Progressive structural modification to a zinc-actuated photoinduced electron transfer (PeT) switch in the context of intracellular zinc imaging

Article abstract of DOI:10.1039/c7ob02235b

Photoinduced electron transfer (PeT)-type fluorescent molecular switches are often applied in ion-selective sensors. Zinc-targeting sensors that contain an anilino-based electron donor (aka, the PeT 'switch') have multiple advantages over those with an al

Full text of DOI:10.1039/c7ob02235b

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DOI: 10.1039/C7OB02235B.
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DOI: 10.1039/C7OB02235B
Progressive Structural Modification to a Zincꢀ
Actuated Photoinduced Electron Transfer (PeT)
Switch in the Context of Intracellular Zinc Imaging
Miguel Macias-Contreras,†,¶ Kirsten L. Daykin,†,¶ J. Tyler Simmons,† John R. Allen,‡ Zachary S.
Hooper,† Michael W. Davidson,‡,§ and Lei Zhu*,†
†Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way,
Tallahassee, Florida 32306ꢀ4390, United States
‡National High Magnetic Field Laboratory and Department of Biological Science, Florida State
University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310, United States
¶These two authors contributed equally.
§Deceased on December 24, 2015
KEYWORDS. Zinc sensor, photoinduced electron transfer, BODIPY, coumarin
ABSTRACT. Photoinduced electron transfer (PeT)ꢀtype fluorescent molecular switches are
often applied in ionꢀselective sensors. Zincꢀtargeting sensors that contain an anilinoꢀbased
electron donor (aka, the PeT ‘switch’) have multiple advantages over those with an aliphatic
amino switch. In addition to the lower pKa value of an aniline than that of a comparably
1

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substituted aliphatic amine, which reduces the interference of pH on the spectral properties of the
attached fluorophore, the oxidation potentials of anilino groups are lower than those of aliphatic
amino counterparts, which make them better electron donors in PeT. The effectiveness of anilino
as a PeT switch is evaluated in a series of zincꢀsensitive sensors that contain different
fluorophores, zincꢀbinding ligands, and alkyl linkers between ligand and fluorophore. The
abilities of these compounds to distinguish high and low intracellular zinc concentrations in
living cells are demonstrated.
Introduction
Despite being considered a trace metal ion, the total cellular concentrations of zinc are in the
range of hundreds of micromolars,1, 2 most of which are tightly associated with proteins. The soꢀ
called ‘free zinc’, which garners tremendous attention in zinc biology due to its functions in
signalling processes, refers to zinc ions that are not tightly bound to proteins and therefore
kinetically mobile for recruitment by proteins and/or for exchange between different targets.3, 4
‘Free’ zinc is actually coordinated to a diverse set of ligands as encountered within the complex
and heterogenous cellular environment that are far from thoroughly characterized.5 It is generally
accepted that ‘free’ zinc concentrations in a cellular context swing between picomolar to
hundreds of micromolar on spatial and temporal coordinates,6ꢀ9 the acquisition of such data is of
interest to researchers in the area of zinc cell biology.
Fluorescence microscopy is a powerful technique that allows not only the visualization of
subcellular structures but also the intracellular distribution and trafficking of substances of
11
interest with the help of targetꢀselective fluorescent sensors.10,
The targets can be
macromolecules, small molecular metabolites, or ions, which include ‘free’ zinc ions.11 The
2

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sensors can be constructed from fluorescent small molecules or genetically encoded fluorescent
proteins.12ꢀ18
A molecular fluorescent sensor contains two domains: a fluorophore that is responsible for
absorbing and emitting photons, and a receptor (or ligand) that selectively interacts with the
target, such as zinc, in the presence of a plethora of potentially interfering species. The receptor
needs also to act as a trigger to alter either the color (wavelength) or brightness (extinction
coefficient at the excitation wavelength and/or fluorescence quantum yield) of the fluorophore
through various signal transduction mechanisms.19, 20 These mechanisms were proposed based on
sound theories long ago, which have since led to the successful constructions of effective small
molecular sensors. For many other sensors that were developed empirically, the working
mechanism of which were later explained by these ideas. However, challenges often emerge
especially when the strongest interaction with an analyte (as often measured by dissociation
constant Kd) and the largest dynamic range of analyteꢀdependent signal (measured by emission
intensity ratio of targetꢀbound and targetꢀfree states) cannot be satisfied simultaneously, with the
consideration that the use of these sensors shall not be deterred by the cost of their syntheses.
Herein, we report such a case in which progressive structural modification on a sensor structure
was carried out to result in zincꢀselective sensors with various combinations of sensitivity and
signal dynamic range.
Fig. 1 A cartoon drawing of a fluorescent PeT sensor. Adapted from the work by de Silva and
coꢀworkers.21
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Many ionꢀselective sensors are made based on the principle of targetꢀactuated photoinduced
electron transfer (PeT) switching, as articulated by de Silva and coꢀworkers in the early days of
fluorescent sensor research.19 The receptor in a PeT switch acts as a fluorescence quencher,
which could be inactivated via the interaction with the target. There is also a linker that
covalently joins the receptor/quencher and the fluorescent reporter (Fig. 1). For the design of
cation sensors with a fluorescence turnꢀon response, in the absence of the target, the receptor is
electronꢀrich and therefore capable of donating an electron to the excited state of the fluorophore,
thus quenching the fluorescence. Upon binding to the target cation, the electronꢀdonating ability
of the receptor is diminished, and consequently the emission intensity of the fluorophore
increases.22
Fig. 2 Four examples of zincꢀselective molecular sensors that operate via PeTꢀswitching
mechanism. Blue – fluorophore; red – linker; green – zinc receptor/electronꢀdonating quencher.
PY = 2ꢀpyridyl; N.R. = not reported.
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Four fluorescent sensors targeting zinc ions are shown in Fig. 2 to illustrate the zincꢀactuated
PeT switching mechanism. The colors used in the drawings match the colors in Fig. 1 that
identify fluorophore (blue), linker (red), and receptor/quencher (green). Polyaza ligands that
include pyridyl and amino groups are commonly employed receptors for zinc ions, such as di(2ꢀ
picolyl)amino (DPA),13, 21 which have higher affinity for zinc than for biologically abundant
alkali and alkaline earth metal ions.23
Compound 1 (Fig. 2) was reported by de Silva and coworkers,24 which is the product of a
straightforward application of the PeTꢀswitch strategy to construct fluorescent sensors for zinc.
The DPA group is connected to the anthryl fluorophore via a methylene linker. Electron transfer
from the tertiary amino group to the excited anthryl fluorophore followed by back electron
transfer relaxes the excited fluorophore nonꢀradiatively, i.e., quenches the fluorescence. This
action is turned off when zinc and the amino group bond coordinatively. DPA also acts as the
electron transfer quencher and zinc receptor in ZP1 (Fig. 2),25, 26 reported by Lippard and
coworkers.
ZnAF2 (Fig. 2) is a fluoresceinꢀcontaining zinc sensor developed by the Nagano group.27, 28
Differing from ZP1, the electron transfer quencher moiety is a substituted anilino group. The
anilino group together with the DPA moiety forms a tetradentate ligand for zinc. Lastly,
Newport Green PDX (Fig. 2)29 also includes an anilino nitrogen for both zinc binding and
fluorescence quenching purposes.
Based on the data on ZnAF2 and Newport Green PDX (Fig. 2), the anilino group is shown to
be an effective electron transfer quencher30, 31 in zinc sensor design.32 However, the affinity
value of Newport Green PDX (Kd = 40 ꢀM) is lower than the aliphatic DPAꢀcontaining zinc
dye 1 (Kd = 5 ꢀM), which has the same tridentate DPA ligand. Furthermore, syntheses of zinc
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sensors containing anilino groups are usually not as straightforward as aliphatic aminoꢀ
containing counterparts. For example, dialkylation of anilino to produce an anilinoꢀDPA moiety
often takes days to complete.33, 34 The underwhelming affinity to zinc and the challenges in
synthesis might have hindered the use of anilinoꢀderived ligands as receptors and quenchers in
zinc sensors.
In our previous work,35ꢀ37 it was shown that a fluorophore and an electronꢀrich zinc ligand can
be conveniently connected via the copperꢀcatalyzed azideꢀalkyne cycloaddition reaction to afford
PeTꢀbased zinc sensors. The compounds we have reported thus far contain fluorophores that are
excited in the UV region, which do not work well within the technical constraints of commercial
laser scanning confocal fluorescence microscopes,10 in particular with the excitation laser
sources in the visible region and the glass optic systems that strongly absorb UV. We began this
study in a naive attempt to structurally modify one of our earlier UVꢀabsorbing compounds by
replacing the fluorophore with a visible lightꢀabsorbing one. Only with the subsequent structural
alternations of the linker and zinc ligand components did we produce PeTꢀbased zinc sensors that
are excitable in the visible range. This work, we believe, offers a robust modular strategy to
produce zinc sensors with various affinities and emission colors, without taxing synthetic efforts.
Results and Discussion
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Fig. 3 Changing the fluorophore of compound 237 from 7ꢀmethoxycoumarin to 7ꢀ(N,Nꢀ
diethylamino)coumarin in compound 3.
1. From 2 to 3 – changing the fluorophore in an existing PeTꢀbased sensor failed to retain its
switching ability. Compound 2 (Fig. 3) was reported by our group to respond to the change of
zinc under neutrally buffered aqueous conditions sensitively.37 The fluorescence quantum yield
of [Zn(2)] (shorthand of the zinc complex of 2 with 1:1 coordination stoichiometry) is 16ꢀfold
over that of 2 alone. [Zn(2)] has a dissociation constant (Kd) of 5.5 nM. The deficiency of
compound 2, in the context of being an agent for imaging zinc in living cells, is the lack of
absorption in the visible region. That makes the technical aspects of imaging challenging,
because the UV excitation light would largely be absorbed by the optical system instead of the
specimen. This is a wellꢀcited motivation to incorporate longꢀwavelength absorbing dyes in
molecular indicators so that their spectral profiles match properly with the optical setup of
commercial microscopes. In an attempt to increase the absorption cross section in the visible
region based on the structure of compound 2, specifically at 405 nm which is a laser line
available to our fluorescence microscope, the UVꢀabsorbing 7ꢀmethoxycoumarin in 2 was
replaced with 7ꢀ(N,Nꢀdiethylamino)coumarin in 3 (Fig. 3), which had been reported for a
different purpose.38 7ꢀ(N,Nꢀdiethylamino)coumarin has an extinction coefficient exceeding 2 ×
104 cmꢀ1·Mꢀ1 at 405 nm. However, this structural alteration completely abrogated the
fluorescence turnꢀon response of compound 2 to zinc that is expected from PeTꢀbased sensors
(Fig. S1).
2. Examining the thermodynamics of PeT in fluorophoreꢀionophore pairs. The failure in
achieving zincꢀswitched PeT in compound 3 prompted us to examine the thermodynamic driving
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forces of photoinduced electron transfer (∆ꢀꢁ°ꢂꢃ) of 3 in its zincꢀfree form, and other hypothetical
pairings of fluorophore and electron donor. ∆ꢀꢁ°ꢂꢃ can be expressed in a combination of 4 terms
using the RehmꢀWeller equation (eq. 1).39
ꢊ
ꢂ
∆ꢀꢁ°ꢂꢃ = ꢄꢅꢆ − ꢄꢇꢂꢈ − ꢄꢉ,ꢉ
−
(1)
(2)
ꢈꢋ
ꢌ∙∙ → ꢌ∙ꢍ + ꢎ
ꢏ + ꢎ → ꢏꢐ∙
ꢏ + ℎꢑ → ꢏ∗
ꢄꢅꢆ
−ꢄꢇꢂꢈ
ꢄꢉ,ꢉ
(3)
(4)
ꢊ
ꢂ
ꢒ
ꢚ
ꢄ ꢓꢔꢕꢖꢗꢘꢙꢓ = − ꢈꢋ
(5)
The energies associated with the formation of radical cation (eq. 2) and radical anion (eq. 3)
from the neutral electron donor and acceptor are ꢄꢅꢆ and −ꢄꢇꢂꢈ, respectively. The photon energy
(0ꢀ0 transition) for exciting the fluorophore (eq. 4), which is the electron acceptor in the cases
under discussion, is represented by ꢄꢉ,ꢉ. The electrostatic potential energy between the radical
cation and anion is expressed using the Coulomb Law (eq. 5), where d is the centerꢀtoꢀcenter
distance between the electron donor and the acceptor, and ε is the dielectric constant of the
solvent. The ꢄꢅꢆ and −ꢄꢇꢂꢈ terms are the cost of PeT, while the other two terms are the gain of
energy.
Based on the analysis of the RehmꢀWeller equation, high energy, short wavelength
fluorophores have large values of ꢄꢉ,ꢉ, therefore contribute favorably to PeT, and in another
word, are relatively easy to be quenched. When the fluorophore is changed from 7ꢀ
methoxycoumarin to 7ꢀ(N,Nꢀdiethylamino)coumarin, the absolute value of ꢄꢇꢂꢈ increases while
that of ꢄꢉ,ꢉ decreases, which collectively reduces the driving force of PeT. Assuming the
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distance between donor and acceptor does not change upon the exchange of fluorophore, in order
to maintain the thermodynamic driving force of PeT, the ꢄꢅꢆ term of the eꢀdonor has to decrease
to offset the unfavorable changes of ꢄꢇꢂꢈ and ꢄꢉ,ꢉ. That means the eꢀdonor must change
accordingly to an electronꢀricher one.
Fig. 4 Electron donors Q1ꢀQ4, and fluorophores F1ꢀF3.
The combination of four electronꢀdonating ligands (Q1ꢀQ4 in Fig. 4) and three electron
accepting fluorophores (F1ꢀF3 in Fig. 4) were studied using cyclic voltammetry to assess their
effectiveness to act as components in zincꢀswitchable PeT systems. Q1 is a tertiary amine, while
Q2ꢀQ4 are anilino electron donors that incorporate zincꢀcoordinating pyridyl and/or triazolyl
groups. Compounds F1 and F2 are differently substituted coumarin fluorophores, while F3 is a
BODIPY fluorophore. The ꢄꢅꢆ of the electron donors Q1ꢀQ4 and the ꢄꢇꢂꢈ of dyes F1ꢀF3 were
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determined via cyclic voltammetry in acetonitrile. The ∆ꢀꢁ°ꢂꢃ values of arbitrary quencher and
fluorophore pairs were estimated using the RehmꢀWeller equation,39 and are listed in Table 1. In
the calculations, the electrostatic term was omitted considering the large relative permittivity of
the solvent under consideration – 37.5 of acetonitrile.
The oxidation and reduction potential values referenced to ferrocene are listed in Table 1. The
tertiary amino electron donor Q1 has an oxidation potential of 0.70 V. The first oxidation of the
anilinoꢀbased Q2 was found at 0.41 V. The first oxidation potential of paraꢀdiaminobenzeneꢀ
based Q3 dropped to ꢀ0.04 V, while the orthoꢀdiaminobenzeneꢀderived Q4 was oxidized at 0.30
V. Q3, therefore, stood out as the most effective electronꢀdonating quencher.
The reduction potential of 7ꢀ(N,Nꢀdiethyl)aminocoumarin (F2 in Fig. 4) is larger than that of
the 7ꢀmethoxycoumarin F1 (Table 1), while the ꢄꢉ,ꢉ of F2 is smaller than that of F1. Neither
term favors F2 in engaging PeT with Q1. The ∆ꢀꢁ°ꢂꢃ value between Q1 and F2 is consequently
positive (Table 1). For F3, although it has a small ꢄꢉ,ꢉ, its reduction potential is also the lowest –
i.e., the best electron acceptor. The collective effect of ꢄꢉ,ꢉ and ꢄꢇꢂꢈ gives a favorable ∆ꢀꢁ°ꢂꢃ
when pairing with the tertiary amino quencher Q1. Not coincidentally, the combination of F3
and DPA was used as a PeTꢀbased zinc sensor.40 Based on the calculated ∆ꢀꢁ°ꢂꢃ values (Table 1),
molecules equipped with an anilinoꢀcontaining ionophore and any of the three fluorophores in
Fig. 4 possess favorable ∆ꢀꢁ°ꢂꢃ values needed to create a zincꢀactuated PeT switch.
Table 1 Summary of cyclic voltammetry data and calculated ∆ꢀꢁ°ꢂꢃ values (in red).
∆ꢀꢁ°ꢂꢃ (eV) |Ered| (V)a
E0,0 (eV)
Q1
Q2
Q3
Q4
Eox (V)a
ꢀ
ꢀ
0.70
ꢀ0.68
0.41
ꢀ0.97
ꢀ0.04
ꢀ1.42
0.30
ꢀ1.08
1.88
3.26
F1
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2.05
1.69
2.74
2.49
+0.01
ꢀ0.10
ꢀ0.28
ꢀ0.39
ꢀ0.73
ꢀ0.84
ꢀ0.39
ꢀ0.50
F2
F3
a. Oxidation and reduction potentials are referenced to ferrocene in acetonitrile. The electrolyte
is tetrabutylammonium perchlorate.
O
O
N
O
N
N
O
N
PY
N
N
N
PY
N
N
PY
3
4
N
PY
Fig. 5 Changing the ionophore of compound 3 from Q1 to Q2 to afford compound 4.
3. The effect of anilino electron donor on the fluorescence and zincꢀresponse of compound 4.
Based on the data in Table 1, a fluorescent sensor that combines an anilinoꢀDPA ionophore such
as the one in Q2 with fluorophore F2 shall function as a PeTꢀbased zinc sensor (compound 4 in
Fig. 5). The absorption of 7ꢀ(N,Nꢀdiethylamino)coumarin corresponds nicely with the 405ꢀnm
laser that is equipped with our laser scanning confocal fluorescence microscope. The synthesis of
compound 4 is depicted in Scheme S1. The fluorescence intensity of 4 increases upon titration of
Zn(ClO4)2 in acetonitrile (Fig. 6). The fluorescence quantum yield of 4 increases by 90ꢀfold upon
the formation of the zinc complex, suggesting that the PeT process is switched ‘off’ after zinc
binding.41
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Fig. 6 Fluorescence spectra of compound 4 (9.5 µM, λex = 420 nm) in the presence of Zn(ClO4)2
(0 – 14 µM) in acetonitrile. The initial and final spectra are coded green and red, respectively.
Compound 4 is excitable by visible light, which is an improvement over compound 2 that is
excited in the UV region. The fluorescence sensitivity of compound 4 to the presence of zinc is
restored from that of compound 3 by changing the PeT quencher, which is also the zinc ligand,
from aliphatic DPA to anilinoꢀDPA. These are the positive attributes of compound 4 with regard
to the potential as a zinc sensor. The affinity of compound 4 to zinc,42 however, likely is similar
to that of Newport Green PDX (Kd(ZnL) = 40 ꢀM, Fig. 2), which is more than a thousand folds
lower than that of the parent compound 2 (Kd(ZnL) = 5.5 nM), the starting point of our
improvement effort. In the next step, we aimed for increasing the zinc affinity while preserving
the fluorescence sensitivity to zinc of 4 via structural modification of the quencher/ionophore
component.
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Fig. 7 Increasing the electron density of the anilinoꢀcontaining quencher/ionophore in 4 to afford
compounds 5 and 6.
4. Increasing the electron density on the anilino ionophore. In compounds 5 and 6 (Fig. 7),
alkoxy and Nꢀmethylamino groups are included at the para position of the anilinoꢀDPA
ionophore, respectively. This modification was aimed to increase the zinc affinity of the
tridentate anilinoꢀDPA ligand, while also easing the synthesis of the dialkylated aniline by
increasing the nucleophilicity of the anilino moiety. The synthesis of compound 6 started with
nucleophilic
aromatic
substitution
between
1ꢀfluoroꢀ4ꢀnitrobenzene
and
Nꢀ
methylpropargylamine to afford compound 7 (Fig. 8). The nitro group was reduced by stannous
chloride to afford 8. Dialkylation of the anilino group of 8 was completed in an overnight
reaction either with 2ꢀpyridinecarboxaldehyde (reductive amination), or with 2ꢀpicolylbromide
(SN2 alkylation), rather than over multiple days as reported in other syntheses of anilinoꢀDPA
moieties.33, 34 The faster anilinoꢀDPA synthesis results from having the additional electronꢀ
donating amino group, which enhances the nucleophilicity of the unsubstituted amino group.
Finally, copper(I)ꢀcatalyzed cycloaddition between azide 1043 and alkyne 9 afforded compound
6.
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Fig. 8 Synthesis of compound 6. Reagents and conditions: a) neat, Nꢀmethylpropargylamine (2
molar equiv.), rt, > 95%, or Nꢀmethylpropargylamine (1 molar equiv.), K2CO3, DMSO, 50 °C,
overnight, 72%; b) SnCl2, HCl (37%, v:v), overnight, 70%; c) 2ꢀpyridinecarboxaldehyde,
NaBH(OAc)3, 1,2ꢀdichloroethane, rt, 75%, or 2ꢀpicolylbromide hydrobromide, Na2CO3, EtOH,
reflux, overnight, 46%; d) CuCl (20 mol%), THF, under argon, overnight, 60%.
The fluorescence quantum yield of compound 6 grew by 4.5ꢀfold upon the formation of a zinc
complex in acetonitrile (Fig. 9a). Fluorescence enhancement of compound 6 was also observed
in a zinc titration experiment conducted in an aqueous buffer at pH 7.4 (Fig. 9b). Fitting the
binding isotherm with a 1:1 stoichiometry yields a dissociation constant (Kd) of ZnL of 3 ꢀM
(Fig. 9c).44 This value is more than 10ꢀfold lower than that of Newport Green PDX (Kd(ZnL)
=
40 ꢀM, Fig. 2), suggesting that paraꢀNꢀmethylamino group installed on the anilinoꢀDPA ligand
increases its affinity to zinc to the extent of an aliphatic DPA ligand as seen in compound 1 (Kd =
5 ꢀM, Fig. 1). The fluorescence of compound 6 selectively responds to the presence of zinc over
alkali and alkaline earth metal ions, as well as a few selected firstꢀrow transition metal ions (Fig.
S3). Cadmium, however, elicits a similar emission enhancement from compound 6. Compound 5
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(synthesis see Scheme S2) was also characterized, and up to 6ꢀfold fluorescence enhancement
upon zinc coordination in acetonitrile was observed (Table 2). Due to the similar aggregation
issues under aqueous conditions to that we experienced with compound 4,42 the Kd(ZnL) of
compound 5 under pH neutral aqueous conditions was not determined.
Fig. 9 Fluorescence spectra of (a) 6 (5 µM, λex = 400 nm) in the presence of Zn(ClO4)2 (0ꢀ8.3
ꢀM) in acetonitrile, and (b) 6 (6 ꢁM, λex = 410 nm) in the presence of Zn(ClO4)2 (0ꢀ7.3 mM) in
an aqueous solution: [HEPES] = 100 mM, [citrate] = 10.0 mM, [KNO3] = 100 mM, pH = 7.4.
The initial and final spectra of each titration experiment are coded green and red, respectively.
(c) Fluorescence intensity at 500 nm measured under conditions in (b) was fit as a function of
‘free’ zinc concentration, using a 1:1 binding model,45 to yield a Kd value of 3 ꢀM.
Fig. 10 Changing the zinc ligand portion of 6 to afford 11 and 12.
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5. Further increasing zinc affinity based on the structure of 6. The effort of structural
modification acted upon compound 6 was directed at increasing the zinc affinity further to a
Kd(ZnL) within or close to single nanomolars. Starting from compound 6, two strategies were
considered: the tridentate di(2ꢀpicolyl)amino (DPA) group in 6 could be replaced by a
tetradentate ligand with an additional amide carbonyl arm46, 47 to afford compound 11 (Fig. 10).
In the second strategy, the DPA group on anilino could be moved to the ortho position in regard
to the other amino group to potentially engage it, and the triazolyl group,35 in binding zinc. Yet
after numerous unsuccessful attempts of incorporating a DPA moiety ortho to an amino group,
we resorted to synthesize compound 12 (Fig. 10), which has an NꢀmethylꢀNꢀ2ꢀpicolylamino
(MPA) group as the zinc binding ligand. The syntheses of 11 and 12 are depicted in Schemes S3ꢀ
4.
Compound 11 exhibited up to 6.4ꢀfold fluorescence enhancement in the presence of zinc
perchlorate in acetonitrile (Fig. 11a). It has been reported that the instalment of an amide scaffold
one methylene away from a DPA moiety results in a tetradentate ligand with zinc affinity in the
nanomolar range.48 The Kd of the zinc complex of compound 11 determined at pH 7.4 was 30
nM (Fig. 11c/d), which is in agreement with other zinc sensors containing the amideꢀDPA
scaffold.49, 50 This value is 100ꢀfold higher than the zinc affinity of compound 6 and thus
validating the strategy outlined earlier. The metal ion response profile of the emission of
compound 11 is similar to that of compound 6 (Fig. S4). Compound 11 had the same λem for both
zincꢀfree and zinc(II)ꢀbound states as that of compound 6. In the case of 12 (MPA as the zincꢀ
binding unit, Fig. 11b), there was a 12ꢀnm redꢀshift from the emission of the free ligand (λem
=
482 nm) to that of the zinc complex (λem = 494 nm). The emission of compound 12 increased by
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4.3ꢀfold upon zinc complex formation in acetonitrile. The Kd of compound 12 under aqueous
conditions was not determined due to the aggregation issues.42
Fig. 11 Fluorescence spectra of (a) 11 (5 ꢁM, λex = 400 nm) in the presence of Zn(ClO4)2 (0ꢀ25
ꢀM), (b) 12 (5 ꢁM, λex = 405 nm) in the presence of Zn(ClO4)2 (0ꢀ57 ꢀM) in acetonitrile, and (c)
11 (6 ꢁM, λex = 410 nm) in the presence of Zn(ClO4)2 (0ꢀ7.9 mM) in an aqueous solution:
[HEPES] = 100 mM, [ADA] = 10.0 mM, [KNO3] = 100 mM, pH = 7.4. The initial and final
spectra are coded green and red, respectively. (d) Fluorescence intensity at 500 nm of 11 was fit
as a function of free zinc concentration, using a 1:1 binding model,45 to yield a Kd value of 30
nM.
Table 2 Summary of photophysical properties of 4-6, and 11-13 in acetonitrile.
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Compound
λ
abs (nm)
λ
em (nm)
φL
φZnL
0.45
0.54
0.09
0.05
0.25
0.02
0.13
0.19
413
385
416
421
407
515
496
494
486
484
499
495
482
523
503
501
0.005
0.09
0.02
0.02
0.07
0.01
0.04
0.06
4
5
6
11
12
13a
13b
13c
Fig. 12 Changing the fluorophore in 6 to afford compounds 13a-c.
6. Effect on the PeTꢀswitch by changing fluorophore, and distance between eꢀdonor and eꢀ
acceptor. We were curious whether the paraꢀaminoꢀanilinoꢀDPA quencher would work well with
other fluorophores to afford zincꢀactuated PeT switches. The BODIPY fluorophore was selected
in this work because of its brightness and photostability in cell imaging.51ꢀ54 This dye has been
incorporated as a reporter in fluorescence sensors for zinc.55, 56 The effectiveness of BODIPY
and anilino group to act as a PeT switch has been demonstrated in one case.57 Compounds 13a-c
(Fig. 12) were prepared (Scheme S5) that differ in the length of the alkyl linker connecting the
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fluorophore to the triazolyl group. By comparing the properties of 13a-c, the effect of the linker
length on the performance of these compounds as zinc sensors was demonstrated.
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Fig. 13 Fluorescence spectra of 13a-c (2 ꢀM) in acetonitrile in the presence of Zn(ClO4)2 (0ꢀ30
ꢀM). (A) 13a (λex = 488 nm), (B) 13b (λex = 465 nm), and (C) 13c (λex = 465 nm). The initial and
final spectra of each titration experiment are coded green and red, respectively.
The zinc sensitivity of the fluorescence of compounds 13a-c were studied (Fig. 13). In all zinc
titration experiments, the addition of Zn(ClO4)2 led to an increase in fluorescence as expected
from a zincꢀactuated PeT switch. The photophysical data of 13a-c are included in Table 2.
Compound 13a has the smallest fluorescence quantum yield values in both free and zincꢀbound
states. The smallest distance (one methylene) between the zincꢀbinding DPA unit and the
fluorophore appears to result in a low quantum yield of the BODIPY fluorophore. Compounds
13b and 13c with longer linkers separating the fluorophore and the quencher have similarly
higher quantum yield values.
7. Comments on the fluorescence quantum yields. In acetonitrile, zinc binding was able to
restore the fluorescence quantum yields of compounds 4 and 5 to 0.45 and 0.54, respectively,
while smaller extents of enhancement were observed for other compounds that contain the paraꢀ
amino anilino quencher component (Table 2). The oxidation potential of zinc complex of Q2,
which is the quencher component in compound 4, was not measurable within the voltage cycling
window in this study, suggesting that zinc complexation with Q2 increased the oxidation
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potential of Q2 to a sufficiently high level so that the PeT process in compound 4 is effectively
stopped. By comparison, the oxidation potential of compound Q3 was increased from ꢀ0.04 V to
0.48 V in the presence of an equal molar amount of Zn(ClO4)2.58 The effect of zinc binding on
increasing the oxidation potential of this quencher is apparent, but not up to even the level of
tertiary amino group in its free form (0.70 V). Therefore, the zinc complex of Q3, and by
analogy the zinc complexes of the quencher components in compounds 6, 11, 12, and 13a-c, are
still sufficiently electronꢀrich to keep the PeT channels open, which diminish the emission of the
fluorophores they are attached to. Compound 12 has the highest quantum yield (0.25) in the zincꢀ
bound form among these compounds. The ability to engage both amino groups in zinc binding
might have hindered the PeT pathway more effectively in the zincꢀbound form than the case of
compound 6.
8. Visualizing zinc ions in live HeLa cells. Fluorescent ligands 4-6 and 11-13 were evaluated
for their potentials in visualizing zinc ions in live HeLa cells. All cells were incubated in a
medium containing 1ꢀ4 micromolars of one of the dyes for 30 min. The medium was then
replaced with fresh medium that contained either basal or enriched level of zinc ions in the form
of ZnCl2. The images were acquired by different experimenters at different time periods of this
project. Therefore, the images taken using the same parameter sets are grouped together, so that
within each group the images are comparable in emission intensity. The fluorescence
microscopic images of cells stained with compounds 4-6 (λex = 405 nm), compounds 11 and 12
(λex = 405 nm), and compounds 13a-c (λex = 488 nm) are shown in Figures 14ꢀ16, respectively.
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Fig. 14 Laser scanning confocal fluorescence microscopic images of compounds 4 (a), 5 (b), and
6 (c) in live HeLa (S3) cells. The incubating concentration was 2 ꢀM each. Images in the top row
were taken in the absence of supplemented ZnCl2. Images in the bottom row were taken in the
presence of 50 µM added ZnCl2. [sodium pyrithione] = 50 ꢀM; λex = 405 nm; scale bar = 10 ꢀm.
The images on the top row of Fig. 14 show cells that were loaded with compounds 4-6 (from
aꢀc) in a media without supplemented zinc. The bottom images show the fluorescence responses
from these compounds after the media was supplemented with 50 µM ZnCl2. All the images
were acquired using the same parameter set. Therefore, the observed intensity of these images is
comparable. The images of cells labeled with 4 (Fig. 14aꢀI) and 6 (Fig. 14cꢀI) show least
fluorescence intensity, likely due to the background fluorescence of the sensors in the unbound
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state. The image associated with 5 (Fig. 14bꢀI) shows a larger fluorescence intensity; but there is
still a large contrast between itself and the corresponding bottom image of cells (Fig. 14bꢀII)
bathed in a media supplemented with zinc. Large fluorescence enhancement is also evident in the
fluorescence images of cells with 4 (Fig. 14aꢀII) and 6 (Fig. 14cꢀII) in zincꢀenriched media.
Fig. 15 Laser scanning confocal fluorescence microscopic images of compounds 11 (a,
incubating concentration 1 ꢀM) and 12 (b, 1 ꢀM) in live HeLa (S3) cells. Images in the top row
were taken in the absence of supplemented ZnCl2. Images in the bottom row were taken in the
presence of 50 µM added ZnCl2. Green channel (λex = 405 nm, emission window 460ꢀ560 nm),
red channel (λex = 488 nm, emission window 590ꢀ630 nm). [sodium pyrithione] = 50 ꢀM.
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Similar to compounds 4ꢀ6, 11 and 12 showed the ability to reflect changes of intramolecular
zinc concentrations in living HeLa (S3) cells, as the intensity of the bottom images is higher than
that on the top row (Fig. 15). The incubating concentrations of the dyes was 1 ꢀM each, despite
the unremarkable fluorescence quantum yield values of these two compounds. These cells were
transfected with mCherryꢀH2Bꢀ6,59 a red fluorescent protein that marks nuclei of mammalian
cells. The emission from mCherryꢀH2Bꢀ6 in the red channel was used as nuclear counter stain.
Fig. 16 Laser confocal fluorescence microscopic images of compounds 13a (a), 13b (b), and 13c
(c) in live HeLa (S3) cells. The incubating concentration was 4 ꢀM each. Images in the top row
were taken in the absence of supplemented ZnCl2. Images in the bottom row were taken in the
presence of 50 µM added ZnCl2. [sodium pyrithione = 50 ꢀM]; λex = 488 nm; scale bar = 10 ꢀm.
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BODIPYꢀderived compounds 13a-c were tested in live HeLa (S3) cells to see if the chain
length affected the performance of the dyes in reporting intracellular zinc alterations. Fig. 16
shows the three compounds imaged in the cell under both basal (no added zinc) or zincꢀ
supplemented (50 µM) conditions. Compounds 13b and 13c both show a larger contrast between
zincꢀfree and supplemented incubation conditions than compound 13a, which afforded overall
dimmer emission than 13b and 13c. These imaging data are consistent with the difference in
fluorescence quantum yields of the three compounds.
Conclusion
The initial attempt to generate visible lightꢀexcitable, PeTꢀbased fluorescent indicators for zinc
ions with an aliphatic amine quencher based on a UVꢀabsorbing precursor was unsuccessful
because a favorable thermodynamic driving force of electron transfer was lost. Cyclic
voltammetry studies showed that anilinoꢀbased ionophores/quenchers would be effective in the
zincꢀactuated PeTꢀswitch. Due to the difficulties associated with synthesizing anilinoꢀDPA
ligands, electronꢀdonating groups were placed para to the anilino nitrogen to increase its
nucleophilicity. The syntheses of the new compounds (5 and 6) were accomplished with less
time, as a result of the additional electronꢀdonating substituents on the anilino moiety. All the
anilinoꢀDPAꢀcontaining indicators undergo various degrees of fluorescence enhancement upon
binding zinc, as anticipated from zincꢀactuatable PeT switches. The installment of electronꢀ
donating groups onto the anilino moiety also increases the zinc affinity of the indicators. Using
paraꢀaminoꢀanilino group as a quencher is effective in pairing up with both 7ꢀN,Nꢀ
diethylaminocoumarin and BODIPY fluorophores. Three BODIPYꢀderived compounds were
made that differ in the linker length between the fluorophore and the quencher/ionophore. The
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one with the shortest linker (13a) suffers with relatively low fluorescence quantum yields in both
zincꢀfree and zincꢀbound forms. All the anilinoꢀDPAꢀcontaining sensors were evaluated for their
potentials as zinc sensors in live cell imaging experiments, and except 13a are all similarly
capable of reporting the changes of intracellular zinc concentrations. These compounds can be
excited at either 405 nm or 488 nm with various affinities to zinc. The strategy of using anilinoꢀ
DPA as a key component of zinc receptor and electron transferꢀbased quencher appears to be
applicable in producing zinc sensitive dyes with various excitation energy, emission colors, and
effective concentration ranges.
ACKNOWLEDGMENT
This work was supported by the National Science Foundation (CHE1566011).
Electronic supplementary information (ESI) available: Synthetic procedures and
characterizations of new compounds, procedures of spectroscopic data acquisition, cyclic
voltammetry, cell culture and fluorescence microscopy, and copies of NMR spectra of new
compounds.
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