PH-dependent optical properties of synthetic fluorescent imidazoles

Article abstract of DOI:10.1002/chem.200801784

An imidazole moiety is often found as an integral part of fluorophores in a variety of fluorescent proteins and many such proteins display pH-dependent light emission. In contrast, synthetic fluorescent compounds with incorporated imidazoles are rare and

Full text of DOI:10.1002/chem.200801784

DOI: 10.1002/chem.200801784
pH-Dependent Optical Properties of Synthetic Fluorescent Imidazoles
[
a]
[b]
[a, c]
Mikhail Y. Berezin, Jeff Kao, and Samuel Achilefu*
Abstract: An imidazole moiety is often
found as an integral part of fluoro-
phores in a variety of fluorescent pro-
teins and many such proteins display
pH-dependent light emission. In con-
trast, synthetic fluorescent compounds
with incorporated imidazoles are rare
and have not been studied as pH
probes. In this report, the richness of
imidazole optical properties, including
pH sensitivity, was demonstrated by
means of a novel imidazole-based fluo-
sponding to protonated, neutral, and
deprotonated imidazoles were identi-
fied in the broad range of pH 1–12.
The absorption and emission bands of
each species were assigned by compa-
rative spectral analysis with synthesized
mono- and di-N-methylated fluorescent
imidazole analogues. pKa analysis in
the ground and the excited states
showed photoacidic properties of the
fluorescent imidazoles due to the excit-
ed state proton transfer (ESPT). This
effect was negligible for substituted
imidazoles. The assessment of a pH-
sensitive center in the imidazole ring
revealed the switching of the pH-sensi-
tive centers from 1-N in the ground
state to 3-N in the excited state. The
effect was attributed to the unique
kind of the excited state charge trans-
fer (ESCT) resulting in a positive
charge swapping between two nitro-
gens.
Keywords: charge transfer · emis-
sion spectroscopy · energy transfer ·
imidazole · pH sensitivity · sensors
rophore
1H-imidazol-5-yl-vinylben-
z[e]indolium. Three species corre-
[
8]
Introduction
and oxazoles. However, in addition to common groups,
some fluorescent proteins also employ imidazole as a pH-
sensitive functionality (class 6 according to Tsien classifica-
Fluorescent molecular pH sensors are widely used to control
and monitor chemical reactions and biological processes. A
majority of the available synthetic fluorescent probes
employ tunable acidity of phenyls (napththols, fluoresceins,
and pyranines) and cyclic aromatic amines (oxazoline, cya-
[9]
tion ). For example, imidazole is a key constituent of the
fluorophore system of red fluorescent proteins EBFP2,
mCherry, and EosFP,
sponsible for their pH sensitivity.
knowledge, there have been no reports on utilizing imida-
zole moieties in synthetic pH probes despite the attractive
optical properties of imidazole-containing fluorophores,
such as high quantum yield and pH dependence.
[7,10]
(Figure 1) and apparently is re-
[11–13]
To the best of our
[1–5]
nines, and porphyrins)
to create pH-sensitive centers. In-
terestingly, fluorescent proteins with a seemingly limited
choice of available pH-sensitive groups utilize mostly the
same set of functionalities: phenyls, aromatic amines,
[6]
[7]
The long standing interest of our laboratory lies in synthe-
sis of novel near-infrared probes and their applications in
biological imaging. Recently, we initiated a screening of or-
ganic functionalities which could be suitable as pH sensors
and synthetically appropriate for incorporation into a poly-
methine skeleton. Here, inspired by the optical properties of
fluorescent proteins, we report our first experimental data
where the new molecular construct was based on incorporat-
ing an imidazole heterocycle via a methine linker into a flu-
orogenic p-conjugated benz[e]indolium core (compound 2,
see Scheme 1). Optical studies on the synthesized molecules
revealed a number of interesting and sometimes unexpected
observations. We noted three modes of pH sensitivity in the
range of pH 1–12 attributed to the protonated, neutral, and
deprotonated forms of imidazole with different degrees of
[
a] Dr. M. Y. Berezin, Prof. Dr. S. Achilefu
Department of Radiology, Washington University School of Medicine
525 Scott Avenue, St. Louis, MO 63110 (USA)
Fax : (+1)314-747-5191
Homepage: http:/www/orl.wustl.edu
E-mail: achilefus@mir.wustl.edu
4
[
b] Dr. J. Kao
Department of Chemistry, Washington University
St. Louis, MO 63130 (USA)
[
c] Prof. Dr. S. Achilefu
Department of Biochemistry & Molecular Biophysics
Washington University School of Medicine, St. Louis, MO 63110
(
USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200801784.
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FULL PAPER
or required protection–depro-
[19,20]
tection schemes,
which re-
sulted in lower yields. Finally,
we chose methylation of 5-imi-
dazole carbaldehyde with
stoichiometric amount
a
of
methyl iodide. The mixture of
-N methyl and 3-N methyl de-
1
1
rivatives (ratio 2:3, H NMR
spectroscopy) was found to be
difficult to separate. After the
aqueous workup, the mixture
[
7]
[7]
[10]
Figure 1. Fluorophores from red-emitting proteins: mCherry, EBFP2, and EosFP.
fluorescence. There existed at least two channels of the ex-
cited state charge transfer (ESCT) at certain pH: within the
imidazole ring and between the imidazole ring and ben-
z[e]indolium. The interpretation of pH sensitivities in both
ground and excited states and the detailed investigation of
the related underlying mechanisms were the goals of this
study.
was allowed to react with benz[e]indolium 1 via electrophilic
substitution in the next step. The reaction mixture contain-
ing two products with the same molecular masses was easily
separated into two constituent compounds by reversed-
phase chromatography. The H NMR and LCMS spectra of
the first, more hydrophilic elutant were identical to the com-
pound alternatively prepared from commercially available
1
1
-N-methylimidazole carbaldehyde. The second, more hy-
drophobic product, is most likely the 3-N-methylimidazole
isomer 5.
Results and Discussion
To confirm their identity, compounds 4 and 5 were fully
1
13
Synthesis of fluorescent imidazoles: To understand the
mechanism of pH sensitivity, we prepared imidazole 2 and
examined its optical properties and compared them with re-
lated compounds such as di-N-methylated imidazolium 3
and mono-N-methylated imidazoles 4 and 5, an approach
often used in the literature to explore the mechanism of
characterized by H and C NMR spectroscopy, including
COSY, HMQC, and HMBC. The final assignment of an N-
1
methyl group position was made by H NOE experiments
(Figure 2 and the Supporting Information). Irradiation of
compound 4 at d=4.17 ppm (H-16) led to the observed
signal enhancements of the two nearest protons, H-12 (d=
8.18 ppm) and H-15 (d=8.92 ppm), confirming that the
methyl group was attached to the 1-N position. In contrast,
irradiation at d=3.89 ppm (H-16) of the second elutant re-
sulted in the enhancement of H-14 (d=8.05 ppm) and H-15
(d=8.22 ppm), suggesting that the methyl group is connect-
ed to the 3-N position and confirming that the second elu-
tant is 3-N-methyl-substituted regioisomer 5. In addition,
the strong NOE between H-12 and H-9 (not shown) and the
absence of a NOE between H-14 and H-12 (crossed dotted
arrow) indicated a trans–cis conformation of compound 4.
In contrast, a strong NOE between H-12 and H-9 (not
shown) and a strong NOE between H-14 and H-12 (dotted
[
14–16]
protonation.
Fluorescent imidazoles 2–4 were prepared
by electrophilic substitution by mixing imidazole-bearing al-
[17]
dehydes prepared in our laboratory or from commercial
sources with a known fluorophore building block 3-(2-car-
boxyethyl)-1,1,2-trimethyl-1H-benz[e]indolium
Scheme 1). The pendant carboxylate group in 1 was selected
for potential further conjugation with bioactive molecules.
With a stoichiometric ratio of the starting materials, the con-
version was nearly quantitative. Compounds 2, 4, and 5
were made as internal salts from the pairing of a carboxylate
ion with the positively charged benzoindolium; compound 3
(1;
[4]
+
[17]
is a BF4 salt.
[21]
While imidazoles 2, 3, and 4 could be easily made from
arrow) indicated trans–trans conformation of 5.
commercially or readily available imidazole carbalde-
[
17]
hydes, the preparation of 3-N-methylimidazole 5 present-
ed a certain challenge due to the lack of regioselective imi-
dazole methylation at the 3-N position. Our initial efforts
Absorption and emission spectra assignment: For the analy-
sis of pH-dependent properties, the spectra were recorded
in water between pH 1.0 and 12.0 using diluted HCl or
NaOH for pH adjustment. At pH 6, compound 2 showed a
broad absorption band at
[18]
based on literature methods led to a mixture of isomers
4
3
30 nm and a smaller band at
60 nm (Figure 3, left). At
lower pH, the absorption of
the visible band became small-
er with a simultaneous increase
of the near-UV band with a
diffuse isosbestic point at
3
70 nm. Below pH 2.9, the
Scheme 1. Synthesis of imidazole(Im)-containing fluorophores.
360 nm band became dominant
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3561

S. Achilefu et al.
of two titration centers and
therefore the presence of the
three states, which we tenta-
tively assigned as the protonat-
ed, neutral, and deprotonated
forms of imidazole (Scheme 2).
In an acidic environment,
compound 2 revealed a broad
and almost structureless fluo-
rescence at 600 nm (Figure 4,
left). Under neutral conditions,
the fluorescence became much
weaker and blue-shifted to
560 nm with an isoemissive
point at 525 nm. Further basifi-
1
Figure 2. Interactions between protons used to assign the position of the methyl group by NOE. H NOE en-
hancement: 4 (left): d=4.17 ppm to d=8.18 and 8.92 ppm: H-16 to H-12 and H-15; 5 (right): d=3.89 ppm to
d=8.05 and 8.22 ppm: H-16 to H-14 and H-15.
Figure 4. Fluorescence spectra of compound 2 at pH from 1.7 to 6.4 (left,
excitation 400 nm) and at pH 7.0–11.51 (right, excitation 460 nm).
Figure 3. Absorption spectra of 2 under acidic (left) and basic (right) con-
ditions in water. Absorbance increases with the increase of pH and shifts
to longer wavelengths.
cation increased the emission intensity upon excitation at
460 nm, accompanied by a pronounced emission shift to
530 nm (Figure 4, right). The change in absorbance and
emission titration curves as a function of pH revealed three
well-separated regions in both the ground and excited states
(Figure 5). Optical properties of the compounds under
acidic, neutral, and basic conditions are given in Table 1.
All species showed similar molar absorptivity values at a
given pH and exhibited a general upward trend with an in-
and no further changes in absorption band were observed
past pH 2.5. Under basic conditions, the absorption band ex-
perienced a red shift to 477 nm with a well-defined isosbes-
tic point at 440 nm (Figure 3, right). The presence of two in-
dependent isosbestic points clearly suggested the existence
ꢀ
1
ꢀ1
crease in pH (from 9000–16000m cm in acidic solutions
ꢀ
1
ꢀ1
to 21000–25500m cm in basic media, Table 1). Similarly,
all compounds exhibited large and pH-dependent Stokes
ꢀ
1
ꢀ1
shifts (ca. 11000 cm at pH 2, ca. 5000 cm at pH 7, and ca.
2
ꢀ
1
000 cm at pH 11). Quantum yields and fluorescence life-
times for the studied molecules were rather low in water but
Scheme 2. Suggested transformations of 2 under acidic, neutral, and basic
conditions. pK
a
and pK
a
* were determined for the ground and excited
Figure 5. Titration absorption spectra of compound 2 (left) and titration
states, respectively.
emission spectra (right, excitation and emission as indicated).
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Optical Properties of Synthetic Fluorescent Imidazoles
FULL PAPER
Table 1. Molar absorptivities and quantum yields of compounds 2–5 in
water at different pH.
(Scheme 2). In the fluorescence spectra of 3, the absence of
the band at 560 nm in spectra for 2 and 2H under the neu-
[
a]
ꢀ
Entry pH 2
pH 7
pH 11
e [m cm
tral and basic conditions correlated well with the absence of
the neutral form of 3. Thus, the emission at 560 nm in neu-
tral pH of 2 was unequivocally assigned to the neutral imi-
dazole (Scheme 2).Basic media: Having assigned protonated
and neutral forms, we turned our attention to compound 2
ꢀ
1
ꢀ1
F[b,f] e [m cm
ꢀ1
ꢀ1
F[c,f]
ꢀ1
ꢀ1
F[c,d]
e [m cm
]
]
]
2
3
4
5
9200
0.014 15400
0.002
0.008
0.001
0.002
25500
degradation
21000
0.004
[
e]
[e]
16000
11400
12100
0.006 15500
0.008 15300
0.013 15100
[
g]
n/f
n/f
21100
ꢀ
under basic conditions (2H ). At a pH greater than 9, the
[
a] e: molar absorptivity obtained at max absorption wavelength. [b] Ex-
absorption spectra showed a dramatic change, which was
characterized by a red shift to 477 nm and a significant in-
crease in absorption intensity (Figure 3, right). Simultane-
ously, the fluorescence experienced a substantial blue shift
to 530 nm (Figure 4, right). These changes were unique to 2
citation at 400 nm. [c] Excitation at 460 nm. [d] Quantum yield F relative
to fluorescein in 0.1n NaOH. [e] pH 6.2, at higher pH, significant decom-
position has been observed. [f] Quantum yield F relative to quinine in
0
2 4
.1m H SO . [g] n/f: non-fluorescent.
among the four studied imidazoles and were attributed to
ꢀ
significantly increased in a constrained environment, such as
highly viscous media, which typically exist inside proteins.
Indeed, relatively weak fluorescence of 2 in aqueous solu-
tions with a fluorescence quantum yield F=0.014 and life-
time t=0.5 ns increased significantly to F=0.15, t=1.19–
the formation of the imidazole anion 2H or its resonance
a
ꢀ
form 2H via the deprotonation of the imidazole ring
b
(Scheme 2). Unlike 2, the absorption spectra of compounds
lacking labile N–H protons such as 4 and 5 under basic con-
ditions showed no bathochromic shifts and no new isosbestic
points (Figure 8), suggesting the absence of the respective
deprotonated forms. Indeed, the absorption spectra of 4 and
5 under basic conditions were similar to the absorption spec-
tra of the neutral imidazole 2 (Figure 3 and Figure 8). Also
the absence of blue-shifted fluorescent peaks under basic
conditions for 4 and 5 at 530 nm (Figure 9) further support-
ed the absence of deprotonation for these molecules. Thus,
the 477 nm band in the absorption spectra of 2 was assigned
1.28 ns in glycerol.
Acidic and neutral media: To assign absorption and emission
bands, we first considered acidic and neutral conditions.
Compound 3, which was previously synthesized in our labo-
[10]
ratory and possesses a permanent cationic charge set due
to the exhaustive N-methylation of imidazole, was used as
an isoelectronic analogue of the protonated imidazolium
+
ꢀ
2
H . As expected, 3 was not pH sensitive (no spectral shift
to their deprotonated forms 2H a,b.
ꢀ
in acidic and neutral pH, Figure 6), which correlated well
The pK of the 2$2H deprotonation in both the ground
a
state and the excited state was determined to be 10.7. Such
a pK value is unusually low for imidazoles since the depro-
a
tonation of imidazoles to their anionic form, imidazolates, is
[23]
typically higher (pK =14.5 ) and necessitates very strong
a
bases. However, in the presence of certain functionalities,
the pK can be lowered significantly. For example, the pres-
a
ence of electron-withdrawing groups might shift the dissoci-
ation constant to lower pK values; that is, for urocanic acid,
a
[20]
a pK value of 13 has been observed. Furthermore, conju-
a
gation of the imidazolate ring to a positively charged group
(
e.g. benz[e]indolium) can lower the pK even more due to
a
Figure 6. Absorption and emission spectra of 3 under acidic and neutral
conditions in water (excitation 400 nm). The overall change in quantum
yield for compound 3 was less than 30%.
the energetically favorable loss of overall charge and the
formation of a neutral conjugated chromophore 2H (a re-
lated case was recently described by Oto et al. ).
ꢀ
b
[24]
Among neutral, protonated, and deprotonated forms of 2,
the protonated form demonstrated the highest fluorescence
(Table 1). The decrease of fluorescence and the blue shift
from 600 nm to 530–560 nm (Figure 4) was apparently
caused by photo-induced electron transfer originating from
an unshared electron pair located in the imidazole nitro-
[
22]
with the lack of “active solvation centers”
cule. The change in molar absorptivity and quantum yield of
from neutral to acidic pH was much smaller than the cor-
in the mole-
3
responding changes in compound 2 (Table 1). The absorp-
tion and the emission spectra of the compound 2H in
acidic media and of 3 in both acidic and neutral solutions
clearly resembled each other, suggesting electronic similarity
between the two entities. Fluorescence lifetime measure-
ments of 2H and 3 showed that both molecules had the
same lifetime of 0.5 ns. The similarities of the spectral pa-
rameters (position, shape, and lifetime) of 2H and 3 sug-
gested that the absorption at 360 nm and emission at 600 nm
+
[25]
gens, which is electronically coupled with a chromophore.
Under acidic conditions, the electron pair was deactivated
via protonation and did not compete with the radiative
decay on the same level. For that reason, the emissions of
+
+
the electronically identical 2H , di-N-methylated 3, and
+
+
+
mono-N-methylated protonated 4H and 5H were found
to be quite similar (Figure 7); in all the structures the elec-
tron pair was blocked.
+
band belonged to the protonated imidazolium 2H
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S. Achilefu et al.
Figure 9. Fluorescence spectra of compounds 4 (left) and 5 (right) (exci-
tation 400 nm). Fluorescence decreases with increasing pH and remains
stable after pH 6. No emission signals at 530–560 nm at basic pH suggests
the absence of deprotonated forms.
Figure 7. Normalized fluorescence spectra of compounds 2–5 at pH 2 (ex-
+
+
citation 400 nm). Emission traces of 2H and 5H completely overlap.
a
Table 2. pK of fluorescent imidazoles in acidic media in the ground and
excited states, calculated from PCA and titration diagrams (in parenthe-
sis).
+
Emission traces of 3 and 4H are shifted 7–8 nm.
+
+
+
2
$2H
4$4H
5$5H
Determination of the pH-sensitive nitrogen center—pKa
analysis: Having established the correlation between imida-
zole structures and their optical properties, we closely exam-
ined the behavior of the protonated molecules to establish
which nitrogen in the imidazole ring of 2 is responsible for
pH sensitivity. Careful analysis of Figure 7 revealed that, de-
spite an obvious similarity in emission profiles between the
ground state, pKa 4.20
4.22
2.68
(4.20ꢁ0.05)
2.82
(3.00ꢁ0.03)
(4.39ꢁ0.03)
(2.75ꢁ0.08)
excited state,
pK
3.89
2.85
a
*
(4.07ꢁ0.05)
(2.72ꢁ0.06)
ground state. Calculated molar ratios for each of the princi-
pal components (from PCA) as a function of pH are shown
in Figure 10. Each of the three panels represents a photolyt-
ic equilibrium of two principal components identified in the
ground and the excited state. The point of the intersection
of both components at each panel corresponds to the pKa
+
studied imidazoliums, the emission profile of 2H fully
+
overlapped only with the emission of 5H , suggesting signif-
icant resemblance between these two excited species. In ad-
dition, detailed pK analysis showed that the sensitivities in
a
the ground state and in the excited state were different: 1-N
was the pH-sensitive center in the ground state and 3-N in
the excited state.
The pK values of the studied compounds in the ground
a
and excited states were determined from the corresponding
absorption and emission spectra (Figure 3, 4, 8, and 9) by
using principal component analysis (PCA), and were vali-
dated with sigmoidal dose-response curve fits (see the Sup-
porting Information) from titration diagrams. The results
are tabulated in Table 2.
+
Imidazole 2H exhibited photoacidic properties, known
[26–29]
as excited-state proton transfer (ESPT).
In the excited
state, the pK was 1.4 pH units lower than the pK of the
a
a
Figure 8. Absorption spectra of 4 (left) and 5 (right) at pH 1.0–11.5. Ab-
sorbance increases with increasing pH. The existence of only one isosbes-
tic point suggests the presence of only two species: protonated and neu-
tral forms.
Figure 10. Calculated molar ratio for each of the components as a func-
tion of pH using PCA. The intersection of the two molar ratio curves cor-
responds to the pK of the protolytic equilibrium.
a
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Chem. Eur. J. 2009, 15, 3560 – 3566

Optical Properties of Synthetic Fluorescent Imidazoles
FULL PAPER
values. The intersections of absorption curves correspond to
electron density in the imidazole ring rearranges in such a
way so that the positive charge moves from the 3-N position
in the ground state to the 1-N position in the excited state,
as shown in a modified Fçrster cycle (Scheme 3). In this
the pK in the ground state, whereas intersections of the
a
fluorescence curves correspond to the pK in the excited
a
state. The top panel describes the equilibrium between the
+
+
+
neutral imidazole 2 and its protonated form 2H (2$2H ).
Clearly, the intersections of absorptions and fluorescence
differed. The pK found for the excited state (pK *=2.82)
scheme, the ground state 2H resembles the ground state of
+
4H (as shown by a double-headed arrow) and the excited
+
+
state 2H resembles 5H (as shown by a double-headed
a
a
was markedly lower than that in the ground state (pK =
arrow). Upon excitation, the redistribution of charges in
2H takes place, resulting in a shift of electron density from
one nitrogen to another.
a
+
4
.20), indicating photoacidic properties. The same approach
+
for imidazole 4H revealed substantially less photoacidity
+
+
than 2H and negligible photoacidity for compound 5H .
The results shown in Figure 10 clearly illustrate which ni-
trogen in the imidazole ring is responsible for the pH sensi-
tivity in the ground and excited states. The dissociation con-
Conclusions
+
stants of 2H in the ground state were identical to the dis-
An imidazole incorporated into a fluorophore showed a
wealth of pH-dependent optical properties. Investigation
into the mechanisms of pH sensitivity was accomplished by
synthesizing the mono- and di-N-methylated analogues and
provided the following findings: 1) Fluorescent imidazole
was found to be pH-sensitive in all three ranges of the pH
scale from 2.0 to 11.0. Three distinct absorption bands at
360, 430, and 477 nm in acidic, neutral and basic conditions
were assigned to the protonated, neutral, and deprotonated
forms of imidazole. The deprotonotated and neutral forms
of fluorescent imidazole emitted at 530 and 560 nm corre-
spondingly; in acidic conditions, the emission shifted to
600 nm. 2) 1-N was determined to be a pH-sensitive center
in the ground state, whereas 3-N was responsible for pH
sensitivity in the excited state. Such switching between the
centers was attributed to the excited-state charge transfer
(ESCT) from one nitrogen to another, resulting in a positive
+
sociation constant of 4H (dashed vertical line on the right-
hand side) in the ground state, suggesting similar mecha-
nisms of protonation/deprotonation. Indeed, it is generally
accepted that the 3-N position in imidazoles is preferably
protonated under acidic conditions due to the lone electron
[30]
pair localized on the 3-N position. The unshared electron
pairs in 2 and 4, which are localized on the 3-N position, are
equally available for protonation and thus apparently have
similar basicity as indicated by their identical pK values,
a
+
which suggests the isoelectronic configuration of 2H and
+
4
H
in the ground state. In contrast to the ground state, the
+
excited state of imidazole 4H showed substantially higher
basicity (pK * 3.87) than 2H in its excited state (pKa*
+
a
2.82), demonstrating the divergence of their electronic con-
figurations in the excited state. In contrast, the dissociation
+
+
constants in the excited states of 2H and 3-N-Me 5H
pK * 2.85) were nearly identical, suggesting isoelectronic
(
charge swapping between two nitrogens. 3) pK analysis in
a
a
+
+
configurations of 2H and 5H in their excited states. Thus,
imidazole 2H is similar to 4H in the ground state and
5
the ground and the excited state showed photoacidic proper-
ties of the studied fluorescent imidazole, which were attrib-
uted to excited-state proton transfer (ESPT); this effect was
found negligible for substituted imidazoles and under basic
conditions. 4) Large Stoke shifts of the dyes in protonated
form and much shorter Stokes shift in the deprotonated
form clearly indicate the presence of pH-dependent twisted
+
+
+
H
in the excited state.
To explain this phenomenon, we propose a mechanism
based on excited-state charge transfer, where the rearrange-
ment in electron density upon excitation leads to a swap of
a positive charge between 1-N to 3-N. Upon excitation, the
+
+
+
Scheme 3. Proposed Fçrster cycle for imidazole compounds, illustrating the similarity between 2H and 4H in the ground state and between 2H and
+
5
H in the excited state.
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3565

S. Achilefu et al.
intramolecular charge transfer (TICT). The mechanism of
this channel as well as theoretical experimental data con-
firming the presence of the process will be the focus of fur-
ther study.
[
[
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(
(
(
2-carboxyethyl)-1,1,2-trimethyl-1H-benzo[e]indolium
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1
bromide
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4
6
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4
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[
2
desired pH was attained by titrating with aqueous HCl, or backwards at
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[
[
[
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[
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solution (10 uL) was added to cuvettes with neutral and acidic glycerol
(
1 mL) (acidified with TFA, total volume of TFA 0.06 vol%). The glycer-
ol solutions were stirred with a small spatula directly in a cuvette and al-
lowed to stand for 2 h in the dark for complete homogenation.
[
[
[
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Acknowledgements
We thank Dr. Yunpeng Ye for providing 3-(2-carboxyethyl)-1,1,2-trimeth-
yl-1H-benz[e]indolium bromide, and Prof. Vladimir Birman for fruitful
discussions. This study was supported in part by the NIH (NIBIB R01
EB7276).
Received: August 29, 2008
Revised: December 18, 2008
Published online: February 11, 2009
3566
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Chem. Eur. J. 2009, 15, 3560 – 3566