Computational and experimental characterization of a fluorescent dye for detection of potassium ion concentration

Article abstract of DOI:10.1021/jp507552q

The fluorescence of the SKC-513 ((E)-N-(9-(4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl)-6-(butyl(3-sulfopropyl)amino)-3H-xanthen-3-ylidene)-N-(3-sulfopropyl)butan-1-aminium) dye is shown experimentally to have high sensitivity to binding of

Full text of DOI:10.1021/jp507552q

Article
pubs.acs.org/JPCA
Computational and Experimental Characterization of a Fluorescent
Dye for Detection of Potassium Ion Concentration
†
‡
§
‡
§
Matteus Tanha, Subhasish K. Chakraborty, Beth Gabris, Alan S. Waggoner, Guy Salama,
,†
and David Yaron*
†
‡
Chemistry Departmentand Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213,
United States
§
Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United
States
*
S Supporting Information
ABSTRACT: The fluorescence of the SKC-513 ((E)-N-(9-
4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl)-
-(butyl(3-sulfopropyl)amino)-3H-xanthen-3-ylidene)-N-(3-
(
6
sulfopropyl)butan-1-aminium) dye is shown experimentally to
+
have high sensitivity to binding of the K ion. Computations
are used to explore the potential origins of this sensitivity and
to make some suggestions regarding structural improvements.
+
In the absence of K , excitation is to two nearly degenerate
states, a neutral (N) excited state with a high oscillator
strength, and a charge-transfer (CT) state with a lower
+
oscillator strength. Binding of K destabilizes the CT state, raising its energy far above the N state. The increase in fluorescence
+
quantum yield upon binding of K is attributed to the increased energy of the CT state suppressing a nonradiative pathway
mediated by the CT state. The near degeneracy of the N and CT excited states can be understood by considering SKC-513 as a
reduced symmetry version of a parent molecule with 3-fold symmetry. Computations show that acceptor−donor substituents can
be used to alter the relative energies of the N and CT state, whereas a methylene spacer between the heterocycle and phenylene
groups can be used to increase the coupling between these states. These modifications provide synthetic handles with which to
+
optimize the dye for K detection.
OVERVIEW
presented here find that the lowest-energy excitations of
SKC-513 correspond to a fluorescent state with predominantly
N character and a less optically intense state with
predominantly CT character. The potassium ion preferentially
destabilizes the CT state, thereby altering the quantum yield,
consistent with the mechanism implicated in other PET
sensors.
■
Florescence imaging provides a set of powerful techniques for
monitoring biological processes in living organisms.
1
−6
These
techniques rely on dye molecules that change their fluorescence
behavior under varying environments. For neural processes, it is
useful to have dyes whose fluorescence tracks the concentration
+
of the K ion. The experimental data presented below for the
A feature of the excited states of SKC-513 that may be
contributing to the high sensitivity of the fluorescence quantum
yield to ion binding is a near degeneracy of the N state, which
mediates the radiative pathway, and the CT state, which+
mediates a nonradiative pathway. The binding of K
substantially raises the energy of the CT state, suppressing
the nonradiative pathway. The near degeneracy of the N and
CT states may appear to be accidental, given that SKC-513 has
only a 2-fold symmetry axis. However, comparison to a 3-fold
symmetric parent reveals a systematic origin. In the parent
molecule, the lowest excited state is doubly degenerate. In
addition, the molecular orbitals involved in these degenerate
excitations have low density in the regions of the molecule that
dye SKC-513 (Figure 1) show a substantial change in quantum
yield as the K concentration varies between 0 and 1000 mM.
The dye has a crown ether portion that binds the potassium ion
and a chromophore that signals the presence of the ion. We use
quantum chemical calculations to explore the mechanism
through which the ion alters the fluorescence of the
chromophore.
+
The computations presented below indicate that SKC-513 is
a photoinduced electron-transfer (PET) sensor, a class of dyes
whose fluorescence quantum yield is known to be sensitive to
4
−6
ion binding.
In these dyes, the change in quantum yield
arises from the competition between a radiative pathway,
emission from a neutral excited state (N) created on
photoexcitation, and a nonradiative pathway, which is mediated
by a charge-transfer (CT) excited state. Ion binding destabilizes
the CT state and alters the branching ratio between the
radiative and nonradiative pathways. The computations
Received: July 27, 2014
Revised: September 8, 2014
Published: September 12, 2014
©
2014 American Chemical Society
9837
dx.doi.org/10.1021/jp507552q | J. Phys. Chem. A 2014, 118, 9837−9843

The Journal of Physical Chemistry A
Article
Figure 1. Primary steps in the synthesis of SKC-513.
must be altered to convert the parent molecule to SKC-513.
The degeneracy of excited states in SKC-513 is therefore not
accidental but can be traced back to degeneracies expected for
this 3-fold symmetric parent.
Our calculations also find that replacing the potassium ion
with a point charge has little effect on the nature of the relevant
excited states. This suggests that changes in the excited states of
the chromophore in SKC-513 arise from field effects of the
potassium ion, as opposed to more specific interactions such as
ligation with the nitrogen atom that connects the crown ether
to the chromophore.
and filtered, the solid was washed with 100 mL of THF. The
combined organic filtrate was concentrated and the oily residue
was subjected to silica gel column chromatography using
diethyl ether as eluent, providing a light yellow oil (10.361g,
1
31% yield) as compound 1. H NMR δ (CDCl3): 3.702 (m,
24H), 6.697 (m, 2H), 7.22 (m, 1H), 7.37 (m, 1H), 7.805 (d,
+
1H). EI MS: m/z 339.73 (M ).
Synthesis of Compound 2: 4-(1,4,7,10,13-Pentaoxa-
16-azacyclooctadecan-16-yl)benzaldehyde. Compound 1
(3.414g) (1 mmol) was dissolved in 45 mL of DMF, and the
solution was cooled to −5 °C using a salt ice bath under
stirring. POCl (18.93 g) was added to the reaction solution
3
SYNTHESIS OF SKC-513
Figure 1 illustrates the primary steps in the synthesis of SKC-
13 described in this section.
over a period of 1 h while the reaction temperature was
maintained between −5 and 0 °C. The solution was removed
from the ice bath, stirred at room temperature for 20 h, warmed
to 70 °C, and stirred for an additional 2 h. The solution was
then cooled and added to 450 g of ice water, basified with
■
5
Synthesis of Compound 1: 16-Phenyl-1,4,7,10,13-
pentaoxa-16-azacyclooctadecane. In a 2 L three-neck
round-bottom flask fitted with a reflux condenser, 8 g of NaH
Na
The combined organic layer was then washed with 3 × 200 mL
water, dried over anhydrous MgSO , and concentrated to
CO to pH 7.5, and extracted with 3 × 300 mL of CHCl .
2
3
3
(
60% in mineral oil, 2 mmol) was added to 400 mL of
anhydrous THF and the mixture was refluxed under argon. N-
Phenyldiethanolamine (18.17 g, 1 mmol) and 50.631 g of
tetra(ethylene glycol)-di-p-tosylate (1 mmol) dissolved in 400
mL of anhydrous THF were added to the refluxed NaH
solution over a period of 8 h, and the resulting mixture was
refluxed for another 24 h. After the reaction mass was cooled
4
provide 5.31 g of crude product. This was purified by silica gel
column chromatography using CH Cl to give 3.687 g of
2
2
1
product (100% yield) as a light yellow oil. H NMR, δ
(CDCl ): 3.67 (m, 24H), 6.743 (d, 2H), 7.73 (d, 2H), 9.73 (s,
3
+
1H, CHO). EI MS: m/z 367.92 (M ).
9
838
dx.doi.org/10.1021/jp507552q | J. Phys. Chem. A 2014, 118, 9837−9843

The Journal of Physical Chemistry A
Article
Synthesis of Compound 3: N-(3-Hydroxyphenyl)-
butyramide. n-Butyric anhydride (20.23 g) was added
dropwise to a solution of 3-aminophenol (10.93g) in 400 mL
of dry THF at 0 °C over a period of 1 h. After stirring at room
temperature for 20 h, the solution was concentrated under
reduced pressure. The residue was resuspended in 300 mL of
EXPERIMENTAL CHARACTERIZATION
The fluorescence intensity of SKC-513 as a function of ion
concentration is shown in Figures 2 and 3. Figure 2 highlights
■
CHCl , and the suspension was stirred at room temperature for
3
2
h, at which point the white solid that formed was filtered and
dried to provide 14.13 g of colorless crystals as compound 3.
1
MW C H NO : 179.0946 g/mol. H NMR, δ (DMSO-d ):
10
13
2
6
0.912 (t, 3H), 1.60 (q, 2H), 2.257 (t, 2H), 6.41 (m, 1H), 6.930
(
1
m, 1H), 7.04 (m, 1H), 7.19 (m, 1H), 9.303 (s, 1H), 9.696 (s,
H). ESI-MS: m/z 179. 34 (M ).
+
Synthesis of Compound 4: 3-(Butylamino)phenol.
NaBH4 (5.03 g) and compound 3 [N-(3-hydroxyphenyl)
butyramide] (10.31 g) were taken in 100 mL of dry THF, and
the mixture was cooled to 0 °C. I (10.57 g) in 80 mL of dry
2
THF was added at 0 °C for 1 h under argon. The mixture was
refluxed for 3 h and cooled to room temperature. HCl (3 M, 30
mL) was added carefully over a period of 30 min. The mixture
was neutralized by 1 M NaOH and extracted with 2 × 250 mL
of ethyl acetate. The combined organic extract was washed with
Figure 2. Experimental data for SKC-513 showing the fluorescence
intensity (in arbitrary units) as a function of the concentration of
various ions in the range from 0 to 1000 mM. SR refers to experiments
done in the presence of a sarcoplasmic reticulum.
brine and water, dried over anhydrous MgSO , and
4
concentrated to dryness, providing a crude colorless oil that
was purified by silica gel column chromatography using n-
hexane/ether (3:1 v/v) to give 7.33 g of product (compound
1
4
) as a colorless solid. MW C H NO: 165.1154 g/mol. H
1
0
15
NMR, δ (CDCl ): 1.007 (t, 3H), 1.46 (m, 2H), 1.62 (m, 2H),
3
3
.11 (t, 2H), 6.16 (m, 1H), 6.22 (m, 2H), 7.037 (t, 1H). ESI-
+
MS: m/z 165.54 (M ).
S y nt h e s is o f Co m p o u n d 5 : 3 - ( B u t yl ( 3 -
hydroxyphenyl)amino)propane-1-sulfonic acid. Com-
pound 4 (3.32g) and 1,3-propanesultone (2.88 g) were
dissolved in 15 mL of dry DMF, and the solution was heated
at 130 °C for 20 h, then cooled to room temperature, and
concentrated. The resulting crude residue was purified by a PR
C-18 column using 50% water in methanol as solvent, to give
3
.23 g of a light brown oily residue that solidified upon standing
1
(
compound 5). MW C H NO S: 287.1191 g/mol. H NMR,
13 21 4
δ (DMSO-d ): 0.83 (m, 3H), 1.25 (m, 4H), 1.75 (m, 4H),
6
3
.41−3.64 (m, 4H), 6.89 (d, 1H), 7.06 (m, 2H); 7.41 (m, 1H).
+
Figure 3. As in Figure 2, but over an ion concentration range 0−40
mM.
ESI-MS: m/z 287.33 (M ).
Synthesis of SKC-513. Compound 2 (370 mg), compound
5
(704 mg), and PTSA (100 mg) were dissolved in 25 mL of
propionic acid, and the solution was stirred at 70 °C for 20 h.
After concentrating, 150 mL of 3 M NaOAc aqueous solution
was added to the residue and the mixture was stirred for 1 h at
room temperature. After the solution was concentrated, the
residue was subjected to silica gel column chromatography
using CH Cl /MeOH (3:1 v/v), providing a purple gummy
residue that was used immediately for subsequent reaction with
tetrachloro-1,4-benzoquinone (530 mg) in a methanol/chloro-
form (1:1) mixture at ambient temperature for 15 h. Excess
tetrachloro-1,4-benzoquinone was removed by filtration, and
the reaction mixture was concentrated under reduced pressure.
The residue was purified twice by Silica gel column
+
how fluorescence intensity increases with K concentration,
saturating at about 1000 mM, giving an enhancement of nearly
an order of magnitude in the fluorescence intensity. Figure 3
shows that the binding is selective over the biologically relevant
range of concentrations, with no change in fluorescence
intensity found for Ca , Mg , or Na . Also shown is the
effects of K on fluorescence intensity in the presence of the
sarcoplasmic reticulum (SR), isolated via the procedure
described in Salama et al. The experiment was conducted in
a solution composed of the isolated SR with 5 μM ion (KCl,
NaCl, CaCl , or MgCl ) in 100 mM dye, 20 mM HEPES, and 1
2+
2+
+
2
2
+
7
2
2
mM gluconic acid.
chromatography using 30% MeOH in CHCl as solvent to
3
COMPUTATIONAL METHODS
get a crimson to dark violet solid as product SKC-513 (113
■
1
mg). H NMR, δ (CD OD): 0.93 (t, 6H), 1.38 (m, 4H), 1.72
Unless otherwise indicated, all calculations used density
functional theory (DFT) for the ground electronic state and
time dependent DFT (TDDFT) for excited states, with the
3
(
m, 4H), 2.02 (t, 4H), 2.93 (m, 4H), 3.54−3.82 (m, 32H), 7.03
(
m, 4H), 7.17 (d, 2H), 7.40 (d, 2H), 7.67 (d, 2H). ESI-MS: m/
8
z 902.51 (M − 2H).
CAM-B3LYP functional and a 6-31G** basis. CAM-B3LYP
9
839
dx.doi.org/10.1021/jp507552q | J. Phys. Chem. A 2014, 118, 9837−9843

The Journal of Physical Chemistry A
Article
has previously been shown to provide good results for
9
excitation energies of conjugated dyes. For all calculations
10
reported below, the polarizable continuum model (PCM), as
11
implemented in Gaussian 09, was used with water as solvent.
The structure of the SKC-513 dye is shown in Figure 1. The
ion binds to the crown ether at the top of the dye. To facilitate
the calculations, the structure was simplified to contain only the
optical chromophore shown in Figure 4, which will be referred
Figure 5. Effects of a point charge on the excited states of SSKC-513.
(The radii of the circles are proportional to the oscillator strength.)
In the absence of a point charge, the lowest-energy state is
the less intense state, which implies the molecule should be
only weakly fluorescent. As the magnitude of the point charge is
increased in the positive direction, the bright state drops below
the less intense state, suggesting that the presence of a cation
such as potassium should substantially enhance the fluores-
cence quantum yield. We next explore the origin of the excited-
state degeneracy and then consider calculations that use an
Figure 4. Structure of the simplified SKC-513 dye (SSKC-513).
to as simplified-SKC-513 (SSKC-513). Effects of ion binding
were modeled either by explicit inclusion of a potassium ion or
by including the electrical potential arising for a point charge
placed at a location corresponding to the center of the crown
ether. The charge was positioned at a distance of 3 Å from the
nitrogen of the crown ether, along an axis connecting the
nitrogen to the oxygen of the heterocycle. The distance of 3 Å
was taken from experimental and theoretical studies on
+
explicit K ion instead of a point charge.
Origin of the Degenerate Excited States. Examination
of the frontier orbitals allows assignment of netural (N) and
charge-transfer (CT) character to the excited states of Figure 5.
These states are essentially pure excitations between the
orbitals shown in Figure 6. For vertical excitation in the absence
12−14
potassium crown ether complexes.
Torsion about the angle θ of Figure 4 is relatively facile with
a minimum at 52° and the range 30° to 80° being populated at
room temperature. Because the energy and oscillator strengths
of the lowest-energy excited states are only weakly dependent
on θ (Supporting Information), the results shown below are for
either the geometry-optimized ground or excited state.
The Supporting Information also includes results from
semiempirical calculations that were used in initial exploration
of the photophysics. The semiempirical and DFT results lead to
similar conclusions regarding the way in which binding of
potassium ion alters the fluorescence properties. This agree-
ment across quite different quantum chemical models indicates
that the conclusions are robust with respect to the level of
theory employed.
COMPUTATIONAL RESULTS
■
Figure 6. Frontier orbitals of SSKC-513. There are two nearly
degenerate HOMOs, one located on the heterocycle and the other on
the phenylene ring. The LUMO is nondegenerate and located on the
heterocycle. Optical intensity relies on good overlap between orbitals,
such that only the transition from HOMO-N to the LUMO carries
large optical intensity.
Effects of a Point Charge. Figure 5 shows the effect of a
point charge placed at a position correponding to the center of
the crown ether in SKC-513. In the absence of a point charge,
there are two nearly degenerate excited states. These states
have different oscillator strengths and relative positions that are
strongly dependent on the sign and magnitude of the charge.
15
Following Kasha’s rule, we expect the molecule to fluoresce
strongly only when the lowest excited electronic state carries
significant optical intensity. This rule assumes that excitation to
the optically intense excited state is followed by relaxation to
the lowest excited electronic state on a time scale that is much
faster than the fluorescence lifetime. If the lowest excited state
has a strong optical transition to the ground state, fluorescence
will occur. As the optical intensity of the lowest excited state
decreases, more population is lost to nonradiative pathways and
the flourescence quantum yield decreases.
of a point charge, the first excited state has 95% HOMO-CT →
LUMO character and the second excited state has 97%
HOMO-N → LUMO character. The lowest unoccupied
molecular orbital (LUMO) resides on the heterocycle.
HOMO-CT resides primarily on the phenylene ring, such
that the HOMO-CT → LUMO corresponds to transfer of
charge from the phenyl ring to the heterocycle, indicating a CT
state. Because there is little overlap between HOMO-CT and
the LUMO, this CT state carries little optical intensity.
9
840
dx.doi.org/10.1021/jp507552q | J. Phys. Chem. A 2014, 118, 9837−9843

The Journal of Physical Chemistry A
Article
HOMO-N resides on the heterocycle, such that HOMO-N →
LUMO corresponds to a neutral (N) excitation, with
substantially more oscillator strength than the CT state.
The effects of the point charge in Figure 5 can also be
understood in terms of the frontier orbitals. Because a positive
charge bound to the crown ether is closer to the phenylene ring
than to the heterocycle, it preferentially lowers the energy of
the HOMO-CT. This raises the energy of the HOMO-CT →
LUMO transition that dominates the CT state. This is in
agreement with Figure 5, which shows that a positive charge
causes the less intense CT state to rise significantly above the
bright N state.
The near degeneracy of the two lowest-excited states
observed in SSKC-513 may be unexpected, because the
excitations have a largely different character, one being a N
excitation on the heterocycle and the other being a CT
excitation from the heterocycle to the phenylene ring.
Furthermore, SSKC-513 has at most 2-fold symmetry, and
degeneracies are not expected for C2 symmetry groups.
However, doubly degenerate states are expected for molecules
rotation of the upper phenylene ring to form SSKC-513 does
not substantially lift the degeneracy: the HOMOs have little
amplitude in the regions of the additional oxygen atoms. The
nearly degenerate HOMOs of SSKC-513 are therefore not an
accidental degeneracy, but rather a result of the 3-fold
symmetry of this parent molecule.
The effects of a point charge on the 3-fold symmetric parent
molecule are shown in Figure 7. The point charge lifts the
degeneracy, similar to the behavior of SSKC-513 in Figure 5.
However, both states retain optical intensity such that binding
of the ion would not be expected to have a strong effect on the
fluorescent behavior. The 3-fold symmetric molecule is
therefore useful for understanding the origin of the degenerate
excited states of SSKC-513, but is not expected to be a useful
chromophore for ion detection.
Effects of a Potassium Ion. Above, the effects of ion
binding were explored by examining the effects of a point
charge located at a position corresponding to the center of the
crown ether. The results suggest that a positive charge
preferentially stabilizes HOMO-CT, which resides primarily
on the phenylene ring (Figure 6). This causes the less intense
CT state to rise above the bright N state (Figure 5), which
rationalizes the observed increase in fluorescence quantum
yield upon binding of a cation.
with 3-fold symmetry, because the C symmetry groups have
3
doubly degenerate representations. The molecule of Figure 7
+
Results from DFT calculations that explicitly include a K ion
(
Figure 9) lead to similar conclusions. Figure 9 shows the
lowest two excited states, S and S , with CT and N character
1
2
assigned on the basis of the predicted optical intensity and
examination of the orbitals that contribute to the excitation
(
Supporting Information). Results are shown for both the
ground- electronic-state geometry, corresponding to vertical
excitation, and the relaxed S geometry, corresponding to
1
fluorescence.
+
In the presence of K (right side of Figure 9), the gap
between S and S is large, with S having strong N character
1
2
1
and so carrying substantial oscillator strength. We therefore+
expect high flourescence quantum yield in the presence of a K
ion, for reasons that are consistent with those obtained above
based on the effects of a point charge.
Figure 7. Effects of a point charge on the excited states of the 3-fold
symmetric parent of SSKC-513. (The radii of the circles are
proportional to oscillator strength.)
+
In the absence of K (left side of Figure 9), the gap between
S and S is small, being 0.05 eV for the ground-state geometry
1
2
and 0.15 eV for the relaxed S geometry. In the ground-state
1
uses bridging oxygen atoms to convert SSKC-513 to a 3-fold
symmetric system. The orbitals of this symmetric parent
molecule are shown in Figure 8. The HOMO is again doubly
degenerate, whereas the LUMO is nondegenerate. The nodal
pattern of the HOMOs also reveals why removal of oxygen and
geometry, the S and S states have the same character as seen
1
2
in the presence of a point charge: S has CT character and
1
carries 2.5 times less intensity than the S state, which has N
2
character. Upon excited-state relaxation, the S and S states are
1
2
predicted to cross: S has N character and carries 2.5 times
1
more intensity than the S state, which now has CT character.
2
+
The weaker flourescence seen in the absence of a K ion can
then be attributed to the near degeneracy of S and S , with the
1
2
energy separation being so close that they cross as the geometry
relaxes from that of the vertical excitation to that of the S state.
1
Because torsions associated with θ of Figure 4 are low-
frequency, it is plausible that the excited-state relaxation is
dominated by motion along this coordinate. However, the
change in torsional angles are relatively small. In the absence of
+
a K ion, the relaxation of the torsonial angle is from 54° to
+
5
7.5° and in the presence of a K ion, the relaxation is from 63°
to 60.7°.
Also of interest is the degree to which the interaction with K+
goes beyond field effects to include more specific chemical
interactions. The molecular orbitals that participate in the
excitation have negligible amplitude on the potassium ion
Figure 8. Frontier molecular orbitals of the 3-fold symmetric parent of
SSKC-513.
9
841
dx.doi.org/10.1021/jp507552q | J. Phys. Chem. A 2014, 118, 9837−9843

The Journal of Physical Chemistry A
Article
+
Figure 9. Electronic states of SSKC-513 (Figure 5) with (right) and without (left) a K ion at a location corresponding to the center of the crown
ether in SKC-513.
(Supporting Information). This, along with the similar behavior
observed for a point charge, suggests that the effects of the ion
on the excited state arise primarily from the electric field of the
ion.
Effects of Electronic Substituents. The suggested
+
mechanism for the effects of K on the fluorescence quantum
yield is that the electric field of the ion causes the molecule to
move from the less-emissive regime on the left of Figure 5 to
the more emissive regime on the right. The computations
reported here are approximate, not only in the methods used
for the electronic structure of the chromophore but also for the
use of a continuum dielectric model for the solvent and the use
of a simplified structure for the dye (Figure 4). Although such
approximations likely do not invalidate the proposed
mechanism, they do suggest that the predicted location of the
crossing point between the less emissive and more emissive
regimes may not be highly reliable. A reasonable target for
synthetic modification to SKC-513 is adding substituents that
alter the crossing point. This can be done by adding electronic
acceptors or donors that alter the relative energies of the
HOMOs in Figure 6.
Figure 10. Effects of Flourine substituents on the two lowest excited
states of SSKC-513. Adding F substituents to the phenyl ring (left)
raises the energy of the CT excited state, and adding F substituents to
the heterocycle has the opposite effect.
Two derivatives of SSKC-513 are compared with SSKC-513
in Figure 10. The results can be understood in terms of the
effects of the substituents on the HOMOs of Figure 6. Addition
of fluorine atoms to the phenylene group stabilizes the
HOMO-CT located on the phenylene ring (the HOMO on
the right in Figure 6), thereby raising the energy of the CT state
relative to that of the N state. Similarly, addition of flourine
atoms to the heterocycle stabilizes the HOMO-N shown on the
left of Figure 6, thereby raising the N state relative to the CT
state.
These results suggest that electronic substituents provide a
handle that may be used to optimize the sensitivity of SSKC-
5
13 fluorescence to ion binding, by altering the relative energies
Figure 11. Two lowest excited states of a modified SSKC-513, in
which a methylene bridges between the phenylene group and the
heterocycle. (The radii of the circles are proportional to the oscillator
strength.)
of the N and CT states.
Effects of a Bridging Methylene. Figure 11 shows the
effects of a point charge on the excited states when an extra
methylene group is placed between the heterocycle and the
phenylene group. The calculations are done for the optimized
geometry, in which the phenylene ring is nearly perpendicular
to the heterocycle. The avoided crossing between N and CT
states suggests a stronger electronic coupling between these
states than is seen in Figure 5 for SSKC-513. A more detailed
examination of the curve crossings suggests a coupling of 0.15
eV for this system, compared to about 0.01 eV for SSKC-513.
The proposed mechanism for ion sensitivity assumes rapid
9
842
dx.doi.org/10.1021/jp507552q | J. Phys. Chem. A 2014, 118, 9837−9843

The Journal of Physical Chemistry A
Article
relaxation between N and CT states. A stronger coupling
between N and CT states may enhance this relaxation, such
that the addition of a bridge methylene group may enhance the
sensitivity of fluorescence to ion binding. The introduction of
a bridging methylene also leads to a substantial decrease in the
oscillator strength of the CT state. This increased contrast
between the N and CT states may also enhance the sensitivity
of fluorescence to ion binding.
(4) de Silva, A. P.; Gunnlaugsson, T.; Rice, T. E. Recent Evolution of
Luminescent Photoinduced Electron Transfer Sensors. A Review.
Analyst 1996, 121, 1759.
16
(5) de Silva, A. P.; Moody, T. S.; Wright, G. D. Fluorescent PET
(
Photoinduced Electron Transfer) Sensors as Potent Analytical Tools.
Analyst 2009, 134, 2385−93.
6) Bissell, R.; Prasanna de Silva, A.; Nimal Gunaratne, H.; Mark
(
Lynch, P.; Maguire, G.; McCoy, C.; Samankumara Sandanayake, K.
Fluorescent PET (Photoinduced Electron Transfer) Sensors. Curr.
Top. Med. Chem. 1993, 168, 223−264.
(7) Salama, G.; Scarpa, A. Enhanced Ca2 Uptake and ATPase
Activity of Sarcoplasmic Reticulum in the Presence of Diethyl Ether. J.
Biol. Chem. 1980, 255, 6525−6528.
+
CONCLUSIONS
■
The quantum chemical calculations presented here suggest a
mechanism for the sensitivity of SKC-513 to ion binding, and a
possible means for enhancing this sensitivity. The mechanism
relates to the near degeneracy of a bright excitation,
corresponding to a N excitation on the heterocycle of SKC-
(
8) Yanai, T.; Tew, D. P.; Handy, N. C. A New Hybrid Exchange-
Correlation Functional Using the Coulomb-Attenuating Method
CAM-B3LYP). Chem. Phys. Lett. 2004, 393, 51−57.
(9) Jacquemin, D.; Perpete, E. A.; Scuseria, G. E.; Ciofini, I.; Adamo,
(
̀
5
13, and a CT excitation corresponding to charge transfer from
C. TD-DFT Performance for the Visible Absorption Spectra of
Organic Dyes: Conventional versus Long-Range Hybrids. J. Chem.
Theory Comput. 2008, 4, 123−135.
+
the phenylene group to the heterocycle. In the absence of K ,
these two states lie close in energy. Binding of K destabilizes
+
(
̀
10) Tomasi, J.; Mennucci, B.; Cances, E. The IEF Version of the
the CT excited state, raising its energy far above the N state. In
+
PCM Solvation Method: an Overview of a New Method Addressed to
the presence of K , the lowest-energy excited state has high
Study Molecular Solutes at the QM Ab Initio Level. J. Mol. Struct.
oscillator strength and is well separated from other electronic
1
999, 464, 211−226.
11) Frisch, M. J.; et al.; Gaussian 09, Revision A.1; Gaussian Inc.:
Wallingford, CT, 2009.
12) Glendening, E. D.; Feller, D.; Thompson, M. A. An Ab Initio
states. This rationalizes the increase in fluorescence intensity
(
+
seen experimentally upon binding of K . Computations suggest
that electronic substituents may be used to alter the relative
location of the N and CT states, whereas the introduction of a
methylene group as a bridge bewteen the heterocycle and the
phenylene group alters the electronic coupling between these
states. Such modifications may therefore provide synthetic
handles with which to optimize the sensitivity of the
fluorescence to ion binding.
(
Investigation of the Structure and Alkali Metal Cation Selectivity of
18-Crown-6. J. Am. Chem. Soc. 1994, 116, 10657−10669.
(13) Thompson, M. A.; Glendening, E. D.; Feller, D. The Nature of
+
K
Crown Ether Interactions: A Hybrid Quantum Mechanical-
Molecular Mechanical Study. J. Phys. Chem. 1994, 98, 10465−10476.
14) Steed, J. W. First- and Second-Sphere Coordination Chemistry
of Alkali Metal Crown Ether Complexes. Coord. Chem. Rev. 2001, 215,
(
1
71−221.
ASSOCIATED CONTENT
■
(15) Kasha, M. Characterization of Electronic Transitions in
*
S
Supporting Information
Complex Molecules. Discuss. Faraday Soc. 1950, 9, 14−19.
16) Marcus, R. A. Electron Transfer Reactions in Chemistry. Theory
and Experiment. Rev. Mod. Phys. 1993, 65, 599−610.
(
Computed torsional potential for SSKC-513, two lowest
excited-state energies of SSKC-513 as a function of torsional
angle, semiempirical calculations of SSKC-513 in the presence
of a point charge, and comparison of molecular orbitals and
electronic excitations of SSKC-513 in the absence and presence
+
of K . This material is available free of charge via the Internet at
http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
D. Yaron. E-mail: yaron@cmu.edu.
■
*
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(
This work was supported by the National Institutes of Health
Grant No. 1R01HL093074-01) and the National Science
Foundation (Grant No. 1027985).
REFERENCES
■
(
1) Ntziachristos, V. Fluorescence Molecular Imaging. Annu. Rev.
Biomed. Eng. 2006, 8, 1−33.
(
̈
2) Wache, N.; Scholten, A.; Kluner, T.; Koch, K.-W.; Christoffers, J.
Turning On Fluorescence with Thiols - Synthetic and Computational
Studies on Diaminoterephthalates and Monitoring the Switch of the
2
+
Ca Sensor Recoverin. Eur. J. Org. Chem. 2012, 2012, 5712−5722.
3) Olsen, S.; Smith, S. C. Radiationless Decay of Red Fluorescent
(
Protein Chromophore Models via Twisted Intramolecular Charge-
Transfer States. J. Am. Chem. Soc. 2007, 129, 2054−65.
9
843
dx.doi.org/10.1021/jp507552q | J. Phys. Chem. A 2014, 118, 9837−9843