 302, and 377 nm), all of which decrease in inten-
sity as the pH increases. The pH dependence of the l=377,
254, and 302 nm absorption maxima allowed for determina-
tion of three pKa values of 4.38, 4.80, and 5.24, respectively
(Table 2 and Figure S3 in the Supporting Information). We
assume that the pKa value of 4.38 is associated with simulta-
neous protonation of both bipyridine units, whereas the two
other values reflect sequential protonation of the two amine
Figure 6. Effect of pH on the fluorescence intensity of 3 (lex =360 nm).
Inset: The effect on the monomer emission at l=455 nm and the excimer
emission at l=510 nm.
groups.
As was the case for 2@1, the spectral dependence of 3@1
on the pH allowed us to determine only the pKa of the bi-
pyridine moiety. Thus, monitoring the intensity of the bands
at l=308 and 371 nm, which are separated by an isosbestic
point at l=341 nm, gave a pKa value of 7.53 (Figure 5).
Again, as was the case for 2@1, the notable pKa shift
(DpKa =3.15) highlights the strong charge–dipole interaction
as the main binding mechanism between 3 and 1.
The emission spectrum of 3, which is far more complex
than the emission spectrum of 2, exhibits dual fluorescence
bands at l=455 and 510 nm (Figure 6). Although the l=
455 nm emission is reminiscent of the broad l=417 nm
band of 2, including the ICT tailing, the additional broad
energy band followed a similar decrease but at higher pH.
Thus, the variation in emission at l=510 nm with pH re-
vealed a pKa value of 6.05Æ0.05, which is probably associat-
ed with protonation of the amine groups.
Excimer emission is totally prevented upon formation of
either the 1:2 or the 1:1 complex of 3@1 because the two
segmers are now shielded from one another by the host mol-
ecule and by the loss of conformational flexibility. Thus, the
emission spectrum of 3@1 exhibits only the monomeric
emission band at l=450 nm (Figure 7), which is similar to
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Cucurbituril–Bipyridine Beacons
FULL PAPER
changed over a broad pH range of between 2 and 7. Unlike
the case of 2, the titration of 3 by 1 indicated a binding stoi-
chiometry of 1:2, as revealed by the Job plot[19] (Figure S6 in
the Supporting Information). To obtain the 3@1 binding
constant we used a model of two identical noninteracting
binding sites[20] to give the overall binding constant of
logb=5.07Æ0.02. Apparently, monoalkylation of the amine
group on going from 2 to 3 introduces steric demands that
diminish the binding efficiency. Moreover, considering the
fact that 1,4-bis(aminomethyl)benzene is a strong binder of
CB,[6,21] our observation that the central portion of 3 is a
weaker binder than its peripheral bipyridine moieties indi-
cates that alkylation of 1,4-bis(aminomethyl)benzene signifi-
cantly reduces its binding affinity to 1.
We used isothermal titration calorimetry (ITC) measure-
ments to independently determine the binding affinity and
maximal binding stoichiometry between 1 and either 2 or 3
(Table 3 and Figures S7 and S8 in the Supporting Informa-
tion). As can be seen from Table 3, the ITC data verified
Figure 7. Effect of pH on the fluorescence intensity of 3@1 (lex =360 nm,
lem =455 nm). Inset: Determination of the pKa value (Table 2) was car-
ried out by using the acid–base isotherm equation given in Figure 3.
the spectrum of 2@1. As in the case of 2@1, the pH depend-
ence of the emission intensity of 3@1 revealed a pKa value
of 6.80Æ0.05 (Table 2) associated with protonation of the
bipyridine unit, which is lower than the pKa value of the
ground state (7.53Æ0.05). This effect can be explained by
excited state acidity enhancement of the bipyridine units
when hosted within 1. It should be noted, however, that the
logarithmic isotherm is not perfectly linear; this reflects
multiple protonations. Because it was not possible to differ-
entiate between the various acid–base equilibria, the ob-
served pKa should be considered as an average value.
Addition of 1 to a buffered aqueous solution (NH4OAc,
pH 7.0) of either 2 or 3 resulted in remarkable fluorescence
enhancement factors of 30 and 24, respectively (Figure 8
and Figure S4 in the Supporting Information). We used the
continuous variation plot[16] to verify the 1:1 binding stoichi-
ometry between 2 and 1 (Figure S5 in the Supporting Infor-
mation). By using the Hill plot[17] for the titration curve of 2
by 1, the host–guest binding constant was obtained (logb=
5.70Æ0.02).[18] This value of binding constant remained un-
Table 3. Overall binding constants (logb), binding stoichiometry (n), en-
thalpy (DH), and entropy (DS) of binding for host–guest complexes 2@1,
3@1, 4@1, 5@1, and 8@1.[a]
Host–Guest Conditions logb[18]
n
DH
DS [eu]
[KcalmolÀ1
]
2@1
A
B
5.49Æ0.02 0.87 À13.79Æ0.01 À20.4Æ0.2
4.87Æ0.01 0.96 À12.18Æ0.01 À18.7Æ0.1
5.34Æ0.04 0.44 À20.64Æ0.03 À43.6Æ0.2
3@1
4@1
5@1
8@1
A
A
A
A
B
4.80Æ0.02 0.96
À8.0Æ0.1
À4.4Æ0.2
4.20Æ0.05 0.45 À15.79Æ0.18 À32.9Æ0.2
6.25Æ0.04 0.91 À11.13Æ0.01 À8.1Æ0.02
6.59Æ0.05 0.86 À10.35Æ0.01 À4.6Æ0.03
[a] [1]=0.13 mm for all experiments. Conditions: A) [Ligand]=1 mm in
NH4Cl buffer (10 mm, pH 4, 303 K). B) [2]=1.6 mm and [8]=1 mm in
NH4OAc buffer (10 mm, pH 7, 298 K).
both binding constants and binding stoichiometry values
(1:1 for 2@1 and 1:2 for 3@1) that were obtained from the
fluorescence enhancement experiments. To better under-
stand the binding mode between 1 and 2, we prepared two
deaza analogues, 4 and 5. Their binding constants to 1 were
determined by using ITC and the data were compared with
the known binding constant of 1,6-diaminohexane (8).[21]
The binding constant of 4 is quite similar to that of 2, which
indicates a double interaction with both portals of 1. In con-
trast, compound 5 binds weakly to 1 and is apparently inca-
pable of interacting with both portals simultaneously
(Table 3 and Figure S9 in the Supporting Information). This
interaction did not produce any notable spectral signature;
no change was observed in the absorption spectrum (lex =
255 nm), and only a 20% increase was noticed in the emis-
sion spectrum (lem =325 nm).
In contrast, the 1H NMR spectra exhibited a significant
shift in the phenyl protons (ꢀ1 ppm upfield) upon binding
to 1, whereas the signals that correspond to the protons
ortho to the amine group were not affected. These observa-
tions reflect an interesting binding mode of 5 in which the
Figure 8. Changes in the emission spectrum of 2 (lex =360 nm, lem
=
455 nm) upon addition of 1. A buffered solution of 1 (NH4OAc, 0.1 mm,
pH 7) was added in portions (20 mL each, 0.1 mm) to a solution of 2
(10 mL, 0.001 mm) at 298 K. Inset: Hill plot analysis that afforded the
value of logb (5.70Æ0.02); y=À3.0028xÀ17.133, R2 =0.9938.
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E. Keinan et al.
phenyl group is incorporated within the cavity of 1, with
preferred interaction between the CB portal and the pyri-
dine function rather than with the amine group. The 1:2
binding stoichiometry between 5 and 1 confirms that one
molecule of 1 interacts with the pyridine ring and another
molecule interacts with the amine group. This unique behav-
ior is consistent with the reported basicity of 3-aminopyri-
dine in aqueous solution, in which the first protonation site
is the pyridine nitrogen atom.[22]
Scheme 3. A series of interconnected equilibria, a chemical food chain
composed of five components.
Formation of host–guest complexes 2@1 and 3@1 was
found to involve not only significant increase in fluorescence
intensity but also a remarkable enhancement of the quan-
tum yields. The quantum yield of free 2 and 3 in the absence
of 1 was found to be 0.89 and 0.45, respectively, whereas the
quantum yields of their inclusion complexes with 1 became
quantitative (1.00; Table 4 and Figure S10 in the Supporting
Table 4. Fluorescence quantum yields (FF) and fluorescence lifetime (t)
values (aqueous HCl, pH 3) for 2, 2@1, 3, and 3@1. The FF values were
determined by applying the comparative method[32] with coumarin-1 as
the standard and correcting for the solvent refractive index ratio (0.959;
see the Supporting Information).
t [ns]
FF
2
7.8
0.89
2@1
3
7.6
7.6
1.00
0.45
1.00
0.70 (EtOH)
Figure 9. A chemical food chain monitored by using fluorescence intensi-
ty (lex =360 nm, lem =455 nm). The concentration scale refers to the se-
quential additions of all four components to a solution of 1 (0.1 mm;
10 mm NH4OAc, pH 7). The blue curve indicates stepwise addition of 2
(up to 0.1 mm). The red curve indicates stepwise addition of Cu+2 acetate
(up to 0.2 mm). The green curve indicates stepwise addition of EDTA
(up to 0.2 mm). The orange curve indicates stepwise addition of In+3 ace-
tate (up to 0.2 mm).
3@1
10.1
4.3 (EtOH)
Coumarin[33]
Information). This effect could probably arise from the re-
stricted motion of the guest molecules within the tight
cavity of 1. It has been previously proposed that the rela-
tively high quantum yields (0.95 and 0.94) observed for 4,4’-
bis[(2,5-dimethoxyphenyl)ethynyl]biphenyl and 4,4’-bis[(2,5-
dimethoxyphenyl)ethynyl]-2,2’-bipyridine, respectively, re-
flected their rigid structures.[23] The enhanced quantum yield
could also result from a strong polar medium effect and de-
solvation of the guest molecule on moving from an aqueous
medium to the hydrophobic environment of the host. In
contrast, very small variations in the fluorescence lifetime of
the free and bound guests were observed that were within
the most usual range of between 1 and 10 ns. The diversity
of the lifetime parameter exhibited only minor fluctuations,
particularly in comparison with other parameters, such as
energy bands, intensities, and quantum yields.
To demonstrate the concept of interconnected equilibria
networks, we constructed a system that was comprised of
five components (A–E) that were added sequentially to a
buffered aqueous solution (10 mm NH4OAc, pH 7.0, 298 K).
The resulting noncovalent binding events were monitored
by measuring the fluorescence intensity (Scheme 3 and
Figure 9). This “chemical food chain” started with the addi-
tion of component B (represented by 2) to a solution that
contained component A (represented by 1) and formation
of the highly fluorescent inclusion complex 2@1. Addition
which indicates quantitative dissociation of 2 from 1 with
concomitant formation of the expected 1:2 complex of CuII
with 2 (for evidence for the 1:2 stoichiometry, see the Sup-
porting Information and Figure S11). This complex forma-
tion was associated with complete quenching even of the in-
dependent, relatively low fluorescence of 2, as was evident
from a control experiment in which addition of CuACTHUNRGTNEUNG(OAc)2 to
an aqueous solution of 2 in the absence of 1 resulted in a de-
crease in the fluorescence intensity by two orders of magni-
tude. Addition of the fourth component (D) represented by
ethylenediaminetetraacetic acid (EDTA), which is a strong
binder of CuII (logb=15.5),[24] resulted in complete release
of 2. Compound 2 was thus once again made available for
interaction with 1 and the maximal fluorescence intensity of
2@1 was recovered. Finally, addition of the fifth component
(E) represented by InACTHNUTRGNEUNG(OAc)3 resulted in quantitative forma-
tion of the expected InIII@EDTA complex (logbꢀ25).[25]
The released CuII ions then became available to regenerate
the BC complex with concomitant dissociation of the AB
(2@1) complex and complete suppression of the fluores-
cence intensity (Scheme 3 and Figure 9).
The complexation-dependent fluorescence phenomenon
provides an opportunity to quantitatively determine the
binding constants between 1 and other ligands through com-
of the third component (C; represented by Cu
sulted in complete suppression of the fluorescence intensity,
ACHTUNGRTENN(UNG OAc)2) re-
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Chem. Eur. J. 2010, 16, 9056 – 9067

Cucurbituril–Bipyridine Beacons
FULL PAPER
petitive binding experiments. This approach allows for moni-
toring “dark” binding events that by themselves are not as-
sociated with any significant change in either UV/Vis ab-
sorption or fluorescence intensity. Typical examples are the
formation of the inclusion complexes between 1 and saturat-
ed diamine compounds, such as putrecine (6), cadaverine
(7), and 1,6-diaminohexane (8).[26,27] To demonstrate this ad-
vantage we carried out displacement titration experiments
with 6–8 (Figures S12 and S13 in the Supporting Information
and Figure 10, respectively). Plotting the competitive bind-
sired quenching by interfering analytes.
Another attractive application of the complexation-de-
pendent fluorescence of 2@1 is the ability to rapidly monitor
biocatalytic reactions, including quantitative determination
of their kinetic parameters.[28] We demonstrated this advant-
age by quantitatively monitoring the hydrolysis of amide 20
as catalyzed by penicillin G acylase (PGA) and the b-elimi-
nation reaction of b-cabamoyloxy ketone 21 as catalyzed by
aldolase antibody 38C2 (Scheme 4).
Substrate 20 was prepared in high yield by dimethylami-
nopyridine-catalyzed acylation of 2 with phenylacetyl chlo-
ride in dichloromethane. Substrate 21 was prepared from
ethyl propioacetate (22; Scheme 5). Protection of the
ketone group followed by LiAlH4 reduction afforded alco-
hol 23, which was converted to active imidazolyl carbamate
24. Transamidation of the latter by using compound 9 af-
forded stable carbamate 25. Stille coupling with 2-(tributyl-
tin)pyridine and palladium(0) catalyst afforded bipyridine
derivative 26. Finally, deprotection of the ketone group af-
forded 21.
Both biocatalytic reactions were carried out in the pres-
ence of excess 1, which did not interfere with the catalytic
activity of either PGA or 38C2 as verified by control experi-
ments. Substrates 20 and 21 were designed to release 2 as
one of the biocatalytic reaction products. The rapid forma-
tion of highly fluorescent complex 2@1 upon release of 2 al-
lowed for fast, quantitative monitoring of the progress of
both reactions by using the appropriate calibration curves of
fluorescence intensity versus [2]. Because the initial rates
within the first 2 min of the reaction were monitored by
using fluorescence intensity (lex =360 nm, lem. =453 nm), it
was possible to carry out all reactions in a very small
Figure 10. Determination of binding constant of 8@1 by competition
binding. The fluorescence intensity of 2 (lex =360 nm, lem =455 nm) was
measured as a function of the stepwise addition of 8 (in 10 mm NH4Cl,
pH 3 buffer) to an aqueous solution of 2@1 (0.03 mm, in 0.1 mm NH4Cl,
pH 3 buffer). Inset: Determination of the binding constant for 8@1 was
obtained from the equation: [1]t/P=0.017Q+1.1, R2 =0.9983, LogK8@1
=
7.47 (for details, see the Supporting Information).
ing data by using the appropriate model (see details in the
Supporting Information) with the above-mentioned binding
constant of 2@1 (logb=5.70)
gave the binding constants of
6@1 (logb=6.9), 7@1 (logb=
8.1), and 8@1 (logb=7.49).
These values are comparable
with the values that were mea-
sured by other methods, such as
1H NMR spectroscopy, for 6@1
(logb=7.3),[26] 7@1 (logb=7.6–
8.2), and 8@1 (logb=6.44–
Scheme 4.
8.46).[21a,26,27] The only prece-
dent for monitoring
a dark
binding event between a UV-in-
active guest and CB[6] has been
reported by Nau[5c] for a carba-
zole fluorophore covalently
linked to diaminoalkane. Be-
cause the fluorophore in this
tethered conjugate only indi-
rectly interacts with the host,
the fluorescence intensities
might be more susceptible to
background noise and to unde-
Scheme 5. Synthesis of substrate 21. Reagents and conditions: a) 1,3-propanediol, p-TsOH (cat.), benzene,
reflux, 20 h; b) LiAlH4, Et2O, reflux, 5 h; c) carbonyldiimidazole, CH2Cl2, RT, 2 h; d) 5-amino-2-bromopyri-
dine, NaH, THF, RT, 16 h; e) 2-(tributyltin)pyridine, [Pd
16 h.
ACHTUGNRTNE(NUNG PPh3)4], toluene, 1208C, 24 h; f) AcOH, H2O, RT,
Chem. Eur. J. 2010, 16, 9056 – 9067
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www.chemeurj.org
9063

E. Keinan et al.
volume (250 mL) by using a 96-well plate and a fluorescence
microplate reader.
Conclusion
The PGA-catalyzed reactions were carried out in phos-
phate-buffered saline (PBS, pH 7.4) at 258C by using a fixed
concentration of the enzyme (7.14 nm) and eight different
concentrations of 20 (0.025–0.075 mm) in the presence of 1
(0.08–0.24 mm). The kinetic parameters (kcat. =6.54ꢂ
104 minÀ1, kcat./kuncat. =2.9ꢂ108, Km =0.884 mm, kcat./Km =7.4ꢂ
104 minÀ1) were obtained by a Lineweaver–Burk analysis of
the measured initial rates (Figure 11). These kinetic parame-
4-Aminobipyridine derivatives form strong inclusion com-
plexes with 1 and exhibit remarkably large enhancements of
fluorescence intensity and quantum yields. The remarkable
complexation-induced pKa shift (DpKa =3.3) highlights the
strong charge–dipole interaction upon binding. This en-
hanced fluorescence phenomenon can be used for the
design of switchable beacons that can be incorporated into
cascades of binding networks. This concept has been demon-
strated herein by three different applications: 1) a switcha-
ble fluorescent beacon for chemical sensing of transition
metals and ligands; 2) direct measurement of binding con-
stants between 1 and other, non-fluorescent guest mole-
cules; and 3) quantitative monitoring of biocatalytic reac-
tions and rapid determination of their kinetic parameters.
Further applications of these phenomena in multiparameter
systems are currently underway in our laboratories, includ-
ing the construction of chemical logic gates, fast screening of
libraries of ligands of transition metals, fast measurement of
metal–ligand binding constants, and further expansion of the
interconnected binding networks.
Experimental Section
Figure 11. The kinetics of the PGA-catalyzed hydrolysis of 9 as moni-
tored by using fluorescence intensity (lex =360 nm, lem =453 nm). The
General methods: All reactions were carried out in anhydrous solvents
under an inert atmosphere. Compounds 9, 10, and 22 were purchased
from Aldrich Chemicals. CB[6] 1 was prepared as described before.[3a]
PGA and catalytic antibody 38C2 were purchased from Aldrich in the
form of lyophilized powder. Flash chromatography was performed on
Merck silica gel 60 (230–400 mesh). 1H and 13C NMR spectra were re-
corded in the solvents indicated by using an AVANCE 500 Bruker spec-
trometer. Chemical shifts (d) are given in ppm relative to TMS. The re-
sidual solvent signals were used as references and the chemical shifts
were converted to the TMS scale: CDCl3: dH =7.26 ppm, dC =77.16 ppm;
D2O/DCl (with trace DMSO): dH =2.71 ppm, dC =39.4 ppm. Mass spectra
were recorded by using either Waters MALDI microMX (TOF) or
Waters LCT Premier microMax spectrometers (TOF-ESI, with MeCN/
H2O 1:1). ITC measurements were performed at either 298 or 303 K by
using a VP-ITC instrument (MicroCal, USA). UV absorption and fluo-
rescence spectra for binding studies were recorded at 258C by using a
SpectraMax M2 spectrometer. Quantum yield measurements were deter-
mined by using a Fluorolog 3-21 fluorimeter equipped with a Hamamat-
zu R928 photomultiplier. Emission spectra were acquired following exci-
tation at the given wavelength and were corrected for the wavelength de-
pendence of the photomultiplier tube. Lifetime measurements were car-
ried out by using a continuum Nd–Yag Laser 7010 Model at the third har-
monic, l=355 nm, as the exciting source and applying a pulse width of
5 ns. The laser was equipped with a Spectra-Pro 275 monochromator, a
Hamamatzu R928 photomultiplier, and a Tekronix TDS 220 scope con-
troller. Data were processed by using LabVIEW software (National In-
struments, USA).
concentrations of
2 (a) 0.075, b) 0.070, c) 0.065, d) 0.060, e) 0.055,
f) 0.045, g) 0.040, h) 0.030, i) 0.025 mm) were determined by using a cali-
bration curve (not shown). All reactions were carried out in PBS, pH 7.4,
258C by using PGA (7.14 nm) varying concentrations of substrate. Inset:
Determination of kcat. (6.54ꢂ104 minÀ1) and Km (884 mm) by using a Line-
weaver–Burk
analysis;
y=1.893x+2.1406,
R2 =0.9908,
Vmax =
0.467 mmminÀ1
.
ters indicate that the PGA-catalyzed hydrolysis of 20 is 40
times faster than that of 6-nitro-3-phenylacetamidobenzoic
acid (NIPAB; kcat. =1.62ꢂ103 minÀ1, Km =11 mm, kcat./Km =
1.47ꢂ102 mmÀ1 sÀ1),[29] which is commonly used for determin-
ing the catalytic activity of PGA samples.
The 38C2-catalyzed reactions were carried out in a similar
fashion by using a fixed concentration of aldolase antibody
38C2 (1.5 mm) and eight different concentrations of 21
(0.025–0.075 mm) and 1 (0.08–0.24 mm) to give the kinetic pa-
rameters
(kcat. =9.9 minÀ1,
kcat./kuncat. =1.5ꢂ103,
Km =
0.04 mm; Figure S14). It is interesting to compare these pa-
rameters with those of similar substrates studied earlier with
the same antibody. Although the value of kcat. of 21 is slight-
ly higher than the value of the best substrate studied so far,
3-(N-phenylcarbamoyl)cyclohexanone (kcat. =8.9 minÀ1, Km =
1.27 mm),[30] the much smaller Km value observed with 21
suggests that aminobipyridine derivatives are tight binders
of aldolase antibody 38C2. Hence, the presence of 1 in the
reaction mixture has a beneficial effect on the catalytic reac-
tion in that it scavenges 2 and thereby prevents product in-
hibition of the catalyst.
1-(2-Bromopyridine-5-yl)-2,5-dimethyl-1H-pyrrole (11): p-TsOH (15 mg,
0.08 mmol) was added to a stirred solution of 9 (1 g, 5.8 mmol) and 2,5-
hexanedione (0.75 mL, 6.4 mmol) in toluene (20 mL), and the mixture
was heated at reflux for 4 h by using a Dean–Stark apparatus. After cool-
ing, the mixture was quenched with saturated aq NaHCO3 and extracted
with toluene (2ꢂ20 mL). The organic phase was washed with H2O and
brine, dried over MgSO4, and the solvent was removed under reduced
pressure. The resulting crude product was purified by column chromatog-
raphy (silica gel, hexane/EtOAc 9:1) to give pure 11 as a yellowish solid
(1.4 g, 97%). 1H NMR (500 MHz, CDCl3): d=8.29 (d, J=2.5 Hz, 1H),
9064
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Cucurbituril–Bipyridine Beacons
FULL PAPER
7.61 (d, J=8 Hz, 1H), 7.44 (dd, J=8, 2.5 Hz, 1H), 5.95 (s, 2H), 2.05 ppm
(s, 6H); 13C NMR (125 MHz, CDCl3): d=149.4, 140.7, 138.0, 135.0, 128.8,
128.4, 107.0, 12.0 ppm; MS (TOF-ES): m/z calcd for C11H11BrN2: 250;
found: 249 [MÀH]+.
1-(4-Iodophenyl)-2,5-dimethyl-1H-pyrrole (12): Synthesis was carried out
by using 10 (1.3 g, 6 mmol), 2,5-hexanedione (0.8 mL, 6.6 mmol), p-
TsOH (20 mg) in toluene and by following the same procedure as de-
scribed for 11. Column chromatography of the crude product (silica gel,
4-(Pyridin-2-yl)benzenamonium chloride (4): The synthesis was per-
formed by using 14 (310 mg, 1.25 mmol), hydroxylamine hydrochloride
(900 mg, 12.5 mmol), Et3N (0.35 mL), EtOH (6 mL), and H2O (2.6 mL),
and by following the above-described procedure for the preparation of 2,
and gave 4 as a light-brown solid (180 mg, 85%). 1H NMR (500 MHz,
D2O/DCl): d=8.75 (d, J=6 Hz, 1H), 8.63 (dt, J=1, 7 Hz, 1H), 8.29 (d,
J=8 Hz, 1H), 8.00 (t, J=7 Hz, 1H), 7.97 (d, J=8.5 Hz, 2H), 7.55 ppm
(d, J=8.5 Hz, 2H); 13C NMR (125 MHz, D2O/DCl): d=152.0, 147.8,
141.8, 137.9, 130.6, 130.0, 126.8, 126.1, 123.4 ppm; MS (TOF-LD): m/z
calcd for C11H10N2: 170; found: 171 [M+H]+.
hexane/EtOAc 9:1) afforded 12 as
a yellowish solid (1.73 g, 97%).
1H NMR (500 MHz, CDCl3): d=7.79 (d, J=8 Hz, 2H), 6.96 (d, J=
8.5 Hz, 2H), 5.9 (s, 2H), 2.02 ppm (s, 6H); MS (TOF-ES): m/z calcd for
C12H12IN: 297; found: 296 [MÀH]+.
Inclusion complex of 1 with 4 (4@1): Complex 4@1 was prepared by
using the same procedure as described for 2@1. 1H NMR (500 MHz,
D2O/DCl): d=8.89 (d, J=8.5 Hz, 1H), 8.53 (d, J=5.5 Hz, 1H), 8.40 (t,
J=8 Hz, 1H), 7.70 (t, J=7 Hz, 1H), 7.10 (d, J=8.5 Hz, 2H), 6.04 (d, J=
8.5 Hz, 1H), 5.69 (dd, J=68.5, 15.5 Hz, 12H), 5.48 (s, 12H), 4.24 ppm
(dd, J=11.5, 15.5 Hz, 12H); MS (TOF-ES): m/z calcd for
C47H48Cl2N26O12: 1240; found: 1168 [MÀ2ClÀH]+.
2-Bromo-5-(bis-Boc)aminopyridine (15): A solution of 9 (1 g, 5.8 mmol)
and Et3N (2.5 mL, 17.4 mmol) in CH2Cl2 (50 mL) was stirred for 30 min
at RT. (Boc)2O (2.8 g, 12.8 mmol) and DMAP (116 mg) in CH2Cl2
(5 mL) were added and the mixture was stirred overnight at RT, then
quenched with aq NH4Cl and extracted with CH2Cl2. The organic phase
was separated and washed with H2O and brine, dried over MgSO4, and
the solvent was removed under reduced pressure. The residue was puri-
fied by flash chromatography (silica gel, hexane/EtOAc 8:2) to give 15 as
a colorless solid (1.84 g, 85%). 1H NMR (300 MHz, CDCl3): d=8.19 (d,
J=2.7 Hz, 1H), 7.50 (d, J=8.4 Hz, 1H), 7.35 (dd, J=5.7, 2.7 Hz, 1H),
1.42 ppm (s, 18H).
1-(2,2’-Bipyridine-5-yl)-2,5-dimethyl-1H-pyrrole (13): 2-(Tributyltin)pyri-
dine[31] (1.6 g, 4.5 mmol) in toluene (15 mL) was added to a stirred solu-
tion of 11 (0.75 g, 3 mmol), followed by addition of [PdACTHUNTRGNE(UNG PPh3)4] (70 mg,
0.06 mmol) under argon at RT. After purging with argon for 15 min, the
mixture was heated at 1308C for 24 h, then quenched with 2n NaOH
and extracted with EtOAc. The organic phase was washed with brine,
dried over Na2SO4, and the solvent was removed under reduced pressure.
Column chromatography of the residue (silica gel, hexane/EtOAc 8:2) af-
forded 13 as a white solid (0.61 g, 82%). 1H NMR (500 MHz, CDCl3):
d=8.71 (d, J=4 Hz, 1H), 8.57 (d, J=2 Hz, 1H), 8.54 (d, J=8.5 Hz, 1H),
8.44 (d, J=8 Hz, 1H), 7.86 (ddd, J=7.5, 8, 1.5 Hz, 1H), 8.68 (dd, J=8.5,
2.5 Hz, 1H), 7.35 (dd, J=7, 5 Hz, 1H), 5.96 (s 2H), 2.09 ppm (s, 6H);
13C NMR (125 MHz, CDCl3): d=155.2, 155.1, 149.3, 148.3, 137.1, 136.3,
135.5, 129.0, 124.0, 121.2, 121.2, 106.7, 13 ppm; MS (TOF-ES): m/z calcd
for C16H15N3: 249; found: 250 [M+H]+.
2-[4-(2,5-Dimethyl-1H-pyrrol-1-yl)phenyl]pyridine (14): The synthesis
was carried out by using compound 12 (700 mg, 2.35 mmol), 2-(tributyl-
tin)pyridine (1.3 g, 3.5 mmol), and [PdACTHNUTRGNEUNG(PPh3)4] (55 mg, 0.047 mmol) in
2-Phenyl-5-(bis-Boc)aminopyridine (16):
A mixture of 15 (750 mg,
2 mmol), phenylboronic acid (365 mg, 3 mmol), CsCO3 (2 g, 6 mmol),
and bis(tri-tert-butyl phosphine)palladium (50 mg, 0.1 mmol) was purged
with argon, then anhydrous dioxane (15 mL) was added. The mixture
was heated to 808C for 24 h, quenched with aq NH4Cl, extracted with
Et2O, washed with H2O and brine, and dried over MgSO4. Removal of
solvent under reduced pressure followed by flash chromatography (silica
gel, hexane/EtOAc 9:1) afforded 16 as white solid (650 mg, 88%).
1H NMR (500 MHz, CDCl3): d=8.47 (d, J=2.5 Hz, 1H), 8.02 (d, J=
7.5 Hz, 2H), 7.74 (d, J=8.5 Hz, 1H), 7.54 (dd, J=5.5, 2.5 Hz, 1H), 7.49–
7.41 (m, 3H), 1.43 ppm (s, 18H); 13C NMR (125 MHz, CDCl3): d=156.0,
151.2, 148.8, 138.5, 135.9, 134.7, 129.2, 128.8, 126.9, 120.0, 83.4, 27.9 ppm;
MS (TOF-LD): m/z calcd for C21H26N2O4: 370; found: 371 [M+H]+.
toluene (15 mL), and by following the same procedure as described for
13. The crude product was purified by column chromatography (silica
gel, hexane/EtOAc 8:2) to give 14 as a white solid (466 mg, 80%).
1H NMR (500 MHz, CDCl3): d=8.63 (d, J=2 Hz, 1H), 8.00 (d, J=
6.5 Hz, 2H), 7.70–7.68 (m, 2H), 7.24 (J=6.5 Hz, 2H), 7.19–7.16 (m, 1H),
5.84 (s, 2H), 1.20 ppm (s, 6H); 13C NMR (125 MHz, CDCl3): d=156.5,
149.7, 139.5, 138.6, 136.9, 128.7, 128.4, 127.6, 122.3, 120.5, 105.9,
13.0 ppm; MS (TOF-ES): m/z calcd for C17H16N2: 248; found: 249
[M+H]+.
5-Amino-2,2’-bipyridine hydrochloride (2): A mixture of 13 (500 mg,
2 mmol), hydroxylamine hydrochloride (1.5 g, 20 mmol), Et3N (0.6 mL),
EtOH (6 mL), and H2O (2.6 mL) was heated at reflux for 24 h. After
being cooled to RT, the mixture was quenched with cold 1n HCl (10 mL)
and washed with iPr2O, then the pH was adjusted to 9–10 by careful addi-
tion of 6n NaOH. The aqueous phase was extracted five times with
CH2Cl2 and the combined organic phases were dried over Na2SO4. After
removal of the solvent, the brown residue was dissolved in MeOH and a
few drops of concd HCl were added to give the product as a white pre-
cipitate. The addition of Et2O resulted in additional precipitation of 2 as
a light-yellow solid (291 mg, 85%). 1H NMR (500 MHz, D2O/DCl): d=
8.67 (d, J=5.5 Hz, 1H), 8.39 (dt, J=1.5, 6.5 Hz, 1H), 8.25 (d, J=8 Hz,
1H), 8.23 (d, J=2.5 Hz, 1H), 8.08 (d, J=9 Hz, 1H), 7.80 (t, J=6 Hz,
1H), 7.55 ppm (dd, J=2.5, 6.5 Hz, 1H); 13C NMR (125 MHz, D2O/DCl):
d=148.3, 145.9, 145.2, 144.3, 137.1, 135.4, 126.9, 126.1, 125.2, 123.4 ppm;
MS (TOF-LD): m/z calcd for C10H9N3: 171; found: 172 [M+H]+.
2-Phenyl-5-aminopyridine dihydrochloride (5): Compound 16 (371 mg,
1 mmol) and 4n HCl (3 mL) in EtOH (10 mL) gave 5 as a white solid
(136 mg, 80%). 1H NMR (500 MHz, D2O/DCl): d=7.93 (d, J=2.5 Hz,
1H), 7.83 (d, J=8.5 Hz, 1H), 7.73 (dd, J=6, 3 Hz, 1H), 7.64–7.62 (m,
2H), 7.56–7.52 ppm (m, 3H); 13C NMR (125 MHz, D2O/DCl): d=146.6,
141.3, 131.9, 131.5, 131.4, 130.3, 127.3, 126.6, 126.3 ppm; MS (TOF-LD):
m/z calcd for C11H10N2: 170; found: 170.79 [M+H]+.
Inclusion complex of 1 with 5 (5@1): Complex 5@1 was prepared by
using the same procedure as described for 2@1. 1H NMR (500 MHz,
D2O/DCl): d=9.17 (d, J=9.5 Hz, 1H), 8.79 (s, 1H), 8.46 (d, J=9 Hz,
1H), 7.28 (d, J=8 Hz, 2H), 6.94 (t, J=7 Hz, 1H), 6.73 (t, J=7.5 Hz,
1H), 5.64 (dd, J=61.5, 15.5 Hz, 12H), 5.49 (s, 12H), 4.27 ppm (t, J=
16 Hz, 12H).
2-Bromo-Boc-5-aminopyridine (17): A solution of 9 (1.73 g, 10 mmol)
and Et3N (2.1 mL, 15 mmol) in CH2Cl2 (50 mL) was stirred for 30 min at
RT. (Boc)2O (2.6 g, 12 mmol) in CH2Cl2 (5 mL) was added and the mix-
ture was stirred overnight at RT, then quenched with aq NH4Cl and ex-
tracted with CH2Cl2. The organic phase was separated and washed with
H2O and brine, dried over MgSO4, and the solvent was removed under
reduced pressure. The residue was purified by flash chromatography
(silica gel, hexane/EtOAc 8:2) to give 17 as a white solid (1.9 g, 70%).
1H NMR (500 MHz, CDCl3): d=8.22 (d, J=3 Hz, 1H), 7.87 (d, J=6 Hz,
1H), 7.39 (d, J=8.5 Hz, 1H), 6.51 (s, NH), 1.49 ppm (s, 9H); MS (TOF-
ES): m/z calcd for C10H13BrN2O2: 272; found: 271 [MÀH]+.
Inclusion complex of 1 with 2 (2@1): A solution of 1 (0.1 mmol) in H2O
(25 mL) was added to a solution of 2 (0.12 mmol) in acidic H2O (25 mL,
pH 3) and stirred at RT for 16 h. The resultant precipitate was removed
by filtration, and the solvent of the filtrate was removed under reduced
pressure. The residue from the filtrate was dissolved in H2O (20 mL),
then acetone (10 mL) was added and the resultant precipitate was
washed with MeOH and Et2O to give complex 2@1 in quantitative yield.
1H NMR (500 MHz, D2O/DCl): d=8.80 (d, J=8.5 Hz, 1H), 8.59 (d, J=
5.5 Hz, 1H), 8.43 (t, J=8 Hz, 1H), 7.76 (t, J=6.5 Hz, 1H), 7.3 (d, J=
3 Hz, 2H), 6.13 (d, J=8.5 Hz, 1H), 5.68 (dd, J=81.5, 15.5 Hz, 12H), 5.46
(s, 12H), 4.21 ppm (dd, J=27, 15.5 Hz, 12H); MS (TOF-ES): m/z calcd
for C46H47Cl2N27O12: 1239; found: 1169 [MÀ2ClÀH]+.
1,4-Phenylene-bis(methylene)bis
N
(18):
NaH (60%, suspended in oil, 400 mg, 9.6 mmol) was added to a stirred
solution of 17 (965 mg, 3.5 mmol) in DMF (10 mL) at 08C, and the mix-
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E. Keinan et al.
ture was stirred at RT for 30 min. 1,4-Bis(bromomethyl)benzene (425 g,
1.6 mmol) was added and the mixture was stirred at RT for 20 h, then
quenched with aq NH4Cl, extracted with EtOAc, washed with brine, and
dried over Na2SO4. Removal of solvent followed by column chromatogra-
phy (silica gel, hexane/EtOAc 8:2) afforded 18 as a light-yellow solid
(1.72 g, 76%). 1H NMR (500 MHz, CDCl3): d=8.16 (s, 2H), 7.36 (d, J=
8.5 Hz, 2H), 7.30 (brs, 2H), 7.13 (s, 4H), 4.80 (s, 4H), 1.38 ppm (s, 18H);
13C NMR (125 MHz, CDCl3): d=154.0, 147.8, 138.5, 138.1, 136.8, 136.2,
127.8, 127.7, 81.9, 53.1, 28.2 ppm; MS (TOF-ES): m/z calcd for
C28H32Br2N4O4: 648; found: 671 [M+Na]+.
under argon. The reaction mixture was heated at reflux for 5 h, then
quenched by slow addition of H2O at 08C and filtered through Celite.
The filtrate was washed with brine, dried over MgSO4, and concentrated
under reduced pressure. Column chromatography (silica gel, hexane/
EtOAc 1:1) afforded 23 as a colorless oil (1.78 g, 78% over two steps).
1H NMR (500 MHz, CDCl3): d=3.94 (td, J=11.5, 3 Hz, 2H), 3.86–3.78
(m, 4H), 3.03 (t, J=5 Hz, OH), 1.93–1.83 (m, 5H), 1.45 (dt, J=13,
3.5 Hz, 1H), 0.86 ppm (t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3):
d=102.0, 59.3, 58.5, 38.0, 37.9, 25.2, 24.1, 7.7 ppm.
2-(2-Ethyl-1,3-dioxan-2-yl)ethyl-1H-imidazole-1-carboxylate (24): Car-
bonyldiimidazole (1.6 g, 10 mmol) was added to a stirred solution of 23
(800 mg, 5 mmol) in CH2Cl2 (20 mL) at 08C and stirred for a further 2 h,
then diluted with CH2Cl2, washed with brine, dried over MgSO4, and con-
centrated under reduced pressure to give crude 24, which was used for
next step without further purification.
1,4-Phenylene-bis-methylene-bis-Boc-5-aminopyridine (19): The synthesis
was performed by using 18 (648 mg, 1 mm), 2-(tributyltin)pyridine (1.1 g,
3 mmol), [Pd
lowing the procedure used for the preparation of 13. Column chromatog-
raphy (silica gel, EtOAc/Et3N 100:2) afforded 19 as white solid
ACHTUNGTRENNUNG(PPh3)4] (46 mg, 0.04 mmol) in toluene (15 mL) and by fol-
a
(496 mg, 77%). 1H NMR (500 MHz, CDCl3): d=8.64 (d, J=4 Hz, 2H),
8.46 (s, 2H), 8.32 (d, J=8 Hz, 2H), 8.30 (d, J=8.5 Hz, 4H), 7.78 (td, J=
6, 1.5 Hz, 2H), 7.55 (d, J=7.5 Hz, 2H), 7.27 (t, J=6 Hz, 2H), 7.17 (s,
4H), 4.86 (s, 4H), 1.42 ppm (s, 18H); 13C NMR (125 MHz, CDCl3): d=
155.6, 154.3, 153.1, 149.1, 146.9, 139.2, 137, 136.9, 134.3, 127.8, 123.5,
121.0, 120.7, 81.4, 53.2, 28.2 ppm; MS (TOF-ES): m/z calcd for
C38H40N6O4: 644; found: 645 [M+H]+.
2-(2-Ethyl-1,3-dioxan-2-yl)ethyl-6-bromopyridin-3-yl carbamate (25):
NaH (60% in mineral oil, 350 mg, 8.7 mmol) was slowly added to a solu-
tion of 24 (815 mg, 3.2 mmol) and 9 (500 mg, 2.9 mmol) in anhydrous
THF (20 mL). The mixture was stirred overnight at RT, then quenched
with aq NH4Cl, extracted with EtOAc, washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. Column chromatogra-
phy (silica gel, hexane/EtOAc 1:1) afforded 25 as a white solid (933 mg,
1
90% over two steps). H NMR (500 MHz, CDCl3): d=8.28 (d, J=2.5 Hz,
N,N’-[1,4-Phenylenebis(methylene)]di-2,2’-bipyridin-5-aminium chloride
(3): Compound 19 (200 mg, 0.31 mmol) was dissolved in EtOH (8 mL)
and 4n HCl (3 mL) and stirred overnight at RT. The solvent was re-
moved under reduced pressure, and the residue was dissolved in small
amount of MeOH. Et2O was added and the resultant yellowish precipi-
tate was collected by filtration and found to be pure 3 (121 mg, 88%).
1H NMR (500 MHz, D2O/DCl): d=8.04 (d, J=5.5 Hz, 2H), 7.83 (td, J=
7.5, 1 Hz, 2H), 7.60 (d, J=3 Hz, 2H), 7.56 (s, 4H), 7.31 (t, J=6.5 Hz,
2H), 7.24 (d, J=8 Hz, 2H), 6.94 (d, J=8.5 Hz, 2H), 6.75 (dd, J=2.5,
6 Hz, 2H), 4.37 ppm (s, 4H); 13C NMR (125 MHz, D2O/DCl): d=147.6,
147.5, 145.1, 143.2, 139.0, 134.2, 132.9, 129.6, 125.1, 124.2, 121.6, 121.4,
45.9 ppm; MS (TOF-LD): m/z calcd for C28H24N6: 444; found: 443
[MÀH]+.
1H), 7.88 (brs, 1H), 7.40 (d, J=9 Hz, 1H), 6.87 (s, NH), 4.34 (t, J=7 Hz,
2H), 3.93–3.82 (m, 4H), 2.06 (t, J=7.5 Hz, 2H), 1.83–1.72 (m, 3H), 1.57–
1.53 (m, H), 0.90 ppm (t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3):
d=153.4, 140.2, 134.9, 134.6, 128.5, 128, 99.6, 62.0, 59.4, 34.0, 25.9, 25.2,
7.6 ppm; MS (TOF-ES): m/z calcd for C14H19N2O4Br: 358/360 (1:1 ratio
79Br/81Br); found: 359/361 [M+H]+.
2-(2-Ethyl-1,3-dioxan-2-yl)ethyl 2,2’-bipyridin-5-yl carbamate (26): This
compound was prepared by using 25 (425 mg, 1.2 mmol), 2-(tributyltin)-
pyridine (480 mg, 1.3 mmol), and [PdACHTNUGTRNEUNG(PPh3)4] (30 mg, 0.024 mmol) in tol-
uene (10 mL) at 1208C and by following the above-described procedure
for the preparation of 13. The crude product was purified by column
chromatography (silica gel, hexane/EtOAc 1:1) to afford 26 as a yellow-
ish solid (279 mg, 65%). 1H NMR (500 MHz, CDCl3): d=8.64 (d, J=
4.5 Hz, 1H), 8.56 (d, J=2 Hz, 1H), 8.35 (d, J=9 Hz, 1H), 8.32 (d, J=
8 Hz, 1H), 8.07 (brd, J=6 Hz, 1H), 7.78 (t, J=8 Hz, 1H), 7.26 (t, J=
6 Hz, 1H), 6.92 (s, 1H), 4.35 (t, J=7.5 Hz, 2H), 3.93–3.84 (m, 4H), 2.09
(t, J=7.5 Hz, 2H), 1.84–1.75 (m, 3H), 1.60–1.55 (m, 1H), 0.91 ppm (t,
J=7.5 Hz, 1H); 13C NMR (125 MHz, CDCl3): d=155.8, 153.5, 151.1,
149.1, 139.4, 136.8, 135.0, 126.2, 123.2, 121.3, 120.6, 99.6, 61.8, 59.4, 33.8,
26.1, 25.1, 7.6 ppm; MS (TOF-ES): m/z calcd for C19H23N3O4: 357; found:
358 [M+H]+ and 356 [MÀH]+.
3-Oxopentyl-2,2’-bipyridin-5-yl carbamate (21): A solution of 26 (60 mg,
0.17 mmol) in AcOH/H2O (1:9, 10 mL) was stirred overnight at RT. The
mixture was quenched with aq NaHCO3, extracted with CH2Cl2, washed
with brine, dried over MgSO4 and concentrated to give 21 as a white
solid (48 mg, 95%). 1H NMR (500 MHz, CDCl3): d=8.64 (d, J=4 Hz,
1H), 8.55 (s, 1H), 8.35 (d, J=8.5 Hz, 1H), 8.30 (d, J=8 Hz, 1H), 8.05
(brs, 1H), 7.78 (t, J=8 Hz, 1H), 7.28–7.26 (m, 1H), 6.93 (s, 1H), 4.49 (t,
J=6 Hz, 1H), 2.82 (t, J=6 Hz, 1H), 2.49 (q, J=7.5, 7 Hz, 1H), 1.09 ppm
(t, J=7.5 Hz, 1H); 13C NMR (125 MHz, CDCl3): d=208.3, 155.7, 153.1,
151.3, 149.1, 139.4, 136.9, 134.7, 126.3, 123.3, 121.3, 120.6, 60.5, 41.2, 36.3,
7.5 ppm; MS (TOF-ES): m/z calcd for C16H17N3O3: 299; found: 300
[M+H]+.
Inclusion complex of 1 with 3 (3@1): Complex 3@1 was prepared by
using the above-described procedure for the preparation of 2@1.
1H NMR (500 MHz, D2O/DCl): d=8.51–6.0 (m, 14H), 5.80–5.54 (m,
24H), 5.45–5.42 (brm, 24H), 4.27–4.14 ppm (m, 28H); MS (TOF-LD):
m/z calcd for
[MÀ4HCl+2K]2+
C
100H100Cl4N54O24
:
2584; found: 1257, 1258:
.
N-(2,2’-Bipyridin-5-yl)-2-phenylacetamide (20): NaH (18 mg, 0.45 mmol,
60% in mineral oil) was added to a solution of 2 (52 mg, 0.3 mmol) in
THF (5 mL), and the mixture was stirred for 30 min at RT. A solution of
benzoyl chloride (52 mg, 0.33 mmol) in THF (1 mL) was added, and the
mixture was stirred for 5 h at RT, then quenched with aq NH4Cl and
THF. The solvent was concentrated under reduced pressure, EtOAc was
added, and the organic layer was separated. The aqueous phase was
washed twice with EtOAc and the combined organic extracts were
washed with brine, dried over Na2SO4, and concentrated under reduced
pressure. Flash chromatography (silica gel, hexane/EtOAc 1:1) afforded
20 as a colorless solid (83 mg, 95%). 1H NMR (500 MHz, CDCl3): d=
8.64 (d, J=5 Hz, 1H), 8.58 (d, J=2.5 Hz, 1H), 8.32 (d, J=8.5 Hz, 1H),
8.29 (d, J=8 Hz, 1H), 8.14 (dd, J=6, 2.5 Hz, 1H), 7.77 (td, J=6, 2 Hz,
1H), 7.42–7.33 (m, 5H), 7.27 (ddd, J=2, 3.5, 1 Hz, 1H), 3.78 ppm (s,
2H); 13C NMR (125 MHz, CDCl3): d=169.5, 155.6, 151.9, 149.1, 140.3,
136.9, 134.6, 133.9, 129.5, 129.3, 127.9, 127.6, 123.4, 121.2, 120.7,
44.7 ppm; MS (TOF-ES): m/z calcd for C18H15N3O: 289; found: 288
[MÀH]+.
2-(2-Ethyl-1,3-dioxan-2-yl)ethanol (23): A mixture of 22 (2 g, 13.9 mmol),
1,3-propandiol (1.3 g, 17 mmol), and p-TsOH (265 mg, 1.4 mmol) in ben-
zene (30 mL) was heated at reflux under azeotropic distillation for 20 h,
then quenched with aq NaHCO3 and extracted with EtOAc. The com-
bined organic extracts were washed with NaHCO3 (3ꢂ), H2O (3ꢂ), and
then with brine. The mixture was dried over MgSO4, concentrated under
reduced pressure, and used in the next step without further purification.
LiAlH4 (725 mg, 18.6 mmol) was slowly added to a solution of the above
crude mixture (2.5 g, 12.4 mmol) in anhydrous Et2O (50 mL) at 08C
Acknowledgements
This study was supported by the Israel Science Foundation, the German-
Israeli Project Cooperation (DIP), the Institute of Catalysis Science and
Technology, Technion, and the Skaggs Institute for Chemical Biology.
E.K. is the incumbent of the Benno Gitter & Ilana Ben-Ami Chair of
Biotechnology, Technion. G.P. thanks the Schulich scholarship for a grad-
uate school fellowship. A.K. was supported in part at the Technion by a
fellowship from the Israel Council for Higher Education.
9066
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 9056 – 9067

Cucurbituril–Bipyridine Beacons
FULL PAPER
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Received: November 7, 2009
Revised: March 25, 2010
Published online: June 22, 2010
Chem. Eur. J. 2010, 16, 9056 – 9067
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9067