Enhancing the photostability of arylvinylenebipyridyl compounds as fluorescent indicators for intracellular zinc(II) ions

Article abstract of DOI:10.1021/acs.joc.5b00503

Arylvinylenebipyridyl (AVB) ligands are bright, zinc(II)-sensitive fluoroionophores. The applicability of AVBs as fluorescent indicators for imaging cellular zinc(II), however, is limited by low photostability, partially attributable to the photoisomeriza

Full text of DOI:10.1021/acs.joc.5b00503

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Article
Enhancing the Photostability of Arylvinylenebipyridyl (AVB)
Compounds as Fluorescent Indicators for Intracellular Zinc(II) Ions
Zhao Yuan, Ali H. Younes, John R. Allen, Michael W. Davidson, and Lei Zhu
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b00503 • Publication Date (Web): 05 May 2015
Downloaded from http://pubs.acs.org on May 18, 2015
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Enhancing the Photostability of
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Arylvinylenebipyridyl (AVB) Compounds as
Fluorescent Indicators for Intracellular Zinc(II) Ions
†
†
‡
,‡
,†
Zhao Yuan, Ali H. Younes, John R. Allen, Michael W. Davidson,* and Lei Zhu*
†
Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way,
Tallahassee, FL 32306ꢀ4390, USA; ‡ National High Magnetic Field Laboratory and Department
of Biological Sciences, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL
32310, USA
ABSTRACT. Arylvinylenebipyridyl (AVB) ligands are bright, zinc(II)ꢀsensitive
fluoroionophores. The applicability of AVBs as fluorescent indicators for imaging cellular
zinc(II), however, is limited by their low photostability, partially attributable to the
photoisomerization of the vinylene functionality. Two configurationally immobilized (i.e.,
“locked”) AVB analogues are prepared in this work. The zinc(II)ꢀsensitive photophysical
properties and zinc(II) affinities of both AVBs and their locked analogues are characterized in
organic and aqueous media. The zinc(II) sensitivity of the emission is attributed to the zinc(II)ꢀ
dependent energies of the charge transfer excited states of these compounds. The
configurationally locked ligands have improved photostability, while maintaining the brightness
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and zinc(II) sensibility of their AVB progenitors. The feasibility of the “locked” AVB analogues
with improved photostability in imaging intracellular Zn(II) of eukaryotic cells using laser
confocal fluorescence microscopy is demonstrated.
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KEYWORDS. internal charge transfer, zinc, bipyridyl, photostability, fluorescent indicator.
Introduction
1
ꢀ3
Zinc(II) ions are involved in a broad spectrum of mammalian physiological processes. The
amount of kinetically labile (sometimes referred as “free”) zinc(II) in an organism is maintained
4
ꢀ6
7,8
in a delicate homeostatic balance, the deviation from which is implicated in many diseases.
The understanding on the cellular and molecular levels the functions of zinc(II) in these diseaseꢀ
causing processes is expected to aid diagnosis and treatment. An indicator that is capable of
selectively recording zinc(II) abundance and trafficking in living cells in preferably a
quantitative manner would be an invaluable tool in zinc(II) biology. The choice of fluorescence
as the signal readout is attributed to the minimally invasive liveꢀcell imaging capabilities and the
9
everꢀincreasing spatial and temporal resolutions of modern fluorescence microscopes. The
10,11
success of fluorescent calcium(II) indicators
is a significant facilitator in the development of
zinc(II) indicators, by providing valuable experiences in the designs of ligands and fluorophores.
1
2ꢀ16
Zinc(II) indicators
based on fluorescence intensity readout can be separated in two
1
7,18
categories.
A fluorescence turnꢀon indicator reports the zinc(II) concentration by a
proportional increase in fluorescence intensity without changing its frequency. The fluorescence
intensity at a single wavelength therefore is a function of zinc(II) concentration. A fluorescence
ratiometric indicator undergoes zinc(II)ꢀdependent emission frequency change without
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significantly altering the fluorescence quantum yield. Therefore, the zinc(II) concentration is the
function of the ratio of emission intensity at two wavelengths, those of the free and zinc(II)ꢀ
bound indicators. A ratiometric readout is sometimes preferred over the absolute intensity
readout of a turnꢀon indicator, because the output dependence on photobleaching and uneven
loading of dyes as well as on instrumental variations are somewhat minimized in the ratiometric
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mode.
bathochromic
shift
hypsochromic
shift
Zn(II)
Zn(II)
"
pull"
"push"
donor
acceptor
acceptor
donor
ground state
excited state
Figure 1. A cartoon of chargeꢀtransfer (or “pushꢀpull”) fluoroionophore, and the effect on
emission via zinc(II) coordination.
From a molecular design point of view, turnꢀon and ratiometric indicators require different
structural considerations. A turnꢀon indicator is a fluorophore of which the major nonꢀradiative
decay pathway is eliminated upon forming a zinc(II) complex, while the wavelength of emission
is unchanged. For a ratiometric indicator, the ground and excited state energies are altered
differently upon binding zinc(II), thus resulting an emission frequency change. A straightforward
method for creating a ratiometric indicator is to exploit the differing electrostatic interactions
between a zinc(II) ion and ground and excited state dipoles of a chargeꢀtransfer fluorophore (also
commonly referred as a donorꢀacceptor, or a “pushꢀpull” fluorophore). Assuming the excited
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state has a larger dipole moment than the ground state, cation coordination at either the positive
or the negative end of the excited state dipole (or the disjoint HOMO or LUMO orbital in a
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quadruple
) would lead to a hypsochromic or a bathochromic shift, respectively, of emission
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and/or excitation (Figure 1). Other frequencyꢀchanging molecular photophysical processes such
as excimer formation, excited state proton transfer, and Förster resonance energy transfer, can
1
5,16
also be rendered zinc(II)ꢀdependent for creating ratiometric zinc(II) indicators.
Our group has studied the fluorescence properties of arylvinylenebipyridyl (AVB) compounds,
2
2,23
which are considered chargeꢀtransfer fluorophores.
An example is compound 1 (Chart 1),
which has a pꢀmethoxyphenyl as the electronꢀdonating group that is conjugated via a vinylene
functionality to the electronꢀwithdrawing bipyridyl. The pushꢀpull AVBs show a positive
emission solvatochromic effect, and bathochromic shifts in both emission and excitation upon
zinc(II) binding at the bipyridyl site. In spite of the brightness and zinc(II) sensitivity of the
2
2
AVBs, such as 1 and 3 (Chart 1), one drawback of this series of compounds is their lack of
photostability resulting from their propensity of photoisomerization. To address this issue,
compounds 2 and 4 (Chart 1) are prepared in the current work, in which the vinylene trans
configurations are frozen in a furan ring. As a result, the zinc(II) sensitivity of their AVB
precursors 1 and 3 is preserved, while the photostability is improved.
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Chart 1. Structures.
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Results and Discussion
1. Synthesis. The synthesis of compound 2 is shown in Scheme 1. HornerꢀWadsworthꢀEmmons
2
4
25
reaction between aldehyde 5 and diethyl phosphonate 6 afforded compound 7 in 80%.
Removal of the methoxymethyl (MOM) group of 7 by TsOH resulted in compound 8, which
26
upon treating with I in the presence of K CO was converted to compound 2.
2
2
3
a
Scheme 1. Synthesis of compound 2.
a. Reagents and conditions: a) tꢀBuOK, THF, 80%; (b) TsOH⋅H O, CH OH, 90%; (c) K CO ,
I2, THF, 95%.
2
3
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The synthesis of compound 4 is outlined in Scheme 2. Compound 11 was prepared from the
commercially available 3ꢀmethoxythiophene (9) in two steps. Protection of the hydroxy group of
1
1 by methoxymethyl bromide (MOMBr) afforded aldehyde 12 in 95%, which underwent the
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HornerꢀWadsworthꢀEmmons reaction with diethyl phosphonate 6 to give compound 13 in 66%.
The MOM group of compound 13 was removed using TsOH to result in 14; the oxidative
cyclization of which using K CO and I afforded compound 4.
2
3
2
a
Scheme 2. Synthesis of compound 4.
S
S
S
S
S
CHO
O
CHO
CHO
N
a
b
c
d
O
O
N
O
OCH3
OCH3
10
OH
11
O
9
12
13
S
EtO
N
f
N
S
e
N
P
OEt
HO
O
N
N
6
N
14
4
a. Reagents and conditions: (a) nBuLi, DMF, 40%; (b) BBr , CH Cl , 75%; (c) MOMBr,
3
2
2
DIPEA, THF, 95%; (d) 6, KHMDS, THF, 66%; (e) TsOH⋅H O, CH OH, 99%; (f) K CO , I ,
2
3
2
3
2
THF, 95%.
2. Solventꢀdependent fluorescence of compounds 2 and 4. Apart from the reinforced transꢀ
configuration in a furan ring, the structure of compound 2 is similar to that of 1. The
fluorescence quantum yield (φ) and lifetime (τ) of compounds 1 and 2 were measured in
different solvents (Table 1). The time constants of radiative (k ) and nonꢀradiative (k ) decays
r
nr
(
Table 1) were calculated based on equations 1 and 2. The φ and τ values of the
configurationallyꢀimmobilized compound 2 are larger than those of the photoisomerizable 1, by
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0% and 100% on average, respectively. Both compounds have similar k values in the same
r
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solvents. However, compound 2 has significantly smaller k than that of 1 in all solvents. These
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data suggest that the configurational stability of 2 does not contribute to a more allowed radiative
transition (a larger k ). Rather, the rate of nonꢀradiative decay is reduced (a smaller k , by 78%
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on average), most likely a consequence of eliminating the photoisomerization pathway.
ꢂ
ꢀꢁ
= ꢃ
(1)
(2)
ꢅꢆꢂ
ꢃ
ꢀꢄꢁ
=
Table 1. Fluorescence quantum yield (φ), lifetime (τ), and radiative and nonꢀradiative decay rate
a
constants (k and k ) of compounds 1 and 2 in different solvents.
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nr
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
Solvent
Hexanes
Dioxane
Benzene
Toluene
CHCl3
EtOH
ε
φ (1) φ (2) τ/ns (1)
τ/ns (2)
1.12
1.47
1.30
1.28
1.57
2.02
2.18
1.84
1.80
1.80
2.48
k /ns (1)
k /ns (2)
k /ns (1)
k /ns (2)
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r
nr
nr
2.02
2.3
2.3
2.4
4.8
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0.60
0.49
0.53
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0.48
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0.84
0.78
0.75
0.71
0.70
0.83
0.79
0.91
0.69
0.82
0.25
0.91
0.84
0.75
0.79
0.70
0.92
0.95
0.60
0.76
0.86
1.11
0.65
0.60
0.71
0.69
0.52
0.39
0.35
0.47
0.48
0.56
0.04
0.75
0.53
0.58
0.55
0.45
0.41
0.36
0.49
0.38
0.46
0.10
0.43
0.59
0.62
0.56
0.90
0.69
0.69
1.20
0.82
0.59
0.86
0.14
0.15
0.19
0.23
0.19
0.08
0.08
0.05
0.17
0.10
0.30
MeOH
MeCN
DMF
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36.6
38.3
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DMSO
b
H O
2
78.5
a. ε: dielectric constant; φ: fluorescence quantum yield; τ: fluorescence lifetime determined via
timeꢀcorrelated singleꢀphoton counting (TCSPC); k and k : radiative and nonꢀradiative decay
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time constants, determined using equations (1) and (2). The table is sorted in the ascending order
of ε; b. an aqueous solution buffered by HEPES (100 mM) at pH 7.4.
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It is noteworthy that the high fluorescence quantum yield (φ) of compound 2 measured in
organic solvents (≥ 0.69) depends little on solvent polarity. The lifetime (τ) of 2 increases
moderately as the solvent polarity grows, which can be attributed to the increasing chargeꢀ
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transfer character of the emissive excited state that likely decreases k . The substantial excited
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state dipole moment of compound 2 is reflected in the solventꢀdependent emission band shape
and wavelength (Figure 2a). A structured fluorescence spectrum was obtained in the nonpolar
hexanes, which do not offer strong solventꢀsolute interactions that would lead to inhomogeneous
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broadening of the spectrum.
As the polarity and/or the hydrogen bond donating ability of the
solvent increases, the strong solventꢀsolute interaction not only stabilizes the dipolar excited state
of 2 but contributes to the inhomogenous broadening, to result in a broad, structureꢀless emission
at a longer wavelength from that in hexanes. The normalized excitation of 2 in all solvents is
independent of emission wavelength (Figures S1ꢀS5), which indicates that solvent relaxation of
the excited state is fast and reaches equilibrium well within the lifetime of the excited
fluorophore. Based on the literature convention, the structured high energy emission of 2 in
hexanes might be referred as the locally excited (LE) emission, whereas the structureꢀless low
energy emission in more polar solvents is considered charge transfer (CT) emission, although the
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meanings of LE and CT could become ambiguous under different contexts.
The emission of
compound 4 has similar solvent dependency; the spectra of which are shown in Figure 2b.
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Figure 2. Normalized emission spectra of (a) 2 (2.2 ꢀM), and (b) 4 (2.5 µM) in different
solvents (shown in the legend in b). λ = 340 nm.
ex
The φ and τ values of compounds 3 and 4 along with the k and k values are listed in Table 2.
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The k values of the configurationally locked 4 in the tested solvents are larger than that of the
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“
unlocked” olefin analog 3. The rates of nonꢀradiative decay (k ) of 4 are significantly smaller
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than those of 3 in all solvents. These observations are consistent with the comparison between
compounds 1 and 2, and support the rationale that eliminating the potential of
photoisomerization in the AVB fluoroionophores by confining the trans double bond in a
heterocycle decreases the rate of nonꢀradiative decay, hence increasing the fluorescence quantum
yield.
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Table 2. Fluorescence quantum yield (φ), lifetime (τ), and radiative and nonꢀradiative decay rate
a
constants (k and k ) of compounds 3 and 4 in different solvents.
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nr
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
Solvent
Hexanes
Dioxane
Benzene
Toluene
CHCl3
EtOH
ε
φ (3)
0.16
0.10
0.10
0.12
0.07
0.05
0.04
0.04
0.06
0.07
0.06
φ (4)
0.50
0.50
0.59
0.58
0.44
0.66
0.65
0.60
0.67
0.76
0.54
τ/ns (3)
0.46
0.41
0.40
0.43
0.34
0.33
0.32
0.23
0.32
0.33
0.30
τ/ns (4)
0.85
0.94
0.85
0.86
0.92
1.63
1.82
1.30
1.34
1.51
2.31
k /ns (3)
k /ns (4)
k /ns (3)
k /ns (4)
r
r
nr
nr
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9
0
2.02
2.3
2.3
2.4
4.8
25
0.35
0.24
0.25
0.28
0.21
0.15
0.13
0.17
0.19
0.21
0.20
0.59
0.53
0.69
0.67
0.48
0.40
0.36
0.46
0.50
0.50
0.23
1.83
2.20
2.25
2.05
2.74
2.88
3.00
4.17
2.94
2.82
3.13
0.59
0.53
0.48
0.49
0.61
0.21
0.19
0.31
0.25
0.16
0.20
MeOH
MeCN
DMF
33
36.6
38.3
47
DMSO
b
H O
2
78.5
a. ε: dielectric constant; φ: fluorescence quantum yield; τ: fluorescence lifetime determined via
timeꢀcorrelated singleꢀphoton counting (TCSPC); k and k : radiative and nonꢀradiative decay
r
nr
constants, determined using equations (1) and (2). The table is sorted in the ascending order of ε;
b. an aqueous solution buffered by HEPES (100 mM) at pH 7.4.
a
Table 3. Photophysical parameters of 1ꢀ4 (L) and their zinc(II) complexes (ZnL) in CH CN.
3
L
λ
abs(L)
λ
abs(ZnL)
φL
φZnL
λ
(nm)
em(L)
λ
em(ZnL)
ꢁꢇꢈ
τ
L
τ
ZnL
k
r(L)
ꢀ1
k
r(ZnL)
ꢀ1
k
nr(L)
ꢀ1
k
nr(ZnL)
ꢀ1
em
ꢀ
1
(
nm)
(nm)
374
381
375
380
(nm)
533
538
504
505
(cm ) (ns) (ns)
(ns ) (ns ) (ns ) (ns )
1
2
3
4
345
347
348
343
0.28 0.46 431
0.91 0.34 435
0.04 0.25 422
0.60 0.55 420
4,440
4,401
3,855
4,008
0.60 3.14 0.47
0.15
0.10
0.13
0.15
1.20
0.05
4.17
0.31
0.17
0.20
0.39
0.12
1.84 3.33 0.49
0.23 1.91 0.17
1.30 3.71 0.46
a. λabs(L) and λabs(ZnL) are wavelengths at absorbance maxima; φ and φZnL are fluorescence
L
quantum yields; λem(L) and λem(ZnL) are wavelengths at emission maxima; ꢁ ꢇꢈ is the zinc(II)ꢀ
em
ꢀ1
binding caused emission frequency shift in cm ; τ and τ are fluorescence lifetimes; k and k
L
ZnL
r
nr
are calculated from equations (1) and (2).
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3
. Fluorescence Titrations of 1ꢀ4 with Zn(ClO ) in CH CN. The fluorescence quantum yields,
4
2
3
lifetime, and emission maxima of compounds 1ꢀ4 under zinc(II)ꢀfree and zinc(II)ꢀsaturated
conditions in CH CN are listed in Table 3. Similar to their configurationally unlocked olefin
3
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counterparts, the binding of zinc(II) (in the form of Zn(ClO ) ) of the locked CT
4
2
fluoroionophores 2 and 4 in CH CN leads to bathochromic shifts of their absorption and
3
emission spectra (Figures S6ꢀS7). As an example, the zinc(II) titration data using compound 4 in
both absorption and emission modes are included as Figure S9. Both lowest energy absorption
and emission bands of 4 were redꢀshifted as zinc(II) titration proceeded. Therefore, this
compound could be used for analyzing zinc(II) in a ratiometric mode, i.e., the ratio of the
absorption or the emission bands of the zinc(II)ꢀfree and the zinc(II)ꢀbound forms, respectively,
is a function of zinc(II) concentration. The pꢀmethoxyphenylꢀcontaining compounds 1 and 2 are
capable of larger zinc(II)ꢀinduced emission bathochromic shifts (ꢁ ꢇꢈ , Table 3) than the thienylꢀ
em
containing 3 and 4, suggesting that the pꢀmethoxyphenyl group is a more effective electronꢀ
donating group than the thienyl in CT type fluorophores. Fluorescence quantum yield (φ) relates
to the rates of both radiative (k ) and nonꢀradiative decays (equation 3). Unlike compounds 1 and
r
3
, the φ values of which grow upon zinc(II) binding due to a more significant decrease in knr
relative to k (Table 3), the metalꢀfree forms of compounds 2 and 4 already have high
r
fluorescence quantum yields, which are slightly reduced upon zinc(II) binding, attributable to the
steeper drop of k relative to k (Table 3).
r
nr
ꢊꢋ
ꢉ =
(3)
ꢊꢋꢌꢊ
ꢍꢋ
4. Fluorescence Titrations of 1ꢀ4 with Zn(ClO ) under Physiologically Relevant Conditions.
4
2
The zinc(II)ꢀdependent fluorescence of 1ꢀ4 was also studied under simulated physiological
conditions to evaluate the applicability of these ligands in imaging zinc(II) ions in biological
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1
1
1
1
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1
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2
2
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12ꢀ16
systems.
The zinc(II)ꢀdependent emission wavelengths and quantum yields of 1ꢀ4 are listed
in Table 4. In a neutral aqueous solution (see caption of Figure 3), the emission maximum of 2
478 nm, φ = 0.25) appears at a longer wavelength than that of 1 (460 nm). The free ligand 1 has
(
0
1
2
3
4
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9
0
1
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0
a fluorescence quantum yield (φ) of 0.05. The titration of Zn(ClO ) leads to the formation of
4 2
bipy/zinc(II) complex, which at saturation results in a φ of 0.31 along with a red shift of 53 nm
ꢀ1
(
ꢁ ꢇꢈ em = 2,246 cm ) of compound 1. Under the same conditions, the zinc(II) complex of the
configurationally rigid 2 had a longer emission band (524 nm, φ = 0.15) with a smaller zinc(II)ꢀ
ꢀ1
effected bathochromic shift (46 nm; 1,837 cm ).
Excited at 405 nm where the zinc(II) complex preferentially absorbs, the zinc(II)ꢀdependent
emission spectra of 2 acquired under different conditions are shown in Figure 3. The emission
spectra of 1, 3, and 4 are included in the Supporting Information (Figures S10ꢀS12). A largely
emission enhancement, instead of a ratiometric change, as [zinc(II)] increases was observed due
34
to the increased absorption from the zinc(II) complex at the excitation wavelength of 405 nm.
The intensity at the emission maxima was plotted versus the free zinc(II) concentration [Zn] (see
f
insets of Figure 3) for assessing the zinc(II) affinity of 2. The [Zn] values were controlled using
f
35
a ligand at millimolar with known zinc(II) affinity (a “metal buffer”). In this case, three metal
buffers were evaluated for their appropriateness in determining the affinity of the ligands to
36
zinc(II), as measured by the apparent 1:1 dissociation constant (K ). Using nitrilotriacetic acid
d
(
NTA, K (Zn) = 14 nM) as the metal buffer, the emission of compound 2 (Figure 3a) grew with
d
increasing [Zn] . However, when the upper limit of the NTA buffering range was reached, the
f
emission of 2 was not yet half saturated, as marked by the red curve. A similar observation was
made when Nꢀ(2ꢀacetamido)iminodiacetic acid (ADA, K (Zn) = 83 nM, Figure 3b) was used as
d
the metal buffer. Although the binding isotherms in both cases are fittable using the 1:1 binding
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equation (see the fitting curves in the insets), in our view, those numbers overestimated the
zinc(II) affinity of 2 (the K values are smaller than the true value). Citrate (K (Zn) = 12 ꢀM)
d
d
allowed the zinc(II) titration to reach saturation (Figure 3c), the fitting of the binding isotherm
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gave a K of 0.8 ꢀM. Although based on the initial large changes in the titration data, citrate is an
d
insufficient zinc(II) buffer for competing zinc(II) with compound 2. Based on these observations,
one would draw the conclusion that the apparent K of the presumed ZnL complex is somewhere
d
3
6
between 20 nM and 800 nM. The estimates of the K values of compounds 1, 3, and 4 were
d
obtained similarly and are listed in Table 4.
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0
Figure 3. Fluorescence spectra of 2 (2.5 ꢂM, λ = 405 nm) in the presence of Zn(ClO ) (0ꢀ2
ex
4 2
mM) in an aqueous solution ([HEPES] = 100 mM, [metal buffer] = 1 mM, [KNO ] = 100 mM,
3
pH 7.4). Metal buffer = NTA (a); ADA (b); and citrate (c). The initial zinc(II)ꢀfree and the final
zinc(II)ꢀsaturated spectra of each titration experiment are coded blue and red, respectively.
Insets: fluorescence intensity at indicated emission wavelength vs free zinc(II) concentration
3
7
[
Zn] . The solid lines are fitting curves using a 1:1 binding model.
f
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Table 4. Photophysical parameters of ligands (L) 1ꢀ4 and their zinc(II) complexes [ZnL](ClO4)2
a
in a neutral aqueous solution.
L
λabs(L)
λabs(ZnL
φL
φZnL
λem(L)
λem(ZnL)
τL
τZnL
k
k
k
k
Kd
pKa
4.5
4.3
r(L)
r(ZnL)
nr(L)
ꢀ1
nr(ZnL)
ꢀ1
ꢀ
1
ꢀ1
(nm)
(nm)
)
(nm)
(nm)
(ns)
(ns)
(ns )
(ns )
(ns ) (ns )
(ꢀM)
10
11
12
13
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1
2
3
4
344
347
350
347
368
373
371
372
0.05 0.31 460
0.25 0.15 478
0.06 0.18 456
0.54 0.44 459
513
524
493
495
1.11 2.69
2.48 2.42
0.30 1.75
2.31 3.29
0.04
0.12
0.86
0.30
3.13
0.20
0.26
0.35
0.47
0.17
0.04ꢀ
2
0.10
0.20
0.23
0.06
0.10
0.13
0.02ꢀ
0.8
0.009 4.3
0.2
ꢀ
0.01ꢀ
.4
4.3
0
a. φ and φ
are the fluorescence quantum yields in zinc(II)ꢀfree and –saturated forms,
L
ZnL
respectively; τ and τ are the fluorescence lifetimes; k and k are calculated from equations
L
ZnL
r
nr
(1) and (2); K is the dissociation constant of a presumed ZnL complex; K is the acidity constant
d
a
of the conjugate acid of the ligand. All data except pK were measured in an aqueous solution
a
buffered by HEPES (100 mM) at pH 7.4.
The pK values of ligands 1ꢀ4 cluster in the narrow range of 4.3ꢀ4.5. Therefore, under pH
a
neutral conditions these compounds are in their neutral base forms. The Zn(II) sensitivity of
these compounds, therefore, shall be free of interference from pH fluctuation in the
physiologically tolerated range (from 4.8 in lysosome to 7.4 of blood). Similar to other bipyꢀ
3
8,39
containing fluorophores that we have reported,
compounds 1ꢀ4 preferentially binds transition
metal ions over alkali and alkaline earth metal ions. As expected, the selectivity of the new
compounds 2 and 4 (Figures S13ꢀS14) roughly follows the predictions of the IrvingꢀWilliams
4
0
order.
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Figure 4. The absorption spectra of compounds (a) 1 and (b) 2 at 5 ꢀM each in 10% DMSOꢀ
containing aqueous solution (HEPES: 100 mM, pH = 7.4, NTA: 1 mM, KCl: 100 mM) acquired
under irradiation from a handheld UV lamp (365 nm, 4 W) for 1 min. The spectra were collected
at 10 s increments from blue to red traces. The changes of absorption spectra of (c) 1 and (d) 2
under the same conditions in the presence of Zn(ClO ) (2 mM).
4
2
The contrast in photostability between transꢀolefin 1 and configurationally locked analogue 2
is apparent in their absorption sensitivity to UV irradiation in an aqueous solution buffered at pH
7
.4. When placing under the irradiation of a handheld UV lamp (λ = 365 nm), the absorption
ex
band of compound 1 drops quite significantly (Figure 4a), consistent with the occurrence of a
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rapid photoisomerization. When a sample of 2 was subjected to the same treatment, the
absorption change was rather minimal (Figure 4b). Similarly superior photostability of 2 over 1
was observed while the ligands were saturated with Zn(ClO ) (Figures 4cꢀd). The saturation of
4
2
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compound 1 with zinc(II) appears to stabilize the compound against photoisomerization by a
moderate extent, an interesting observation that is worthy of further investigation.
5. Laser confocal fluorescence microscopy. The ability of 1ꢀ4 to image intracellular free
zinc(II) was evaluated in live eukaryotic cells. HeLa S cells were incubated in culture medium
3
in the presence of a ligand (2 ꢂM) for 30 min. The medium was removed, and the cells were
rinsed twice with fresh, indicatorꢀfree medium. The cells were further incubated in the presence
of sodium pyrithione (50 ꢂM), an ionophore for facilitating zinc(II) entry through the cell
4
1
membrane, and either 0 or 50 ꢂM ZnCl for 10 min. Cell morphology was monitored by
2
acquiring brightfield differential interference contrast (DIC) images in parallel with the
fluorescence images. The lack of change in cell morphology was an indication that the indicator
did not exert toxicity during the course of the incubation and the imaging experiment, which
lasted over 1 h.
All fluorescence images were acquired on a confocal laser pointꢀscanning fluorescence
microscope under the excitation of a 405ꢀnm diode laser. An emission window of 530ꢀ570 nm
was selected via application of a variable bandpass filter. The images of cells stained by the
configurationally locked compounds 2 or 4 are shown in Figure 5 in both fluorescence (bottom)
and DIC modes (top). Using the identical instrumental parameter set, the fluorescence intensity
of cells stained with either 2 or 4 in the presence of 50 ꢀM of ZnCl is much higher than that of
2
the cells incubated in medium without zinc(II) supplementation. This data demonstrates the
abilities of 2 and 4 in imaging zinc(II) ions in live cells. The difference in the distribution
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patterns highlights the inability of predicting and controlling the intracellular destinations after
4
2,43
uptake of synthetic indicators, which has been pointed out by other researchers.
0
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8
9
0
Figure 5. Images of HeLa S cells incubated with 2 (a, b) or 4 (c, d) at 2 ꢂM for 30 min at 37 ºC.
3
The upper and lower panels are the differential interference contrast (DIC) and the fluorescence
images (λ = 405 nm, λ 530ꢀ570 nm), respectively. Panels (a) and (c) are images taken in the
ex
em
absence of supplemental zinc(II) ([ZnCl ] = 0), while images in (b) and (d) are taken under
2
zinc(II)ꢀenriched conditions ([ZnCl ] = 50 ꢂM). [sodium pyrithione] = 50 ꢂM. The scale bar is
2
20 ꢂm.
6. Timeꢀlapse experiments for assessing photostability. The photostability of the
photoisomerizable compound 1 and 3 relative to that of their configurationally locked
counterparts 2 and 4 in live cell fluorescence imaging experiments was assessed in the timeꢀlapse
video mode, while the fluorescence intensity of the samples was recorded over time under
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constant 405ꢀnm diode laser irradiation. Fluorescence images were selected from the video data
at defined time intervals (Figures 6 and 7). The timeꢀlapse data of cells incubated with
compounds 1 and 2 under zinc(II)ꢀenriched conditions (50 ꢀM in the culture medium) were
collected using the same set of instrumental parameters. Therefore, the decays of the
fluorescence intensity originated from 1 and 2, averaged over a number of regions of interest
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(ROIs), can be compared for assessing the relative photostability of these two compounds.
Figure 6. Fluorescence images of HeLa S cells (λ = 405 nm, emission 530ꢀ570 nm) after
3
ex
incubated with “unlocked” 1 (upper) or “locked” 2 (lower) at 2 ꢀM, in the presence of ZnCl (50
2
ꢂM) and sodium pyrithione (50 ꢂM) over time under continuous laser irradiation (images shown
every 20 frames, approximately every 1 min from left to right). The scale bar is 20 ꢂm.
For both 1 and 2, the fluorescence intensity decayed over time by visual examination (Figure
6). The intensity as a function of time is plotted in Figure 8a. It is apparent that the emission
from cells loaded with compound 2 under zinc(II)ꢀenriched conditions (50 ꢀM ZnCl in the
2
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growth medium) decayed slower than the cells stained with compound 1 under the identical
conditions. The fluorescence images and intensity decay traces from the timeꢀlapse experiments
using 3 and 4 were shown in Figures 7 and 8b. Similarly, the configurationally immobilized 4
shows moderately but consistently higher photostability than the isomerizable counterpart 3.
0
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Figure 7. Fluorescence images of HeLa S cells (λ = 405 nm, emission 530ꢀ570 nm) after
3
ex
incubated with “unlocked” 3 (upper) or “locked” 4 (lower) at 2 ꢀM, in the presence of ZnCl (50
2
ꢂM) and sodium pyrithione (50 ꢂM) over time under continuous laser irradiation (images shown
every 20 frames, approximately every 1 min from left to right). The scale bar is 20 ꢂm.
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Figure 8. (a) Intracellular intensity of HeLa S cells (averaged over 21 cells, λ = 405 nm, λ
em
3
ex
530ꢀ570 nm) over time when incubated with 1 (green) or 2 (blue) at 2 ꢀM, in the presence of
ZnCl (50 ꢀM) and sodium pyrithione (50 ꢀM). (b) Same comparison between 3 (green) and 4
2
(blue).
Conclusion
In summary, the photostability of the chargeꢀtransfer fluoroionophore arylvinylenebipyridyls 1
and 3 is improved in their configurationally immobilized analogues 2 and 4, respectively. The
fluorescence quantum yield and lifetime measurements attribute the enhanced photostability to
the reduction of the rate of nonꢀradiative decay, presumably by blocking the photoisomerization
pathway of the excited state. The brightness and zinc(II) sensitivity of the arylvinylenebipyridyls
are maintained, which enable the configurationally locked compounds 2 and 4 to act as
fluorescent indicators for zinc(II) ions in laser confocal microscopic imaging experiments.
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2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
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5
5
5
5
5
5
5
5
5
6
Experimental Section
a) Materials and general methods. Reagents and solvents were purchased from various
(
commercial sources and used without further purification unless otherwise stated. Spectroscopic
grade solvents were used in the UVꢀvis and fluorescence spectroscopic measurements. All
reactions were carried out in ovenꢀ or flameꢀdried glassware in an inert atmosphere of argon.
Analytical thinꢀlayer chromatography (TLC) was performed using preꢀcoated TLC plates with
silica gel 60 F254. Flash column chromatography was performed using silica (230ꢀ400 mesh) gel
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
as the stationary phases. H and C NMR spectra were acquired on a 300 MHz and a 500 MHz
instruments, with frequencies indicated in the synthetic procedures. All chemical shifts were
reported in δ units relative to tetramethylsilane. CDCl was treated with alumina gel prior to use
3
to remove trace acids. High resolution mass spectra (HRMS) were obtained using a timeꢀofꢀ
flight (TOF) analyzer.
(b) Syntheses and characterizations.
Compound 7. In the presence of tꢀBuOK (29 mg, 0.26 mmol), compounds 5 (271 mg, 1.38
mmol) and 6 (424 mg, 1.32 mmol) were dissolved in dry THF (10 mL) in a roundꢀbottom flask
equipped with a magnetic stir bar. The reaction mixture was stirred overnight. Afterwards, water
was added to quench the reaction and the solution was partitioned between water and EtOAc.
The organic layer was dried over Na SO and then concentrated under reduced pressure. The
2
4
crude product was fractionated on a silica gel column and the product 7 was isolated in a 80%
1
yield (384 mg) using CH Cl /EtOAc (0ꢀ30%) as the eluent. H NMR (300 MHz, CDCl ) δ/ppm
2
2
3
8.72 (d, J = 2.3 Hz, 1H), 8.49 (s, 1H), 8.33 (d, J = 8.3 Hz, 1H), 8.28 (d, J = 8.1 Hz, 1H), 7.95
(dd, J = 8.4, 2.3 Hz, 1H), 7.60 (dd, J = 8.2, 2.2 Hz, 1H), 7.57ꢀ7.47 (m, 2H), 7.01 (d, J = 16.6 Hz,
1H), 6.73 (d, J = 2.5 Hz, 1H), 6.59 (dd, J = 8.6, 2.5 Hz, 1H), 5.26 (s, 2H), 3.81 (s, 3H), 3.52 (s,
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9
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3
1
5
H), 2.38 (s, 3H); C NMR (75 MHz, CDCl ) δ/ppm 161.0, 156.1, 154.7, 153.7, 149.8, 148.1,
3
37.6, 133.8, 133.3, 133.2, 127.5, 125.4, 123.2, 120.7, 120.6, 119.7, 107.5, 101.7, 95.1, 56.4,
+
5.6, 18.5; HRMS (CI+): calcd. 363.1709 (C H O N + H ), found 363.1709.
2
2
23
3
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound 8. TsOH⋅H O (312 mg, 1.64 mmol) was added to a solution of compound 7 (300
2
mg, 0.828 mmol) in MeOH (10 mL) in a roundꢀbottom flask equipped with a magnetic stir bar.
The reaction mixture was stirred for overnight. The solvent was removed under reduced pressure
and the residue was washed with water, followed by trituration with ether. Compound 8 was
1
isolated in a 90% yield (237 mg) without further purification. H NMR (500 MHz, DMSOꢀd )
6
δ/ppm 9.98 (s, 1H), 8.74 (s, 1H), 8.52 (s, 1H), 8.34 (d, J = 8.3 Hz, 1H), 8.29 (d, J = 8.1 Hz, 1H),
8.10 (d, J = 8.5 Hz, 1H), 7.76 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 9.1 Hz, 1H), 7.50 (d, J = 16.3 Hz,
1
3
1
H), 7.19 (d, J = 16.6 Hz, 1H), 6.47 (s, 2H), 3.74 (s, 3H), 2.37 (s, 3H); C NMR (125 MHz,
DMSOꢀd ) δ/ppm 160.8, 157.0, 153.8, 153.1, 150.0, 148.1, 138.0, 133.8, 133.2, 128.5, 126.4,
6
122.0, 120.5, 120.2, 117.0, 106.1, 101.7, 55.5, 18.3; HRMS (EI+): calcd. 318.1368 (C H N O )
20
18
2
2
found 318.1347.
Compound 2. To a solution of 8 (300 mg, 0.942 mmol) in THF (50 mL) in a roundꢀbottom
flask equipped with a magnetic stir bar, anhydrous K CO (651 mg, 4.71 mmol) and I (1.2 g, 4.7
2
3
2
mmol) were added. The mixture was stirred at rt for 5 h. The resulting solution was poured into a
saturated NaHCO solution (20 mL) and treated with a saturated Na S O solution to remove the
3
2
2
3
unreacted iodine. Extraction with EtOAc afforded the organic layer that was dried and
concentrated. The crude solid was chromatographed on a silica gel column using EtOAc as the
1
eluent. Compound 2 was isolated in a 95% yield (283 mg). H NMR (300 MHz, CDCl ) δ/ppm
3
9
.10 (d, J = 2.1 Hz, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.44 (d, J = 8.4 Hz, 1H), 8.33 (d, J = 8.1 Hz,
H), 8.18 (dd, J = 8.3, 2.2 Hz, 1H), 7.64 (dd, J = 8.0, 2.2 Hz, 1H), 7.48 (dd, J = 8.6, 1.3 Hz, 1H),
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
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4
4
4
4
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5
5
5
5
5
5
5
5
5
6
13
7
.10 (s, 2H), 6.90 (dd, J = 8.5, 2.2 Hz, 1H), 3.89 (s, 3H), 2.41 (s, 3H); C NMR (75 MHz,
CDCl ) δ/ppm 158.7, 156.4, 155.4, 153.3, 152.5, 149.8, 145.4, 137.6, 133.6, 132.2, 126.4, 122.3,
3
1
21.4, 120.8, 120.7, 112.6, 102.9, 96.0, 55.9, 18.5; HRMS (EI+): calcd. 316.1212 (C H N O )
20 16 2 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
found 316.1204.
27
Compound 10. To a solution of 3ꢀmethoxythiophene 9 (1.0 g, 8.8 mmol) in THF (10 mL) at
ꢀ
78 °C, nꢀBuLi (3.9 mL of 2.5 M solution in hexanes, 9.7 mmol) was added. After stirring for 1
h, DMF (0.8 mL, 0.01 mol) was added. The solution was stirred for 1 h at rt and water was
added. The crude product of 3ꢀmethoxythiopheneꢀ2ꢀcarbaldehyde 10 was obtained by extraction
with CH Cl . The organic portions were dried over Na SO before concentrated under vacuum.
2
2
2
4
The residue was chromatographed on silica gel using CH Cl to obtain a pure product as a
2
2
1
yellow solid in a 40% yield (500 mg). H NMR (300 MHz, CDCl ): δ/ppm 9.96 (s, 1H), 7.63 (d,
3
J = 5.4 Hz, 1H), 6.85 (d, J = 5.4 Hz, 1H), 3.97 (s, 1H).
27
Compound 11. To a solution of 3ꢀmethoxythiopheneꢀ2ꢀcarbaldehyde 10 (0.50 g, 3.5 mmol)
in CH Cl (20 mL) at 0 °C, BBr (4.2 mL of 1 M solution in heptane, 4 mmol) was added slowly.
2
2
3
After being stirred for one day at rt, water was added to the reaction mixture. Product was
extracted by CH Cl . After silica column chromatography, the pure product was obtained as a
2
2
1
brown amorphous solid in a 75% yield (336 mg). H NMR (300 MHz, CDCl ): δ/ppm 10.72 (s,
3
1H), 9.64 (s, 1H), 7.60 (d, J = 5.3 Hz, 1H), 6.78 (d, J = 5.3 Hz, 1H).
Compound 12. To a solution of 3ꢀhydroxythiopheneꢀ2ꢀcarbaldehyde 11 (336 mg, 2.63 mmol)
and DIPEA (596 µL, 3.42 mmol) in THF (5 mL) at 0 °C was added MOMBr (279 µL, 3.42
mmol) over a 5 min period. The solution was stirred overnight, then diluted with EtOAc (25
mL), washed with brine (3 × 25 mL), dried over Na SO and filtered. The solvent was removed
2
4
under vacuum to yield an orange oil. Silica chromatography (eluted by CH Cl , isocratic) yielded
2
2
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the pure aldehyde 12 as a yellow oil in a 95% yield (430 mg). H NMR (500 MHz, CDCl )
3
δ/ppm 10.05 (s, 1H), 7.62 (d, J = 5.4 Hz, 1H), 7.00 (d, J = 5.4 Hz, 1H), 5.25 (s, 2H), 3.54 (s,
1
3
3
H); C NMR (125 MHz, CDCl ) δ/ppm 181.7, 162.3, 135.0, 123.2, 118.4, 96.4, 56.9; HRMS
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
(
ESI+): calcd. 195.0092 (C H O S + Na ) found 195.0100.
7
8
3
Compound 13. A flameꢀdried flask was charged with dry THF (20 mL) and 6 (577 mg, 1.80
mmol), and cooled to ꢀ78 °C. Compound 12 (310 mg, 1.80 mmol) and KHMDS (4.0 mL of 0.5
M in toluene, 2 mmol) were added dropwise sequentially. The mixture turned dark reddish after
1
5 min. The temperature was then slowly raised to rt in 2 h. The reaction mixture was poured
into icy saturated brine and extracted with CH Cl . The organic portions were washed with
2
2
saturated brine and dried over Na SO . The olefin product 13 as a yellow solid was obtained
2
4
after fractionation on a silica column eluted by 50% EtOAc in CH Cl in a 66% yield (404 mg).
2
2
1
H NMR (500 MHz, CDCl ) δ/ppm 8.69 (d, J = 2.2 Hz, 1H), 8.51 (dd, J = 1.5, 0.7 Hz, 1H), 8.33
3
(d, J = 8.4 Hz, 1H), 8.29 (d, J = 8.1 Hz, 1H), 7.93 (dd, J = 8.4, 2.3 Hz, 1H), 7.62 (dd, J = 8.1, 2.2
Hz, 1H), 7.43 (d, J = 16.3 Hz, 1H), 7.13 (d, J = 5.5 Hz, 1H), 6.96 (d, J = 5.5 Hz, 1H), 6.83 (d, J =
1
3
1
6.3 Hz, 1H), 5.18 (s, 2H), 3.54 (s, 3H), 2.40 (s, 3H); C NMR (125 MHz, CDCl ) δ/ppm 154.8,
3
153.7, 153.1, 149.8, 147.9, 137.6, 133.4, 133.2, 133.0, 123.6, 122.3, 122.1, 120.8, 120.7, 119.3,
+
9
6.6, 56.5, 18.6; HRMS (ESI+): calcd. 361.0987 (C H N O S + Na ) found 361.0980.
19
18
2
2
Compound 14. TsOH⋅H O (456 mg, 2.40 mmol) was added to a solution of compound 13 (404
2
mg, 1.20 mmol) in MeOH (5 mL). The reaction mixture was stirred for overnight. The solvent
was removed under reduced pressure and the residue was diluted with CH Cl followed by
2
2
neutralization by a saturated NaHCO solution. The organic layer was collected and concentrated
3
under vacuum. Compound 14 was isolated via silica chromatography (eluted by 1% MeOH in
1
CH Cl ) as a brown solid in a 99% yield (349 mg). H NMR (500 MHz, DMSOꢀd ) δ/ppm 10.13
2
2
6
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2
2
2
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(
s, 1H), 8.72 (d, J = 2.2 Hz, 1H), 8.51 (d, J = 2.2 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.27 (d, J =
8
.1 Hz, 1H), 8.07 (dd, J = 8.4, 2.2 Hz, 1H), 7.74 (dd, J = 8.1, 2.2 Hz, 1H), 7.53 (d, J = 16.2 Hz,
H), 7.34 (d, J = 5.4 Hz, 1H), 6.74 (d, J = 16.2 Hz, 1H), 6.70 (d, J = 5.4 Hz, 1H), 2.36 (s, 3H);
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
C NMR (125 MHz, DMSOꢀd ) δ/ppm 154.1, 153.3, 152.6, 149.5, 147.4, 137.5, 133.4, 133.3,
6
132.5, 124.4, 121.4, 120.8, 120.1, 119.8, 119.4, 115.6, 17.8; HRMS (ESI+): calcd. 295.0905
+
(C H N OS + H ) found 295.0894.
1
7
14
2
Compound 4. To a solution of compound 14 (156 mg, 0.531 mmol) in THF (10 mL),
anhydrous K CO (375 mg, 2.72 mmol) and I (691 mg, 2.72 mmol) were added. The mixture
2
3
2
was stirred at rt for 5 h. The resulting solution was poured into a saturated NaHCO solution (20
3
mL) and treated with a saturated Na S O solution to remove the unreacted iodine. Extraction
2
2
3
with EtOAc afforded the organic layer that was dried and concentrated. The crude solid was
chromatographed on a silica gel column using EtOAc as the eluent. Compound 4 was isolated as
1
light yellow solid in a 95% yield (147 mg). H NMR (500 MHz, CDCl ) δ/ppm 9.05 (d, J = 2.2
3
Hz, 1H), 8.53 (d, J = 2.2 Hz, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.32 (d, J = 8.1 Hz, 1H), 8.12 (dd, J =
8
.4, 2.2 Hz, 1H), 7.65 (dd, J = 8.1, 2.2 Hz, 1H), 7.26 (d, J = 5.3 Hz, 1H), 7.15 (s, 1H), 7.14 (d, J
1
3
= 5.3 Hz, 1H), 2.42 (s, 3H); C NMR (75 MHz, CDCl ) δ/ppm 158.5, 155.2, 154.9, 153.4,
3
1
49.9, 144.9, 137.7, 133.7, 131.8, 126.9, 126.6, 125.3, 120.9, 120.8, 111.1, 102.8, 18.6; HRMS
+
(ESI+): calcd. 293.0749 (C H N OS + H ) found 293.0750.
17
12
2
(c) Zinc(II) titration in absorption and emission modes. Spectrophotometric and fluorometric
titrations were conducted on a UVꢀvis spectrophotometer and a fluorometer, respectively, with a
ꢀcm semiꢀmicro quartz cuvette capped with a septum. The previously published zinc(II)
1
4
4
titration procedure was followed. The pH titration also followed a previously published
44
procedure.
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(
d) Fluorescence quantum yield measurements. All samples were degassed by bubbling argon
through before recording the absorption and emission. Absorbance at the excitation wavelength
was kept below 0.1 to minimize the innerꢀfilter effect. The fluorescence quantum yields were
determined by comparing the integrated area of the corrected emission spectrum with that of the
10
11
12
13
14
15
16
17
18
19
20
21
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23
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4
5
reference – quinine bisulfate (φ = 0.54 in 1 N H SO ).
f
2
4
(
e) Fluorescence lifetime measurements. The lifetime values were collected using the timeꢀ
correlated single photon counting (TCSPC) technique. Compounds 1ꢀ4 and their zinc(II)
complexes were excited using the 370ꢀnm LED. The LED operated at a repetition rate of 1 MHz.
The data were recorded with a bandwidth of 4 nm and 10,000 counts in the peak channel. The
timescale of the experiment was 100 ns (115.3 ps/channel).
(
f) Cell culture and confocal fluorescence microscopy. HeLa S cells were seeded and grown
3
directly in Delta T open culture dishes for at least 24 h to approximately 70ꢀ80% confluency.
The culture medium was a 1:1 mixture of DMEM and Ham’s Fꢀ12; supplemented with 12.5%
cosmic calf serum, 0.01% (w/v) sodium pyruvate, 0.05% (w/v) Lꢀglutamine, 0.12% (w/v)
sodium bicarbonate, 1% (v/v) penicillin/streptomycin, and finally adjusted to a pH of 7.9. In
preparation for imaging, cells were rinsed twice with fresh culture medium then incubated with
the appropriate dye at a concentration of 2 µM, diluted in fresh culture medium, for 30 min at 5%
CO , 95% humidity, and 37 °C. Following incubation with dye, the cells were quickly rinsed
2
twice in fresh culture medium then incubated with 50 µM sodium pyrithione and either 50 or 0
µM ZnCl diluted in fresh culture medium for an additional 10 min. During this time the Delta T
2
culture vessel is loaded into the microscope incubation chamber stage insert.
Confocal fluorescence microscopy was performed using a confocal laser pointꢀscanning
system on an inverted microscope stand. Imaging was performed using an NA = 1.4 oil
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immersion objective and 405ꢀnm diode laser line. Green fluorescence from the tested compounds
was detected between 530ꢀ570 nm using a variable bandpass filter to isolate the waveband of
interest from spectrallyꢀdispersed emission light. Video microscopy was performed using
continuous acquisition at constant laser power and other settings, including the laser excitation
power, PMT voltage, gain register, pixel dwell time, pixel depth, and offset. Photobleaching was
evaluated by drawing an ROI around each cell; with cell boundaries determined using the
brightfield DIC image and not fluorescence. Background fluorescence was determined using an
ROI over part of the image not occupied by any cells, and subsequently subtracted. Next, the
change in absolute intensity in each ROI (N = 21) was determined and averaged for each time
point, with representative images provided by Figures 6 and 7, and plotted in Figure 8.
0
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ASSOCIATED CONTENT
1
13
Supporting Information. Additional spectra, H and C NMR spectra of new compounds. This
material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
davidson@magnet.fsu.edu; lzhu@chem.fsu.edu
Present Addresses
†
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
6610 Prague 6, Czech Republic
1
Author Contributions
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The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
ACKNOWLEDGMENT
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This work was supported by the National Institute of General Medical Sciences
(R01GM081382).
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