A fluorescent sensor for 2,3-bisphosphoglycerate using a europium tetra-N-oxide bis-bipyridine complex for both binding and signaling purposes

Article abstract of DOI:10.1002/chem.200390003

Host 1 was designed and synthesized as a fluorescent sensor for 2,3-bisphosphoglycerate (BPG, 3). The design features a tris-functionalized triethylbenzene core to preorganize binding groups. The three cationic moieties, a tetra-N-oxide bipyridine-europiu

Full text of DOI:10.1002/chem.200390003

FULL PAPER
A Fluorescent Sensor for 2,3-Bisphosphoglycerate Using a
Europium Tetra-N-oxide Bis-bipyridine Complex for
Both Binding and Signaling Purposes
Michael D. Best and Eric V. Anslyn*[a]
Abstract: Host 1 was designed and
synthesized as a fluorescent sensor for
2,3-bisphosphoglycerate (BPG, 3). The
design features a tris-functionalized trie-
thylbenzene core to preorganize binding
groups. The three cationic moieties, a
europium complex was used to signal
binding of the guest through modifica-
tion of the charge transfer emission. A
1:1 complex with BPG was determined
in 50% methanol/acetonitrile with a Ka
of 6.7 Â 105 molÀ1 by monitoring the
reduction of the fluorescence signal
upon guest addition. In the titration of
related glycolytic intermediates lacking
a second phosphate (4 6) into host 1,
2:1 host to guest binding was observed.
Similarly, control compound 2, which
lacks the ammonium groups, binds BPG
and 4 6 in a 2:1 fashion. Also, phenyl-
phosphate 7 binds to host 1 in a 1:1
stoichiometry with a Ka over three times
less than 3.
tetra-N-oxide
bipyridine europium
complex and two ammonium groups,
were included to complement the three
anionic functionalities on the guest.
Beyond acting as a binding site, the
Keywords: bipyridines ¥ europium
¥ host guest systems ¥ lanthanide ¥
receptors ¥ sensors
for BPG, we sought to benefit from the sensitivity of
fluorescent measurements, and to eventually develop a
resin-bound sensor for incorporation into a multiple compo-
nent sensing ensemble for the analysis of complex mixtures.[8]
Complexes between europium and bipyridine-derived
macrocyclic structures have been the subject of extensive
investigation, particularly due to their photophysical proper-
ties.[9] The excitation of the complex leads to multiple charge
Introduction
2,3-Bisphosphoglycerate (BPG, 3) is a glycolytic intermediate
formed in the interconversion between 3-phosphoglycerate
(3-PG, 4) and 2-phosphoglycerate (2-PG, 5) by phosphogly-
cerate mutase.[1] BPG plays an important role in the
regulation of oxygen transport by binding to hemoglobin
and decreasing its affinity for oxygen.[2] As a result, inherited
diseases exist in which abnor-
mal concentrations of BPG
lead to altered levels of oxygen
N
N
N
transport.[3] Also, fetal hemo-
globin has been found to ex-
hibit higher oxygen affinity
than adult hemoglobin due to
a lower binding affinity to-
wards BPG.[4] This has been
attributed to a different N-ter-
minal sequence on the b-chain
of hemoglobin F.[5] BPG has
O
O
O
N
N
N
N
O
O
O
O
EuIII(OAc)3
O
N
O
EuIII(OAc)3
O
N
OAc
N
NH3
NH
O
OAc
NH3
OMe
O
1
2
2-
OPO3
2-
2-
OH
OPO3
OPO3
OPO3
OH
2-
been
previously
detected
2-
OPO3
2-
OPO3
OOC
OOC
through the use of HPLC[6]
and enzymatic assays.[7] In de-
veloping a fluorescent sensor
OOC
OOC
3
4
5
6
7
transfer emission signals with line-like properties. However,
the fluorescence of the complex is often unstable in aqueous
solution due to non-radiative decay into vibrational states of
ligated waters. As a result, much work has focused on the
development of cage-like bipyridine-based ligands to shield
[a] Prof. E. V. Anslyn, M. D. Best
The University of Texas at Austin
Austin, TX 78712-1167 (USA)
Fax : (‡1)512-471-7791
E-mail: anslyn@ccwf.cc.utexas.edu
Chem. Eur. J. 2003, 9, No. 1
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51

E. V. Anslyn and M. D. Best
FULL PAPER
the europium from solvent and thus
increase emission intensity.[10] Due to
the increased affinity of N-oxidized
bipyridine ligands towards europi-
um[11], macrocyclic ligands for euro-
pium have also been developed con-
taining these subunits.[12] Furthermore,
O
S
O
N
N
N
N
NaHN
N
N
N
N
N
N
1) 90 % H2SO4
2) NH4OH
N
O S O
DMF
H
N
Cl
polypyridine europium
complexes
10
8
9
and other derivatives of lanthanides
have been used for the fluorescent
detection of anions and other analy-
tes.[13]
72 %
95 %
O
N
N
N
N
O
N
N
N
O
O
N
The core design of host 1 is based on
the tris-functionalized triethylbenzene
scaffold. Due to steric repulsions be-
tween adjacent benzylic substituents,
this structure preferably adopts a stag-
gered conformation, placing the three
binding groups towards the same face
of the benzene ring, effectively forming
a binding cavity.[14] We have used this
11
CH3OH
KOH
N
N
O
DCC, HOBT
CH2Cl2
O
O
O
O
O
12
97 %
13
95 %
Scheme 1. Attachment of bipyridines to the succinate linker.
scaffold to develop receptors for the detection of citrate[15]
,
the tosyl group was removed with sulfuric acid in the
formation of 10. Following this, monomethylsuccinate (11)
was coupled to the free amine of 10, yielding 12. Here,
succinate was installed as a linker to increase the flexibility of
the bipyridine ligands by distancing them from the relatively
bulky triethylbenzene core. Succinate was specifically used as
a linker such that all nitrogens were incorporated within
amides to avoid their oxidation upon bipyridine N-oxidation
later in the synthesis. Next, the methyl ester of 11 was
hydrolyzed to afford 13.
tartrate,[16] and gallate-like molecules[17] for the analysis of
sports drinks, grape-derived beverages, and scotch whiskies,
respectively. Other applications of this scaffold have been
recently reviewed.[18] For host 1, a tetra-N-oxide bipyridine
group was included to form a stable complex with europium.
The europium and ammonium groups were used to provide
three cationic groups for binding of the three complementary
anionic groups on the BPG.
In the preparation of the triethylbenzene scaffold towards
host 1, two of the amines of tris(aminomethyl) structure 14[15]
were protected with tert-butyloxycarbonyl (Boc) groups,
yielding 15 (Scheme 2). This was then coupled to carboxylate
13 using a standard peptide coupling protocol in the formation
of 16. Next, the bipyridines were oxidized using m-chloro-
Results and Discussion
Synthesis:The synthetic scheme for host 1 begins with
6-chloromethyl-2,2'-bipyridine (8, Scheme 1).[19] This was
allowed to react with sodium tosylamide leading to 9.[20] Next,
N
N
N
O
O
N
DCC
NH2
NH2
NHBoc
NHBoc
O
O
O
H2N
H2N
HOBT
O
N
CH2Cl2
CH2Cl2
NHBco
NHBoc
13
NH
O
15
14
16
23 %
97 %
N
N
N
N
N
O
O
O
O
O
N
N
O
O
O
N
EuIII(OAc)3
mCPBA
TFA
O
O
1
N
N
CH2Cl2
CH2Cl2
OAc
OAc
NHBoc
NHBoc
NH3
NH3
NH
NH
O
O
18
95 %
17
69 %
Scheme 2. Synthesis of host 1.
52
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Chem. Eur. J. 2003, 9, No. 1

A Fluorescent Sensor
51 57
perbenzoic acid to afford compound 17. Finally, deprotection
of the Boc groups with trifluoroacetic acid followed by anion-
exchange chromatography yielded ligand 18. Europium com-
plex 1 was then obtained by reaction of 18 with europium
yielded a Ka of 6.7 Â 105 molÀ1 (Æ 10%). A representative
example of the fluorescence change upon BPG addition,
along with the corresponding binding isotherm and 1:1 curve
fit are illustrated in Figures 1 and 2, respectively.
‡
acetate hydrate and was characterized by the [1 À OAc]
peaks at m/z 1017 and 1019 in the ESI-MS. Control compound
2, which lacks the triethylbenzene scaffold and ammonium
groups of 1, was synthesized using the same strategy. Here, 12
was oxidized to 19, which was then metallated to obtain model
compound 2 (Scheme 3).
N
N
N
O
O
O
O
EuIII(OAc)3
N
mCPBA
12
2
O
CH2Cl2
N
OMe
O
19
Figure 1. Fluorescence modulation upon introduction of BPG to 1.
Scheme 3. Synthesis of model 2.
Photophysical studies: The absorbance spectra of host 1
exhibited a lmax of 260 nm for the complex. Upon exciting at
this wavelength in methanolic solution, a fluorescence spectra
was obtained which agreed with previous reports of the
photophysical properties of similar bipyridine-N-oxide euro-
pium complexes.[12] This spectra features six line-like emission
peaks at 577, 590, the largest peak at 610, 650, 690 and 699 nm.
When the solvent was changed to greater than 5% water/
methanol solutions, though, the fluorescence signal was
completely quenched. However, with less than 5% water in
the solvent, we were able to obtain reproducible results in the
titration experiments.
Upon addition of 2,3-bisphosphoglycerate (3) to host 1 in
methanol, a decrease in the fluorescent signals of the complex
was observed. This was repeated with small percentages of
water to ensure that hydrates on the guests were not the cause
of the emission changes. In the titrations of 1 with BPG in
methanol, the inflection point of mole ratio plots were at
0.5 equivalents of added guest and the data could not be fit to
a 1:1 binding isotherm. This indicated that the host guest
complex which was formed in this solvent system consists of
two hosts bound to one guest. Our hypothesis was that this
stoichiometry might occur because the ammoniums of host 1
were not involved in complexation, and thus BPG binds the
europiums of two separate hosts. As a result, we decided to
decrease the polarity of the solvent to minimize the competi-
tion for hydrogen bonding and electrostatic interactions and
thus drive potential interactions between BPG and the
ammoniums of 1.
Upon changing to 50% or less methanol in acetonitrile, the
fluorescence decrease upon BPG addition to 1 fit a 1:1
binding curve. Also, in these solvents, the inflection of mol
ratio plots consistently occurred at one equivalent of added
guest, illustrating a discrete change in the binding mode. To
obtain a 1-BPG binding constant in 50% methanol/acetoni-
trile, several titrations were performed, the average of which
Figure 2. Binding isotherm and curve fit for 1-BPG.
In addition to this fluorescence change, a very small shift in
the excitation spectra of host 1 was observed upon addition of
3 (Figure 3). The presence of an isosbestic point in this shift
was further evidence for the formation of a 1:1 complex in this
solvent system. We also titrated BPG into control compound 2
in this solvent system, in which 2:1 host to guest binding was
observed. This result indicates the importance of the ammo-
nium groups of host 1 in the 1:1 complexation of BPG under
these conditions as the removal of these groups causes the
system to undergo this change in binding mode. Based on
these results, a suggested binding mode for BPG in the
complex (20) is presented in Scheme 4.
Next, we attempted titrations of other glycolysis intermedi-
ates: 3-phosphoglycerate (3-PG, 4), 2-phosphoglycerate (2-
PG, 5), and phosphoenolpyruvate (PEP, 6). Titrations of
solutions of each of these compounds into solutions of 1 and 2
Chem. Eur. J. 2003, 9, No. 1
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E. V. Anslyn and M. D. Best
FULL PAPER
Table 1. Host guest and model guest titration results in 50% MeOH/
MeCN.
Guest
Ka with host 1 (Â105 mÀ1
)
Result with host 2
[b]
[b]
[b]
[b]
[d]
[d]
2,3-BPG (3)
3-PG (4)
2-PG (5)
PEP (6)
phenylphosphate (7)
acetate
6.7[a]
[b]
[b]
×[b]
2.0[a]
[c]
[a] Æ 15%. [b] Undergoes 2 to 1 host-to-guest binding. [c] No change in
emission spectra observed. [d] Not attempted.
niums to model 2 leads to 2:1 host to guest binding. This shows
that the ammoniums of 1 play an important role in the binding
of BPG in a 1:1 stoichiometry. Related glycolytic intermedi-
ates which lack the second phosphate undergo 2:1 host to
guest binding, similar to 2-BPG, most likely because they do
not interact with the ammoniums of 1. Phenylphosphate, with
only one phosphate binding
Figure 3. Shift in absorbance spectra of 1 with BPG addition.
site, exhibits decreased affinity
to the host, again probably due
to the lack of interaction with
N
N
N
N
N
N
O
P
O
O
O
O
O
P
O
O
O
O
O
N
N
O
O
O
O
the ammoniums of 1. Thus,
BPG showed the highest affin-
ity towards 1 and was the only
guest of the glycolytic inter-
mediates to undergo 1:1 bind-
ing. However, problems with
the host include the inability
to detect binding in > 5%
aqueous solutions due to
Eu3+
O
3+Eu
O
O
O
O
O
O
O
O
P
O
N
N
O
O
O
H
P
O
O
NH3
NH3
NH2
3
NH
NH
H
NH2
O
O
1
20
Scheme 4. Hypothetical binding of BPG to host 1 based on titration evidence.
in solvent systems ranging from 10% methanol in acetonitrile
to 100% methanol were performed. In all of these experi-
ments, the inflection points in mol ratio plots pointed to the
association of two hosts to one guest molecule. These results
suggest that the extra phosphate on BPG is instrumental in
forming a 1:1 complex with host 1, the only guest from this
group which forms a 1:1 complex. Any potential selectivity for
BPG over 4 through 6 could not be directly quantified,
though, due to the change in binding mode of the analytes.
The titration of phenylphosphate (7) was also performed.
The data involving the change in emission upon introduction
of 7 to host 1 in 50% methanol/acetonitrile could be fit to a 1:1
binding curve as was anticipated due to the presence of only
one phosphate binding group. The Ka of this association was
determined to be 2.0 Â 105 molÀ1 (Æ 10%), a little more than
three times less than that of the 1-BPG complex. These data
suggest a strong interaction of phosphates with the metal center
of 1, with only a small increase in affinity for BPG due to
interactions with the ammonium groups. Results from the titra-
tions of each guest in 50% methanol in acetonitrile are summa-
rized in Table 1. Therefore the ammoniums in1 influence the
binding stoichiometry but hardly influence the binding affinity.
quenching, and an inability to dramatically increase the
affinity for BPG by changing the binding site functional
groups.
Currently, we are attempting to develop other techniques
for the detection of 1-BPG binding in water, circumventing
the quenching of the europium fluorescence in this solvent.
Experimental Section
General: Methanol was heated under reflux over magnesium, tetrahydro-
fuan over sodium and benzophenone, and triethylamine and dichloro-
methane over calcium hydride, and distilled when noted. NMR tubes were
dried in an oven at 1258C for at least 24 hours prior to use. Products were
placed under high vacuum overnight before spectra and masses were
obtained. Starting materials were generally purchased from Aldrich. 1H
and 13C NMR spectra were collected at 300 and 75 MHz, respectively, on a
Varian Unity Plus spectrometer. High resolution mass spectra were
recorded on a Finnigan VG analytical ZAB2-E spectrometer.
N,N-Bis-(6-(2,2'-bipyridyl)methyl)-N-tosylamine (9): 6-Chloromethyl-2,2'-
bipyridine (8, 2.29 g, 11.1 mmol) was dissolved in N,N'-dimethylformamide
(75 mL) in a flame dried 250 mL round-bottomed flask. Sodium tosylamide
(1.08 g, 5.57 mmol) and potassium carbonate (2.31 g, 16.7 mmol) were then
added and the reaction heated to 1008C for 6 h. The solvent was removed
through rotary evaporation and high vacuum pumping. Next, the crude was
dissolved in dichloromethane (100 mL) and washed with water (3 Â
100 mL). The organic layer was then dried with magnesium sulfate, filtered
and the solvent removed by rotary evaporation and a high vacuum pump.
The crude material was purified using column chromatography (silica gel,
70% ethyl acetate/hexanes). This yielded 9 as a white solid (2.04 g, 72%).
M.p. 112 1148C; 1H NMR (300 MHz, CDCl3): d ˆ 8.56 (d, 2H, J ˆ
4.5 Hz), 8.10 (m, 4H), 7.62 (m, 6H), 7.31 (d, 2H, J ˆ 7.8 Hz), 7.18 (m,
Conclusion
Host 1 forms a 1:1 complex with 2,3-bisphosphoglycerate in
50% methanol/acetonitrile, while the removal of the ammo-
54
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A Fluorescent Sensor
51 57
1
2H), 7.06 (d, 2H, J ˆ 8.1 Hz), 4.73 (s, 4H), 2.19 (s, 3H); 13C NMR (75 MHz,
CDCl3): d ˆ 155.24, 155.09, 154.74, 148.43, 142.62, 136.85, 136.56, 136.06,
(3.52 g, 40%). M.p. 142 1448C; H NMR (300 MHz, CDCl3) (free based
compound): d ˆ 4.48 (s, 2H), 4.26 (s, 4H), 3.79 (s, 2H), 2.68 (q, 6H, J ˆ
7.5 Hz), 1.37 (s, 18H), 1.13 (t, 9H, J ˆ 7.5 Hz); 13C NMR (75 MHz, CDCl3):
d ˆ 155.16, 142.38, 142.28, 136.91, 131.96, 78.97, 39.09, 38.49, 28.11, 22.55,
‡
128.97, 126.63, 123.11, 122.09, 120.52, 118.96, 53.23, 20.79; HRMS-CI : m/z:
calcd for C29H26N5O2S: 508.1807; found: 508.1808.
‡
22.40, 16.36, 16.23; HRMS-CI : m/z: calcd for C25H44N3O4: 450.3332;
N,N-Bis-(6-(2,2'-bipyridyl)methyl)amine (10): Compound
9
(1.54 g,
found: 450.3332.
3.0 mmol) was dissolved in 90% sulfuric acid (40 mL) in a 250 mL
round-bottom flask. This was heated to 1208C for 2 h under a condenser.
After cooling to room temperature, the reaction was brought to pH 10
through addition of 1m sodium hydroxide, followed by concentrated
ammonium hydroxide to yield a white precipitate. The solution was
extracted with dichloromethane (3 Â 200 mL), after which the extracts
were combined, dried with magnesium sulfate, filtered and the solvent
removed by rotary evaporation and a high vacuum pump. This yielded 10 as
a brown oil (1.02 g, 95%). 1H NMR (300 MHz, CDCl3): d ˆ 8.65 (d, 2H,
J ˆ 4.8 Hz), 8.46 (d, 2H, J ˆ 8.1 Hz), 8.25 (d, 2H, J ˆ 7.8 Hz), 7.87 7.69 (m,
4H), 7.35 (d, 2H, J ˆ 7.5 Hz), 7.27 (t, 2H, J ˆ 6.3 Hz), 4.09 (s, 4H), 3.22 (s,
1H); 13C NMR (300 MHz, CDCl3): d ˆ 158.81, 155.96, 155.23, 148.82,
1-[(3-(N,N-Bis-(6-(2,2'-bipyridyl)methyl)carbamoyl)propamoyl)amino-
methyl]-3,5-bis[N-((tert-butoxy)carbonyl)aminomethyl]-2,4,6-triethylben-
zene (16): Compound 15 was free based by addition of dichloromethane
and concentrated ammonium hydroxide, extraction of the dichlorome-
thane, washing of the aqueous layer, combination of the organic layers,
drying and solvent removal. After placement on the high vacuum pump
overnight, this compound (483 mg, 1.08 mmol) was dissolved in distilled
dichloromethane (20 mL) in a 100 mL round-bottomed flask. The flask was
then cooled to 08C and placed under argon. To this was added 13 (493 mg,
1.08 mmol), dicyclohexylcarbodiimide (270 mg, 1.29 mmol) and hydroxy-
benzotriazole (177 mg, 1.29 mmol). The reaction was then warmed to room
temperature and stirred for 14 hours. Solvent removal yielded a brown oil,
which was purified by column chromatography over silica gel with gradient
elution from 0.5 to 1.5% methanol/dichloromethane. This yielded 16 as a
yellow solid (965 mg, 97%). M.p. 108 1128C; 1H NMR (300 MHz,
CDCl3): d ˆ 8.55 (d, 2H, J ˆ 4.8 Hz), 8.29 8.13 (m, 4H), 7.75 7.61 (m,
4H), 7.25 7.14 (m, 4H), 6.25 (s, 1H), 4.83 (s, 2H), 4.76 (s, 2H), 4.62 (s, 2H),
4.33 (s, 2H), 4.21 (s, 4H), 2.97 (t, 2H, J ˆ 6.0 Hz), 2.69 2.55 (m, 8H), 1.08
(s, 18H), 1.05 1.03 (m, 9H); 13C NMR (300 MHz, CDCl3): d ˆ 173.06,
171.95, 157.11, 156.42, 155.87, 155.63, 155.40, 155.23, 148.85, 143.49, 137.64,
137.41, 136.85, 136.68, 132.06, 131.67, 123.68, 123.50, 121.96, 121.11, 121.03,
120.99, 119.66, 119.39, 79.19, 33.65, 31.26, 29.44, 28.92, 53.27, 53.03, 51.48,
‡
137.03, 136.48, 123.33, 121.99, 120.92, 118.94, 54.40; HRMS-CI : m/z: calcd
for C22H20N5: 354.1719; found: 354.1712.
Methyl-3-[N,N-bis-(6-(2,2'-bipyridyl)methyl)carbamoyl] propanoate (12):
Dicyclohexylcarbodiimide (527 mg, 2.55 mmol), hydroxybenzotriazole
(345 mg, 2.55 mmol) and monomethyl succinate (337 mg, 2.55 mmol) were
dissolved in 5 mL of distilled dichloromethane in a flame dried 100 mL
round-bottom flask. This was allowed to stir at 08C under an argon balloon
for 30 minutes. Compound 11 (752 mg, 2.13 mmol) was separately dis-
solved in 20 mL of distilled dichloromethane and then added to the
reaction flask. The reaction mixture was allowed to stir at 08C for
30 minutes and then at room temperature for 6 h. Next, the crude material
was directly added to a silica gel column and purified using gradient elution
from 1 to 3% methanol/dichloromethane. This afforded 12 as a white solid
(963 mg, 97%). M.p. 88 908C; 1H NMR (300 MHz, CDCl3): d ˆ 8.59 (t,
2H, J ˆ 4.2 Hz), 8.18 8.36 (m, 4H), 7.71 (m, 4H), 7.30 7.17 (m, 4H), 4.78
(s, 2H), 4.76 (s, 2H), 3.53 (s, 3H), 2.79 2.62 (m, 4H); 13C NMR (300 MHz,
CDCl3): d ˆ 173.35, 172.28, 156.56, 155.92, 155.82, 155.61, 155.49, 155.21,
148.92, 148.87, 137.59, 137.40, 136.76, 136.57, 123.68, 123.43, 122.18, 121.00,
‡
38.53, 37.84, 28.20, 22.63, 16.17; HRMS-CI : m/z: calcd for C51H65N8O6:
885.5027; found: 885.5028.
1-[(3-(N,N-Bis-(6-(2,2'-bipyridyl-1,1'-dioxide)methyl)carbamoyl)propa-
moyl)aminomethyl]-3,5-bis-[N-((tert-butoxy)carbonyl)aminomethyl]-
2,4,6-triethylbenzene (17): Compound 16 (167 mg, 188 mmol) was dissolved
in chloroform (2 mL) in a 50 mL round-bottomed flask, which was then
placed at 08C. m-Chloroperbenzoic acid (233 mg, 943 mmol) was dissolved
in chloroform (3 mL) and added to the reaction mixture. The reaction flask
was then brought to room temperature with stirring for 3 h. A second
portion of mCPBA (233 mg, 43 mmol) was dissolved in chloroform (3 mL)
and added to the reaction, followed by stirring for 17 more hours. Following
solvent removal by rotary evaporation, the crude mixture was purified
through column chromatography over silica gel. Here, methanol (1 L) was
passed through the column to elute all biproducts. Then, ammonia
saturated methanol (1 L) was used to remove 17 (123 mg, 69%). M.p.
‡
120.91, 119.52, 119.28, 52.99, 51.63, 51.53, 29.07, 28.08; HRMS-CI : m/z:
calcd for C27H26N5O3: 468.2036; found: 468.2032.
3-[N,N-Bis-(6-(2,2'-bipyridyl)methyl)carbamoyl] propanoate (13): Com-
pound 12 (765 mg, 1.64 mmol) was dissolved in methanol (25 mL) and 1m
KOH (25 mL) in a 250 mL round-bottomed flask. The reaction was heated
to 908C for 5 h. The solvent was then removed through rotary evaporation
and a high vacuum pump to afford an orange oil. The crude material was
then dissolved in chloroform, dried with magnesium sulfate, filtered and
the solvent removed. Purification was performed using column chroma-
tography on silica gel with 20% ammonia sat. methanol/dichloromethane
as eluant, yielding 13 as a brown solid (740 mg, 95%). M.p. 66 688C;
1H NMR (300 MHz, CDCl3): d ˆ 8.48 (s, 2H), 8.21 (d, 1H, J ˆ 8.1 Hz), 8.06
(t, 2H, J ˆ 8.1 Hz), 7.93 (d, 2H, J ˆ 8.1 Hz), 7.63 7.47 (m, 4H), 7.09 7.07
(m, 4H), 4.67 (s, 2H), 4.62 (s, 2H), 2.68 (s, 2H), 2.43 (s, 2H); 13C NMR
(300 MHz, CDCl3): d ˆ 177.55, 173.83, 156.59, 155.90, 155.80, 155.57, 155.23,
149.00, 148.96, 137.77, 137.64, 137.05, 136.86, 123.83, 123.60, 122.39, 121.24,
1
173 1758C; H NMR (300 MHz, CDCl3): d ˆ 8.26 (s, 2H), 7.50 7.30 (m,
12H), 5.87 (s, 1H), 4.99 (s, 2H), 4.86 (s, 2H), 4.54 (s, 2H), 4.40 (s, 2H), 4.31
(s, 4H), 2.87 (t, 2H, J ˆ 6.0 Hz), 2.64 (m, 6H), 2.48 (t, 2H, J ˆ 6.0 Hz), 1.39
(s, 18H), 1.11 (m, 9H); 13C NMR (300 MHz, CDCl3): d ˆ 173.56, 171.59,
155.28, 147.56, 146.84, 143.67, 142.83, 142.74, 142.52, 142.39, 139.81, 132.32,
131.88, 128.12, 127.03, 126.57, 126.41, 126.28, 125.21, 125.02, 124.95, 124.83,
48.52, 45.76, 38.64, 37.96, 30.73, 30.19, 28.29, 25.03, 22.78, 16.34; HRMS-
‡
CI : m/z: calcd for C51H65N8O10: 949.4824; found: 949.4827.
‡
121.18, 119.71, 119.53, 53.16, 51.69, 50.54, 30.68, 28.78; HRMS-CI : m/z:
1-[(3-(N,N-Bis-(6-(2,2'-bipyridyl-1,1'-dioxide)methyl)carbamoyl)propa-
moyl)aminomethyl]-3,5-bis(aminomethyl)-2,4,6-triethylbenzene bis(acetic
acid) salt (18): Compound 17 (140 mg, 148 mmol) was dissolved in distilled
dichloromethane (2 mL) and trifluoroacetic acid (2 mL). The reaction was
stirred for 3 h, at which time solvent removal yielded an orange oil. The
crude was then run down an Amberlite IRA-400 (ClÀ) anion exchange
column which had been treated with ammonium acetate. The resulting salt
was lyophilized and then redissolved in water and lyophilized twice more to
yield 18 as a brown solid (122 mg, 95%). M.p. >2008C; 1H NMR
(300 MHz, CD3OD): d ˆ 8.36 (s, 2H), 7.62 7.48 (m, 13H), 4.97 (s, 2H), 4.74
(s, 2H), 4.35 (s, 2H), 3.98 (s, 4H), 3.21 (s, 4H), 2.91 2.44 (m, 6H), 1.80 (s,
6H), 1.07 (m, 9H); 13C NMR (300 MHz, CDCl3): d ˆ 176.07, 174.37, 161.35,
160.83, 160.31, 159.79, 150.52, 149.35, 147.56, 145.95, 144.43, 144.25, 143.92,
141.73, 141.56, 134.36, 133.90, 132.81, 131.68, 130.97, 130.64, 129.68,
129.56, 129.04, 127.84, 122.52, 118.72, 114.92, 111.11, 38.59, 37.84, 31.35,
calcd for C26H24N5O3: 454.1880; found: 454.1871.
1,3-Bis[N-((tert-butoxy)carbonyl)aminomethyl]-5-(aminomethyl)-2,4,6-
triethylbenzene (15): 1,3,5-Tris(aminomethyl)-2,4,6-triethylbenzene (14,
8.85 g, 35.5 mmol) was dissolved in distilled dichloromethane (300 mL) in
a 1 L round-bottomed flask and was placed under nitrogen. Boc2O (8.53 g,
39.1 mmol) was dissolved in distilled dichloromethane (150 mL) and placed
under nitrogen in an addition funnel on the reaction flask. This solution was
dropped into the reaction slowly over 2 h. Upon addition, the solution
turned cloudy. The solvent was removed by rotary evaporation after 21 h,
and the resulting milky white solid was purified by column chromatography
(silica gel, gradiant elution from 1% to 30% ammonia sat. methanol/
dichloromethane). TLC analysis of the fractions was performed using
ninhydrin spray, which stained the otherwise unidentifiable spots of the
amine products. The first band yielded the tris-Boc protected compound as
a white solid after evaporation (1.47 g, 8%). The next band was the bis-Boc
protected amine (15), a white fluffy solid (3.62 g, 23%), and the third band
afforded the mono-Boc protected product also as a white, fluffy solid
(2.65 g, 21%). The unreacted amine was then removed using 30%
ammonia sat. methanol/dichloromethane, and obtained as a yellow solid
‡
29.25, 24.18, 16.29; HRMS-CI : m/z: calcd for C41H49N8O6: 749.3775;
found: 749.3784.
1-[(3-(N,N-Bis-(6-(2,2'-bipyridyl-1,1'-dioxide)methyl)carbamoyl)propa-
moyl)aminomethyl]-3,5-bis(aminomethyl)-2,4,6-triethylbenzene bis(acetic
acid) europium(iii) trisacetate (1): Compound 18 (26.7 mg, 30.8 mmol)
Chem. Eur. J. 2003, 9, No. 1
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55

E. V. Anslyn and M. D. Best
FULL PAPER
was placed in a 50 mL round-bottomed flask and dissolved in spectroscopic
grade methanol (5 mL). Europium acetate hexahydrate (13.4 mg,
30.8 mmol) was then added and the solution heated to 608C
under a condenser for 3 h. Following the reaction, the mixture was
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diluted into
purification.
a
stock solution and used for studies without further
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[8] a) J. J. Lavigne, S. Savoy, M. B. Clevenger, J. E. Ritchie, B. McDoniel,
S. J. Yoo, E. V. Anslyn, J. T. McDevitt, J. B. Shear, D. Neikirk, J. Am.
Chem. Soc. 1998, 120, 6429 6430; b) D. P. Neikirk, S. M. Savoy, J. J.
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Savoy, M. Rodriguez, T. Curey, A. Tsao, G. Simmons, J. Wright, S. J.
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Tsao, J. Lavigne, Y. Sohn, J. T. McDevitt, E. V. Anslyn, D. Neikirk,
J. B. Shear, Anal. Biochem. 2001, 293, 178 184.
Methyl-3-[N,N-bis-(6-(2,2'-bipyridyl-1,1'-dioxide)methyl)carbamoyl]pro-
panoate (19): Structure 12 (183 mg, 391 mmol) was dissolved in chloroform
(2 mL) in a 100 mL round-bottomed flask, which was placed at 08C. m-
Chloroperbenzoic acid (482 mg, 1.96 mmol) was then dissolved in chloro-
form (3 mL) and added to the reaction flask through a syringe. The reaction
was stirred at 08C for 15 min and then at room temperature for 4 h. At this
point, the reaction was returned to 08C, and another portion of mCPBA
(482 mg, 1.96 mmol) dissolved in chloroform (5 mL) was added. After
15 minutes, the reaction was again allowed to warm to room temperature,
and was then allowed to stir for 15 h. Next, the reaction was directly loaded
onto a silica gel column. First, the m-CPBA and resultant meta-chloro-
benzoic acid were removed using 100% methanol as eluant. This was
followed by the removal of the methyl-3-(N,N-bis-(6-(2,2'-bipyridyl-1,1'-
dioxide)carbamoyl))propanoate using 25% ammonia saturated methanol/
dichloromethane as eluant. The product 19 was obtained as a light yellow
1
solid (208 mg, 97%). M.p. 115 1178C; H NMR (300 MHz, CDCl3): d ˆ
8.27 (t, 2H, J ˆ 6.0 Hz), 7.50 7.28 (m, 12H), 4.97 (s, 2H), 4.87 (s, 2H),
3.65 (s, 3H), 2.82 (t, 2H, J ˆ 6.0 Hz), 2.67 (t, 2H, 6.0 Hz); 13C NMR
(300 MHz, CDCl3): d ˆ 173.35, 173.10, 147.57, 146.92, 142.87, 142.48, 139.96,
139.90, 128.18, 127.06, 126.61, 126.45, 125.53, 124.95, 124.91, 124.81, 51.75,
[9] N. Sabbatini, M. Guardigli, J.-M. Lehn, Coord. Chem. Rev. 1993, 123,
201 228.
‡
48.55, 45.71, 28.84, 27.88; HRMS-CI : m/z: calcd for C27H26N5O7: 532.1832;
[10] a) V. Balzani, E. Berghmans, J.-M. Lehn, N. Sabbatini, R. Terˆrde, R.
Ziessel, Helv. Chim. Acta 1990, 73, 2083 2089; b) R. Ziessel, M.
Maestri, L. Prodi, V. Balzani, A. Van Dorsselaer, Inorg. Chem. 1993,
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found: 532.1840.
Methyl-3-[(N,N-bis-(6-(2,2'-bipyridyl-1,1'-dioxide)methyl)carbamoyl)pro-
panoate] europium(iii) trisacetate (2): Molecule 19 (26.0 mg, 48.7 mmol)
was dissolved in spectroscopic grade methanol (5 mL) in a 50 mL round-
bottomed flask. To this was added europium acetate hexahydrate (21.4 mg,
48.7 mmol). The reaction was heated to 708C under a condenser. Following
this, the solution was diluted into a stock solution and used for studies
‡
‡
without further purification. MS-ESI [2 À OAc] : found: 800, 802; MS-
‡
‡
‡
FAB [2 À OAc] : found: 800, 802; HRMS-FAB : m/z: calcd for
C31H31N5O11Eu: 802.1233; found: 802.1223.
Titrations of guests into host 1 and model 2: Two solutions were freshly
made for each titration, the cuvette solution consisting of only host, and the
titrant having the same concentration of host with a concentration of guest
four to five times larger. The cuvette solution was formed by adding an
unbuffered host stock solution (100 mL) and diluting with 5mm tris-buffer
(4.9 mL), which was adjusted to pH 7.4 through methoxide addition, and
spectroscopic grade acetonitrile (5 mL). Thus, the final solution contained
5.52nm host and 2.45mm buffer in 50% methanol/acetonitrile. The guest
solution contained 100 mL of the host as well, plus, in the case of BPG,
700 mL of guest buffered to pH 7.4 with 5mM tris in methanol, 4.3 mL of
5 mm Tris-buffered methanol and acetonitrile (5 mL). The resulting guest
solution contained 5.52nm host, 22.9nm BPG and 2.45 mm tris buffer in
50% methanol/acetonitrile. Titrations were performed by adding the host
solution (2 mL) to a quartz cuvette and adding guest solution (50 mL) for
each point, leading to a first addition [BPG]/[host] ratio of around 0.1.
Titrations were carried out to around three equivalents of guest. Binding
affinities were calculated through curve fitting of the changes in fluo-
rescence. This was performed through iterative manipulation of the binding
constant and change in molar absorptivity until the best fit of the data with
the theoretical curve was recorded.
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¡
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Acknowledgements
We greatly fully acknowledge the National Institutes of Health for support
of this project (GM57306 04A1).
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56
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Chem. Eur. J. 2003, 9, No. 1

A Fluorescent Sensor
51 57
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Received: July 8, 2002 [F4234]
Chem. Eur. J. 2003, 9, No. 1
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57