Photochemically stable fluorescent heteroditopic ligands for zinc ion

Article abstract of DOI:10.1021/jo8015083

(Chemical Equation Presented) Photochemically stable fluorescent heteroditopic ligands (9 and 10) for zinc ion were prepared and studied. Two independent metal coordination-driven photophysical processes, chelation-enhanced fluorescence (CHEF) and interna

Full text of DOI:10.1021/jo8015083

Photochemically Stable Fluorescent Heteroditopic Ligands for Zinc
Ion
Lu Zhang and Lei Zhu*
Department of Chemistry and Biochemistry, Florida State UniVersity, Tallahassee, Florida 32306-4390
lzhu@chem.fsu.edu
ReceiVed July 10, 2008
Photochemically stable fluorescent heteroditopic ligands (9 and 10) for zinc ion were prepared and studied.
Two independent metal coordination-driven photophysical processes, chelation-enhanced fluorescence
(CHEF) and internal (or intramolecular) charge transfer (ICT), were designed into our heteroditopic ligand
framework. This strategy successfully relates three coordination states of a ligand, non-, mono-, and
dicoordinated, to three fluorescence states, fluorescence OFF, ON at one wavelength, and ON at another
wavelength. This ligand platform has provided chemical foundation for applications such as the
quantification of zinc concentration over broad ranges (Zhang, L.; Clark, R. J.; Zhu, L. Chem.-Eur. J.
2008, 14, 2894-2903) and molecular logic functions (Zhang, L.; Whitfield, W. A.; Zhu, L. Chem.
Commun. 2008, 1880-1882). The binding stoichiometries of dipicolylamino and 2,2′-bipyridyl, the two
binding sites featured in heteroditopic ligands 7-10, were studied in acetonitrile using both Job’s method
of continuous variation and isothermal titration calorimetry (ITC). The fluorescence enhancement of 7-10
upon the formation of monozinc complexes (defined as the fluorescence quantum yield ratio of monozinc
complex and free ligand) is qualitatively related to the highest occupied molecular orbital (HOMO) energy
levels of their fluorophores. This is consistent with our hypothesis on the thermodynamics of the
coordination-driven photophysical processes embodied in the designed heteroditopic system, which was
supported by cyclic voltammetry studies. In conclusion, compounds 9 and 10 not only possess better
photochemical stability but also display a higher degree of fluorescence turn-on upon formation of
monozinc complexes than their vinyl counterparts 7 and 8.
Introduction
conventional, macroscopic manipulation of matter.5-7 Equally
important, the studies of supramolecular systems advance our
understanding on the fundamental chemical and physical
principles underlying their unique, often surprising stimulus-
dependent properties.
The development of chemical systems whose properties
(mechanical, optical, electronic, etc.) can be modulated through
fast, reversible interactions (noncovalent or “dynamic” cova-
lent1) with external chemical stimuli2 is a major objective in
the field of supramolecular chemistry.3,4 These endeavors are
envisaged to offer new tools such as sensors and machines
within a nanometer size regime that cannot be developed via
A typical supramolecular system is heteroditopic metal
coordination ligands that were first developed in the late 1970s8
(5) Stoddart, J. F. Acc. Chem. Res. 2001, 34, 410–411.
(6) Balzani, V.; Venturi, M.; Credi, A. In Molecular DeVices and Machines -
A Journey into the Nanoworld; Wiley-VCH: Weinheim, 2003.
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72–191.
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Soc. 1979, 101, 4333–4337.
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J. F. Angew. Chem., Int. Ed. 2002, 41, 898–952.
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10.1021/jo8015083 CCC: $40.75
Published on Web 10/14/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 8321–8330 8321

Zhang and Zhu
FIGURE 1. Compounds 1-4: Fluorescent heteroditopic ligands for metal ions (refs 18-23). Red-coded atoms: electron donor sites; blue coded
atoms: electron acceptor sites. 5: A chromophoric ditopic ligand developed for Hg2+ sensing (ref 27). 6: A fluorescent ligand capable of forming
multinuclear complexes with Zn2+, Sc3+, and other metal ions (refs 28 and 29).
as model systems for allosterism found in Nature.9 In this case,
the external stimulus, a metal ion, is recognized by a metal
coordination site (the “allosteric site”) within the ditopic ligand.
This interaction is transduced into a conformational change of
the other metal coordination site (the “active site”), which is
manifested as altered binding10-15 and/or catalytic16,17 properties
of the “active site” of the heteroditopic ligand.
More recently, fluorescent ditopic ligands have been devel-
oped for transducing metal coordination events into fluorescence
spectral shifts and/or intensity modulations.18-23 On the basis
of the identities and/or quantity of the metal ion present, a
fluorescent ditopic ligand is capable of offering at least three
coordination states, non-, mono-, and dicoordinated, which give
rise to three distinct fluorescence states. Hence, fluorescent
ditopic systems provide platforms for designing supramolecular
systems to achieve practical goals that are unattainable via
monotopic systems. Typical examples include the simultaneous
detection of two different metal ions and imaging and quanti-
fication of a metal ion over large concentration ranges. The
fundamental chemical challenge in all these applications is to
achieve large fluorescence contrast of the three coordination
states of a fluorescent ditopic ligand based on thorough
understanding of its coordination-driven photophysical properties.
The coordination-modulated internal charge transfer (ICT)24
and excited-state intramolecular proton transfer (ESIPT)25,26 of
donor-acceptor type fluorophores or chromophores have been
a prominent theme in the development of fluorescent ditopic
ligands with three different fluorescence states. Several examples
are shown in Figure 1. The coordinating atoms that are critically
impacting the fluorescence of the ligands are color-coded and
bolded. The metal-coordination at the electron donor (red) and
the electron acceptor (blue) atoms result in a hypsochromic and
bathochromic shifts of emission, respectively.24 Therefore,
mono- and dicoordinated ligands have shorter or longer emission
bands than that of the unbound ligand, depending on whether
the donor or the acceptor atom is preferentially coordinated.
The coordination-modulated ICT or ESIPT has been applied to
achieve three fluorescence states in compounds 2-4. The
fluorophore of 1 does not have strong donor-acceptor charac-
teristics; therefore, only small spectral shifts were observed when
Ag+ and Na+ were introduced in the system.18 The three
coordination states of 5 achieved upon coordinating Hg2+ have
distinct absorption bands.27 However, their fluorescence proper-
ties were not reported. Although not a typical ditopic ligand,
the ability of 6 to form multinuclear complexes with different
photophysical properties was explored to achieve the same
objective.28,29
(9) Kobe, B.; Kemp, B. E. Nature 1999, 402, 373–376.
(10) Rebek, J., Jr.; Wattley, R. V. J. Am. Chem. Soc. 1980, 102, 4853–4854.
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Chem. Soc. 2003, 125, 28–29.
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C. R. Angew. Chem., Int. Ed. 2004, 2004, 4515–4518.
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Fluorescent Heteroditopic Ligands for Zinc Ion
nated ligands adopt three fluorescence statessfluorescence OFF
(Figure 2A), ON at one wavelength (Figure 2B), and ON at
another wavelength (Figure 2C). State “A” has low fluorescence
quantum yield (ΦF) due to PET; States “B” and “C” can be
designed to have large, comparable ΦF, however they fluoresce
at different wavelengths. On the basis of this unique ligand
platform, applications such as metal ion quantification over large
concentration ranges32 and development of molecular logic
functions35 can be achieved (for overviews of the molecular
logic area see refs 6 and 36-40).
FIGURE 2. Three coordination states (non-, mono-, and dicoordinated)
of a fluorescent heteroditopic ligand are related to three fluorescence
states (OFF, ON at λ1, and ON at λ2). M1 and M2, two metal ions;
CHEF, chelation-enhanced fluorescence; ICT, internal (or intramolecu-
lar) charge transfer.
In implementing this design, a series of fluoroionophores for
Zn2+ with moderate affinities which are capable of Zn2+
-
coordination modulated ICT will be studied first. The ligands
with high fluorescence quantum yields in both free and Zn2+
bound forms will be selected for installation of a PET/CHEF
switching unit, which is also designed as the high-affinity Zn2+
-
-
These early examples advanced our understanding of fluo-
rescent heteroditopic ligands; however accomplishing three
different fluorescence states with large contrast is anything but
trivial. Spectral shift was not observed in compounds 1 and 6
due to their inherently weak donor-acceptor characteristics. The
coordination of compounds 2-4 resulted in fluorescence states
with three different emission wavelengths for each ligand based
on the rationale articulated earlier. However, one empirical,
under-controlled factor is that metal coordination may open up
extra or close existing nonradiative pathways, thus changing
fluorescence intensity in a rather unpredictable manner. There-
fore, we seek another design strategy so that control over both
fluorescence intensity and spectral shift can be achieved to afford
three fluorescence states with maximal contrast.
Our group is interested in developing fluorescence probes for
zinc ion (Zn2+) quantification and imaging with low detection
limit, high sensitivity, and large effective concentration
ranges.30-32 We explore the heteroditopic platform to achieve
the coverage of large concentration ranges in Zn2+ quantification
applications.32 In our work, two coordination-driven photo-
physical processes, chelation-enhanced fluorescence (CHEF) and
coordination-modulated internal (or intramolecular) charge
transfer (ICT), were engineered into a compact heteroditopic
ligand framework for achieving three distinct coordination-
dependent fluorescence states (Figure 2). The ligand itself was
designed to have a nonradiative relaxation pathway where
photoinduced electron transfer (PET) occurs from the electron-
rich, high-affinity (the rectangles in Figure 2) Zn2+-binding
moiety to the excited fluorophore. Preferential coordination of
Zn2+ to the high-affinity binding site raises its oxidation potential
so that the PET process becomes thermodynamically disfavored.
Consequently, the fluorescence is restored. This coordination-
driven process has been referred to as chelation-enhanced
fluorescence (CHEF).33,34
binding moiety. The theoretical ground for the choice of the
PET/CHEF switch to achieve sensitive fluorescence turn-on
upon coordinating Zn2+ will be detailed in the section of “Cyclic
Voltammetry Studies”. In summary, two independent coordina-
tion-driven photophysical processes (CHEF and ICT) are
incorporated in one heteroditopic ligand framework. The
sequential activation of CHEF and ICT processes is controlled
by the relative affinities of the two Zn2+-binding moieties
associated with respective processes. By applying this design
principle, control over both emission wavelengths and quantum
yields of the three coordination-dependent fluorescence states
of a fluorescent heteroditopic ligand can be accomplished.
A fluorescent heteroditopic ligand platform for Zn2+, repre-
sented by compounds 7 and 8, has been developed in our
laboratory based on this design principle.32 In the absence of
Zn2+, 7 and 8 are only weakly fluorescent (see ΦF values in
Table 1) because nonradiative relaxation via PET from the
tertiary amino group in either 7 or 8 to the excited arylvinyl-
bipy (bipy ) 2,2′-bipyridyl) fluorophore is operating.41 In the
presence of Zn2+ at low concentration, preferential coordination
to the presumptive high-affinity dipicolylamino group occurs
to result in CHEF. When the concentration of Zn2+ is high
enough to bind bipy, the presumptive low-affinity binding site,
the charge-transferred excited fluorophore is stabilized to result
in a bathochromic shift of the emission band.
Although the rational design of 7 and 8 is satisfactory, we
seek to further expand the scope and practicality of our design
of fluorescent heteroditopic ligands by fully understanding the
fundamental coordination chemistry and photophysical processes
embodied in this system. Herein, we report our progress in (1)
preparing fluorescent heteroditopic systems that are photochemi-
cally stable, (2) studying the binding stoichiometries and relative
affinities of dipicolylamino and bipy, which are the two binding
motifs in this series of heteroditopic ligands (7-10), to Zn2+
,
As the concentration of Zn2+ ([Zn]) is high enough to occupy
the low-affinity site (the ovals in Figure 2), a charge-transferred,
dipolar excited state (ICT state) of the ditopic ligand is expected
to be stabilized (in the system reported in this paper) to result
in a bathochromic shift of emission.24 In summary, depending
on the metal ion concentration, the non-, mono, and dicoordi-
and (3) achieving large fluorescence contrasts between the three
fluorescence states upon further understanding of the thermo-
dynamics of the electron transfer processes.
(35) Zhang, L.; Whitfield, W. A.; Zhu, L. Chem. Commun. 2008, 1880–
1882.
(36) Brown, G. J.; de Silva, A. P.; Pagliari, S. Chem. Commun. 2002, 2461–
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(30) Zhang, L.; Dong, S.; Zhu, L. Chem. Commun. 2007, 1891–1893.
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(32) Zhang, L.; Clark, R. J.; Zhu, L. Chem.-Eur. J. 2008, 14, 2894–2903.
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Zhang and Zhu
TABLE 1. Fluorescence Quantum Yields of Free Ligands (ΦF), Monozinc Complex (ΦZnL), and Dizinc Complex (ΦZn2L), Emission Band
Maxima (λ1, λ2) of Ligands and Their Zn2+ Complexes, and Anodic Peak Potentials of the Free Ligands in MeCN
ΦF
ΦZnL
ΦZnL/ΦF
ΦZn2L
λ1/nm
λ2/nm
(λ2-λ1)/nm
Epa/mV
7
8
9
10
11
12
13
14
0.024 ( 0.0008
0.038 ( 0.001
0.0087 ( 0.0006
0.0092 ( 0.001
0.51 ( 0.02
0.039 ( 0.002
0.20 ( 0.002
0.014 ( 0.0006
0.35a
15
2.5
33
5.0
1.3
6.7
4.0
3.0
0.70 ( 0.01
0.32 ( 0.007
0.63 ( 0.035
0.074 ( 0.0025
N. A.
N. A.
N. A.
N. A.
392b
419b
367
392
392
423b
364
390
454b
496b
415
472
459
504b
420
480
62
77
48
80
67
81
56
90
1636, 1114
1385, 1161
1890, 1120
1698, 1178
1567 ( 4
1356 ( 14
1886 ( 6
1660 ( 4
0.095a
0.29a
0.046a
0.67 ( 0.006
0.26 ( 0.02
0.81 ( 0.025
0.042 ( 0.0012
a The monozinc complex of a heteroditopic ligand is considered as the species with ligand:zinc ratio that maximizes the shorter emission band (at λ1).
The ΦF was determined using the corresponding free ligand as the reference. b Data were taken from ref 32.
Structures
FIGURE 3. Absorption spectra of 7 (red, 20 µM) and 9 (blue, 20 µM)
in MeCN. Samples were exposed to ambient light. The absorption
spectra were collected every 30 min.
and 10 while maintaining the coordination-driven fluorescence
switching functions of 7 and 8.46
Synthesis. The synthesis of 9 is shown in Scheme 1. Radical
bromination of 5,5′-dimethyl-2,2′-dipyridyl (17) with 1 equiv
NBS followed immediately (without purification) with an
Arbuzov phosphonate synthesis afforded 20. Phosphonate 20
was easily isolatable, in contrast to its monobrominated precur-
sor, in 48% yield over two steps. The Horner-Wadsworth-
Emmons reaction between 20 and 21 gave 22, which underwent
a two-step sequence of dibromination-didehydrobromination47-49
to afford alkyne 23. Upon acidic deprotection, reductive
amination between 24 and di-(2-picolyl)amine gave rise to
compound 9 to complete an overall efficient synthesis. The
syntheses and characterizations of other new compounds are
described in the Supporting Information.
Photochemical Stability. Under ambient irradiation over 6 h,
the absorption spectrum of alkynyl compound 9 did not change
Results and Discussion
while the absorption band centered at 339 nm of vinyl compound
7 decreased readily over time (Figure 3), characteristic of a
transfcis photoisomerization.50 Compound 8 isomerized more
Compounds 7 and 8 undergo trans f cis photoisomerization
readily upon ambient irradiation (Figure 3). Furthermore,
stilbenoid structures such as 7 and 8 are known to have different
emissive conformers in their excited states,42-45 which may
complicate the later data analysis. A conservative chemical
modification which replaces the isomerizable double bonds with
triple bonds was envisioned to afford photochemically stable 9
1
readily than 7 as shown by absorption scans and H NMR
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Vedernikov, A. I.; Churakov, A. V.; Kuz’mina, L. G.; Howard, J. A. K.;
Bossmann, S.; Braun, A.; Woerner, M.; Sears, D. F., Jr.; Saltiel, J. J. Am. Chem.
Soc. 1999, 121, 4992–5000.
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J. Chem. 1997, 21, 405–408.
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8324 J. Org. Chem. Vol. 73, No. 21, 2008

Fluorescent Heteroditopic Ligands for Zinc Ion
SCHEME 1a
a (a) NBS, AIBN (cat.), CCl4, reflux, 2.5 h; (b) (EtO)3P, 125 °C, 3 h, 48%, two steps; (c) 21, NaH, rt, 4 h, 75%; (d) Br2, DCM, rt, 3 h; (e) tBuOK, THF,
rt, 14 h, 25%, two steps; (f) HCl/H2O/THF, rt, 14 h; (g) di-(2-picolyl)amine, NaBH(OAc)3, rt, 6 h, 73%, two steps.
(Supporting Information), presumably due to its larger molar
absorptivity in the visible spectrum than that of 7.
Absorption and Fluorescence Titration Studies. The Zn2+
titration experiments of compounds 9 and 10 were carried out
in MeCN. The absorption and fluorescence spectra at different
Zn2+ concentrations ([Zn]) were collected (Figures 4 and 5,
binding isotherms in the Supporting Information). Both ligands
display weak fluorescence (blue spectra) in their free forms due
to PET from the tertiary nitrogen atoms to the excited aryla-
lkynyl-bipy fluorophores. Upon increasing [Zn], fluorescence
intensity of both compounds are greatly enhanced, presumably
due to CHEF, followed by bathochromic shifts of emission
bands as the [Zn] is high enough to coordinate with bipy. The
experiments that independently verified the sequential coordina-
tion of Zn2+ by heteroditopic ligands 7-10 in solution will be
presented in the section of “Binding Studies”. The overall
fluorescence responses of 9 and 10 to Zn2+ are similar to those
of 7 and 8;32 however, the fluorescence enhancements upon
coordinating the first Zn2+ by 9 and 10 are much greater than
those of 7 and 8. The enhanced sensitivity in fluorescence turn-
on of 9 and 10 upon binding the first Zn2+ is expected to expend
the utility of this heteroditopic system in various applications.
Fluorescence Quantum Yield Measurements. The fluores-
cence quantum yields (ΦF) of free ligands 7-14 and the dizinc
complexes of 7-10 were measured by a relative method using
either 2-aminopyridine or quinine bisulfate in 0.05 M sulfuric
acid as references.51 The dizinc samples were prepared using 2
µM of the respective ligands and 6 or 10 equiv of zinc so that
full saturations of the ligands as dizinc complexes were ensured
based on the binding isotherms of 7-10.
The ΦF measurements of monozinc complexes of 7-10
required special considerations. A solution sample containing
only monozinc species cannot be prepared by using equal molar
of a heteroditopic ligand and Zn2+ due to competitive equilibria
with the free ligand and the dizinc complex (Figure 2).
Furthermore, the species distribution at equal molar of ligand
and Zn2+ is expected to be dependent on the concentration of
the ligand. At a fixed ligand concentration (e.g., 2 µM), the
emission intensity and band profile (shape and position) are
highly sensitive to [Zn] when the ratio of ligand and Zn2+ is
FIGURE 4. Absorption (A) and fluorescence (B, λex ) 321 nm) spectra
of 9 (2.0 µM) in MeCN (TBAP: 5 mM; DIPEA: 2.0 µM; DMSO: 0.1%)
upon addition of Zn(ClO4)2 (0-7.9 µM and 0-8.8 µM, respectively).
The blue arrows represent the initial spectral changes; the red arrows
represent the following bathochromic shifts. Blue spectra were taken
in the absence of Zn2+; the red spectra were taken in the presence of
7.9 µM (A) and 8.8 µM (B), respectively, of Zn2+
.
(50) Kopecky, J. Organic Photochemistry:
Publishers, Inc.: New York, 1992.
A Visual Approach; VCH
close to 1:1. The measured ΦF values varied greatly with only
(51) Fery-Forgues, S.; Lavabre, D. J. Chem. Educ. 1999, 76, 1260–1264.
minimal standard deviation of [Zn]. Therefore, it became clear
J. Org. Chem. Vol. 73, No. 21, 2008 8325

Zhang and Zhu
FIGURE 6. Job plots of association between 13 and Zn(ClO4)2 in
MeCN. The total molar concentrations of 13 and Zn(ClO4)2 are 10 µM
(0) and 60 µM ()). The mole fraction (X) of 13 was varied continuously
from 0 to 1.
are other nonradiative pathways available than photoinduced
electron transfer in the free ligands. On the other hand, 8 and
10 display slightly larger emission spectral shift upon coordinat-
ing the second Zn2+ than the benzene-derived 7 and 9;
presumably because thiophene is a stronger electron-donor than
benzene to afford more efficient internal charge transfer
characters in the excited states of 8 and 10 (also in 12 and 14).
Based on the data collected in acetonitrile, we conclude that
the overall fluorescence contrasts between different coordination
states of 7 and 9 are better suited for the design outlined in
Figure 2, where a large fluorescence turn-on upon the first Zn2+
coordination and a large emission spectral shift upon the second
Zn2+ coordination are desired. To our knowledge, this is the
first time that such large fluorescence contrast of three coordina-
tion states of a heteroditopic ligand has been reported. Studies
of these ligands in other media are in progress.
Binding Studies. The conversion from vinyl to alkynyl
substituents does not appear to affect the affinity of substituted
bipy to Zn2+ under the conditions used in our studies, as
supported by the almost overlapping spectrophotometric binding
isotherms of 12 and 14 (Figure S10, Supporting Information).
In the previous study,32 the preferential coordination of
dipicolylamino over bipy to Zn2+ in MeCN was inferred from
the X-ray single crystal structure of a mono-Zn2+ complex
([Zn(19)Cl2] of a model ditopic ligand (19). The apparent
association constants extracted from the binding isotherms using
a 1:1 model supported that dipicolylamino group binds Zn2+
stronger than bipy.32 However, the relatively small difference
(within 1 order of magnitude) observed in the previous study
prompted us to examine the binding of dipicolylamino and bipy
to Zn2+ in MeCN in more detail.
FIGURE 5. Absorption (A) and fluorescence (B, λex ) 335 nm) spectra
of 10 (2.0 µM) in MeCN (TBAP: 5 mM; DIPEA: 2.0 µM; DMSO:
0.1%) upon addition of Zn(ClO4)2 (0-7.9 µM and 0-8.8 µM,
respectively). The blue arrows represent the initial spectral changes;
the red arrows represent the following bathochromic shifts. Blue spectra
were taken in the absence of Zn2+; the red spectra were taken in the
presence of 7.9 µM (A) and 8.8 µM (B), respectively, of Zn2+
.
to us that determination of ΦF of a monozinc complex using a
sample of equal molar ligand and Zn2+ not only does not reflect
the ΦF of the monozinc complex as a unimolecular entity, but
also gives rise to poor reproducibility. In light of those
observations, we decided that the ΦF of a monozinc complex
was best represented as the ΦF of the sample that gave rise to
the highest fluorescence intensity at the shorter emission
wavelength. In this case, the free ligand was used as the
reference.
The emission wavelengths and fluorescence quantum yields
of 7-14 and their zinc complexes are compiled in Table 1. The
two parameters ΦZnL/ΦF and ∆λ (λ1-λ2), representing fluores-
cence enhancement upon first Zn2+ coordination and emission
spectral shift upon second Zn2+ coordination, define the
“fluorescence contrast” of the three coordination states of a
heteroditopic ligand. The benzene-derived 7 and 9 achieved
much higher fluorescence turn-on upon coordinating the first
Zn2+ as evidenced by large ΦZnL/ΦF values (15 and 33,
respectively) than the thiophene-derived 8 and 10. The fluo-
rescence quantum yields of both free ligands and monozinc
complexes of 8 and 10 are low (<0.1), suggesting that there
In this study, the binding stoichiometry between bipy and
Zn2+ was investigated first. A number of studies using 2,2′-
bipy-based fluorescent ligands as Zn2+ sensors reported 1:1
binding stoichiometry;52-55 other work suggested 2:1 or 3:1
(ligand:Zn2+) stoichiometry.56,57 In our study, Job’s method of
(52) Ajayaghosh, A.; Carol, P.; Sreejith, S. J. Am. Chem. Soc. 2005, 127,
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8326 J. Org. Chem. Vol. 73, No. 21, 2008

Fluorescent Heteroditopic Ligands for Zinc Ion
FIGURE 7. ITC data of (A) a 3:1 (13:Zn2+) association (n ) 0.345, ∆H° ) -29.96 kcal/mol) and (B) a two-step (18:Zn2+) association (n1 )
0.474, ∆H°(1) ) -32.26 kcal/mol; n2 ) 0.484, ∆H°(2) ) -2.839 kcal/mol) in MeCN at 298 K.
continuous variation58 (Figure 6) revealed a 2:1 binding
stoichiometry between 13 and Zn2+ at a total concentration of
10 µM ([13] + [Zn]). However, when the total concentration
was increased to 60 µM, the binding stoichiometry shifted
toward 3:1 (13:Zn2+). To study the binding stoichiometry at
higher concentrations, isothermal titration calorimetry (ITC) was
applied.
Traditionally used for the determination of binding enthalpy
(∆H°), free energy (∆G°), entropy (∆S°), and stoichiometry (n)
of protein-receptor interactions,59-62 isothermal titration calo-
rimetry (ITC) has become increasingly important in studying
supramolecular systems.63-69 However, the examples of using
ITC in studying metal-ligand coordination in organic solVents
are rare,70,71 partly because ∆G° of metal coordination in organic
solvents is usually larger than what may be accurately measured
by ITC. However, we note that the ∆H° and n values can still
be reliably determined from ITC experiments.59,70
The ITC study of the association between 13 and Zn2+
revealed a 3:1 (13:Zn2+) stoichiometry when [13] is 300 µM
(Figure 7A). Combining with the results from the Job’s plots,
it is evident that the binding stoichiometry between bipy and
Zn2+ is concentration-dependent: high concentration favors 3:1
stoichiometry, same as that observed between 17 and Zn2+
(Supporting Information), while at low concentration, 2:1 and
1:1 associations become feasible. Other factors that impact
binding stoichiometry between bipy derivatives and transition
metals are solvent, counterions, and substituent groups as
suggested by other studies.53
Contrary to a simple 1:1 binding in MeCN reported in
literature,72 the titration of dipicolylamino group (represented
by 18) with Zn2+ in MeCN shows two calorimetrically distinct
processes (Figure 7B), suggesting 2:1 (18:Zn2+) binding fol-
lowed by breaking off the 2:1 complex to a 1:1 complex as
[Zn]/[18] increases.70,71 Our study suggests that caution be
applied when binding stoichiometry is interpreted using either
Job’s method or ITC alone. A full account on the studies of
binding stoichiometry between polydentate ligands and Zn2+
using both ITC and Job plots will be reported at a later time.
A competitive binding experiment between 14 and 15 was
carried out for demonstrating the difference in affinity between
the dipicolylamino group and bipy in MeCN. As shown in
Figure 8, the addition of Zn2+ in a solution of 14 and 15 at 2
(57) Senechal, K.; Maury, O.; Le Bozec, H.; Ledoux, I.; Zyss, J. J. Am. Chem.
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Complex Stability; John Wiley and Sons: New York, 1987.
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1989, 179, 131–137.
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959A.
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(63) Berger, M.; Schmidtchen, F. P. Angew. Chem., Int. Ed. 1998, 37, 2694–
2696.
(64) Haj-Zaroubi, M.; Mitzel, N. W.; Schmidtchen, F. P. Angew. Chem., Int.
Ed. 2002, 41, 104–107.
(65) Schmidtchen, F. P. Org. Lett. 2002, 4, 431–434.
(66) Otto, S.; Kubik, S. J. Am. Chem. Soc. 2003, 125, 7804–7805.
(67) Haj-Zaroubi, M.; Schmidtchen, F. P. ChemPhysChem 2005, 6, 1181–
1186.
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F. P.; Bates, G. W.; Light, M. E.; Gale, P. A. J. Am. Chem. Soc. 2006, 128,
12281–12288.
(69) Jadhav, V. D.; Schmidtchen, F. P. J. Org. Chem. 2008, 73, 1077–1087.
(70) Dobrawa, R.; Lysetska, M.; Ballester, P.; Grune, M.; Wurthner, F.
Macromolecules 2005, 38, 1315–1325.
(71) Shunmugam, R.; Gabriel, G. J.; Smith, C. E.; Aamer, K. A.; Tew, G. N.
Chem.-Eur. J. 2008, 14, 3904–3907.
(72) de Silva, S. A.; Zavaleta, A.; Baron, D. E.; Allam, O.; Isidor, E. V.;
Kashimura, N.; Percapio, J. M. Tetrahedron Lett. 1997, 38, 2237–2240.
J. Org. Chem. Vol. 73, No. 21, 2008 8327

Zhang and Zhu
FIGURE 9. Frontier molecular orbitals of a PET system. The HOMO
and LUMO of the excited fluorophore are on the left. The HOMO of
the electron donor, in this study the dipicolylamino group, is on the
right. When the dipicolylamino group is coordinated with Zn2+ (blue),
the HOMO of the e-donor is lowered so that PET is not thermodynami-
cally feasible.
to an efficient PET in the unbound ligand. Based on the Marcus
equation, the efficiency (or rate) of PET is a function of the
thermodynamic driving force (∆GPET) of PET.73-76 The height-
ened fluorescence enhancements (ΦZnL/ΦF, Table 1) of 9 and
10 over that of 7 and 8 upon coordinating the first Zn2+ are the
results of increased ∆GPET upon conversion from olefins to
alkynes. In 7-10 where the excited fluorophores serve as
electron acceptors in PET, as shown in Figure 9, the ∆GPET is
approximated as the energy gap between the highest occupied
molecular orbitals (HOMOs) of the electron donors (in this study
the dipicolylamino group) and the electron acceptors (various
excited fluorophores). When the electron donors remain constant,
the comparison of ∆GPET in 7-10 can be made by comparing
the HOMO levels of their fluorophores.73,75
∆GPET ) Eox(D) - Ered(A) - E0,0 - wp
Eox(A) ≈ Ered(A) + E0,0
(1)
(2)
(3)
(4)
∆GPET ≈ Eox(D) - Eox(A) - wp
∆GPET(A2) - ∆GPET(A1) ≈ Eox(A1) - Eox(A2)
The analysis of the Rehm-Weller equation (eq 1, all terms
are in units of eV)77 also concludes that the differences in ∆GPET
among 7-10 resulted primarily from the differences in the
HOMO energy levels of their fluorophores. Oxidation (Eox) and
reduction (Ered) potentials of a molecule are proportional to its
HOMO and LUMO energies, respectively. Therefore, the sum
of Ered of a fluorophore and its excitation energy E0,0 can be
approximated as Eox (eq 2). By substituting eq 2 into eq 1, eq
3 is obtained, which is the relationship that was reached by
analyzing Figure 9 if wp (the work term for the charge-separated
state) is negligible. Therefore, when the electron donors are
identical as in 7-10, the difference in thermodynamic driving
forces of PET (∆∆GPET) of two ligands can be represented as
the difference in Eox (eq 4) of their fluorophores.
FIGURE 8. (A) Fluorescence spectra (λex ) 348 nm) of the mixture
of 14 (2.0 µM) and 15 (2.0 µM) in MeCN (TBAP: 5 mM; DIPEA: 2
µM) with incremental addition of Zn(ClO4)2 (0-12 µM). Blue arrow
and blue spectra follow the initial increase of fluorescence of 15 upon
Zn2+ addition; red arrows and red spectra follow the delayed fluores-
cence modulation of 14 upon further Zn2+ addition. (B) 0: fluorescence
intensity of 14 at 380 nm (2 µM) with increasing [Zn]; ): fluorescence
intensity of 14 and 15 at 380 nm (2 µM each) with increasing [Zn].
µM each immediately enhances the fluorescence of 15 (blue
arrow in Figure 8A) before the occurrence of spectral change
of 14 (red arrows in Figure 8A). The clear delay of spectral
change of 14 in the presence of 15 (2 µM, Figure 8B)
independently confirms our initial hypothesis that in MeCN,
Zn2+ binds with dipicolyl group preferentially over bipy. Due
to the dependence of apparent association constants and binding
stoichiometries of dipicolylamino and bipy to Zn2+ on ligand
concentration, solvent, and other factors, we prefer using the
competitive binding experiment over comparing the apparent
association constants of the two binding sites measured inde-
pendently for evaluating the binding preference of zinc in a
ditopic ligand.
Cyclic voltammetry was applied to determine the Eox of
7-14. All compounds were oxidized irreversibly. The peaks
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T.; Nagano, T. J. Am. Chem. Soc. 2001, 123, 2530–2536.
(74) Fahrni, C. J.; Yang, L.; VanDerveer, D. G. J. Am. Chem. Soc. 2003,
125, 3799–3812.
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J. Am. Chem. Soc. 2005, 127, 4888–4894.
(76) Ueno, S.; Tsukamoto, M.; Hirano, T.; Kikuchi, K.; Yamada, M. K.;
Nishiyama, N.; Nagano, T.; Matuski, N.; Ikegaya, Y. J. Cell. Biol. 2002, 158,
215–220.
Cyclic Voltammetry Studies. In a system of CHEF, a large
fluorescence enhancement upon metal coordination is attributed
(77) Rehm, D.; Weller, A. Isr. J. Chem. 1970, 8, 259–271.
8328 J. Org. Chem. Vol. 73, No. 21, 2008

Fluorescent Heteroditopic Ligands for Zinc Ion
FIGURE 12. Correlation between fluorescence enhancements of ditopic
ligands (7-10) upon coordination the first Zn2+ and thermodynamic
driving force of PET (∆Epa) derived from the cyclic voltammograms
of 7-10.
FIGURE 10. Cyclic voltammograms of 11, 16, and 7 in MeCN.
Bu4NPF6 (0.1 M) was used as the supporting electrolyte. Working
electrode: glassy carbon electrode; scan rate: 100 mV/s; reference
electrode: Ag/AgCl.
those of their vinyl counterparts 7 and 8, respectively. Conse-
quently, upon Zn2+ coordination at the PET donor site dipico-
lylamino, 9 and 10 enjoy a greater extent of fluorescence
restoration than that of 7 and 8.
The fluorescence enhancements (ΦZnL/ΦL) of heteroditopic
ligands 7-10 over the first step coordination were plotted as a
function of anodic potential differences (∆Epa) between the
fluorophore and the dipicolylamino group (represented by 16),
which is considered as ∆GPET in the unbound ligands. Evidently,
fluorescence enhancement upon Zn2+ coordination increases
with larger ∆GPET. The substituent effect on the fluorescence
quantum yields of arylvinyl-bipy and arylalkynyl-bipy systems
is a topic of current study.
Conclusion
Photochemically stable fluorescent heteroditopic ligands 9 and
10 were prepared. The three coordination states in the presence
or absence of Zn2+, non-, mono-, and dicoordinated, are related
to three distinct fluorescence states, OFF, ON at λ1, and ON at
λ2. The binding stoichiometry of dipicolylamino and 2,2′-bipy
to Zn2+ in MeCN were studied using Job’s method of continuous
variation and ITC, and were shown to be concentration
dependent. The higher binding affinity of dipicolylamino to Zn2+
than that of 2,2′-bipy was demonstrated using a competitive
binding experiment. Cyclic voltammetry studies indicated that
the thermodynamic driving forces for PET (∆GPET) in unbound
9 and 10 are larger than those of free 7 and 8, leading to larger
FIGURE 11. Cyclic voltammograms of 11-14 and 16 in MeCN.
Bu4NPF6 (0.1 M) was used as the supporting electrolyte. Working
electrode: glassy carbon electrode; scan rate: 100 mV/s; reference
electrode: Ag/AgCl.
of 7 at 1636 mV and 1114 mV are assigned to the oxidation
(anodic) peaks of the phenylvinyl-bipy fluorophore and the
dipicolylamino group, respectively, based on the comparison
with the cyclic voltammograms of monotopic ligands 11 and
16 (Figure 10). The higher oxidation potential of the fluorophore
than that of the dipicolylamino group is consistent with the
efficient PET observed in 7. The observation of the two
oxidation peaks of 7 supports our rationale where the HOMOs
of e-donor and e-acceptor in an intramolecular PET system can
be analyzed separately (Figure 9). The cyclic voltammograms
of 8-10 are in the Supporting Information.
Monotopic ligands 11-14 and 16 were used for the com-
parison of oxidation potentials, hence HOMO levels, of different
e-acceptors and an e-donor. Fluorophores 11-14 have oxidation
potentials higher than that of 16, N-methyldipicolylamine, which
explains the thermodynamically favorable PET processes ob-
served in 7-10 (Figure 11, anodic peak potentials are listed in
Table 1). On the other hand, the alkynyl compounds (13, 14)
have lower HOMO levels than their vinyl analogs (11, 12),
which leads to more efficient PET processes in 9 and 10 than
fluorescence enhancements of 9 and 10 upon coordinating Zn2+
.
It was demonstrated in this study that fluorescence contrast
between three coordination states of a fluorescent heteroditopic
ligand can be rationally tuned by altering the structure of the
fluorophore. Our dipicolylamino and 2,2′-bipy-based heterodi-
topic system also provides insight into binding stoichiometry
of these commonly used bidentate and tridentate metal coor-
dination ligands. The combination of Job’s method and ITC
may provide a wealth of information that would not be
uncovered by either approach alone. The structure-function
relationship pertaining to the coordination chemistry and pho-
tophysical processes in our heteroditopic systems will be applied
in future rational design of fluorescent heteroditopic ligands
tailor-made for specific applications.
J. Org. Chem. Vol. 73, No. 21, 2008 8329

Zhang and Zhu
) 1/6/7. The solution was stirred overnight before partitioned
between basic brine (pH > 9) and EtOAc (3 × 50 mL). The
organic portions were dried over Na2SO4 before concentrated
under vacuum. The crude product was analyzed by TLC (silica,
EtOAc, Rf ≈ 0.6). The purity of the product judging by TLC
and NMR (1H and 13C) spectra is sufficient for the next step.
Experimental Section
Representative procedures for preparing 9 and 10.
Compound 22. NaH (60% in mineral oil, 4 mmol, 160 mg) was
added at 0 °C to a solution of 21 (2.0 mmol, 356 mg) in dry
dimethoxyethane (4.0 mL) in a flame-dried round-bottom flask. The
suspension was stirred for 5 min. A solution of 20 (2.0 mmol, 641
mg) in dry dimethoxyethane (4.0 mL) was added dropwise to the
flask while stirring at 0 °C. Following addition, the stirring was
continued for 3 h while the temperature was brought to rt. The
reaction mixture was then cooled to 0 °C before brine (2 mL) was
added. The reaction mixture was stirred for another 5 min before
partitioned between DCM and water. The aqueous layer was washed
with DCM (50 mL × 3) and the organic portions were combined.
The organic portions were dried over Na2SO4 followed by solvent
removal under vacuum. The crude product was analyzed by TLC
(silica, EtOAc, Rf ≈ 0.7, an intensely blue fluorescent tailing spot).
Compound 22 was isolated using silica chromatography eluted by
EtOAc in DCM (gradient 0-30%). The isolated yield was 75%.
1H NMR (300 MHz, CDCl3): δ/ppm 8.76 (d, J ) 1.8 Hz, 1H),
8.52 (s, 1H), 8.37 (d, J ) 8.4 Hz, 1H), 8.30 (d, J ) 8.4 Hz, 1H),
7.98 (dd, J ) 2.4, 8.4 Hz, 1H), 7.65-7.49 (m, 5H), 7.41 (d, J )
8.4 Hz, 1H), 7.15 (d, J ) 8.4 Hz, 1H), 5.84 (s, 1H), 4.19-4.04 (m,
4H), 2.41 (s, 3H); 13C NMR (75 MHz, CDCl3): δ/ppm 155.8, 153.5,
149.9, 148.3, 137.8, 137.6, 133.6, 132.7, 130.4, 127.1, 126.9, 126.6,
125.6, 120.8, 120.8, 103.7, 65.5, 29.9, 18.6; HRMS (ESI+): calcd.
(C22H20N2O2 + Na+) 367.1422, found 367.1418.
1
The yield was quantitative. H NMR (300 MHz, CDCl3): δ/ppm
10.04 (s, 1H), 8.82 (s, 1H), 8.52 (s, 1H), 8.41 (d, J ) 8.1 Hz, 1H),
8.32 (d, J ) 8.1 Hz, 1H), 7.95 (dd, J ) 2.1, 8.4 Hz, 1H), 7.91 (d,
J ) 8.4 Hz, 2H), 7.72 (d, J ) 8.4 Hz, 2H), 7.65 (dd, J ) 2.1, 8.1
Hz, 1H), 2.41 (s, 3H); 13C NMR (75 MHz, CDCl3): δ/ppm 191.5,
155.8, 153.0, 151.9, 150.0, 139.7, 134.2, 137.7, 132.4, 129.8, 129.1,
121.2, 120.3, 119.3, 92.4, 90.6, 18.6; HRMS (ESI+): calcd.
(C20H14N2O + Na+) 321.1004, found 321.1003.
Compound 9. Di-(2-picolyl)amine (38 µL, 0.21 mmol) was
added dropwise to an anhydrous 1,2-dichloroethane (0.84 mL)
solution of 24 (62 mg, 0.21 mmol). The mixture was stirred
overnight before NaBH(OAc)3 (89 mg, 0.42 mmol) was added. The
mixture was stirred for another 5 h before the solvent was removed
under vacuum. The residue was washed with basic brine (pH >
11) and extracted with DCM (3 × 50 mL). The organic portions
were dried over K2CO3 before concentrated under vacuum.
Compound 9 was isolated by alumina chromatography eluted by
EtOAc in DCM (gradient 0-30%). The isolated yield was 73%.
1H NMR (300 MHz, CDCl3): δ/ppm 8.78 (s, 1H), 8.54-8.51 (m,
3H), 8.36 (d, J ) 8.4 Hz, 1H), 8.30 (d, J ) 8.4 Hz, 1H), 7.91 (dd,
J ) 1.8, 8.4 Hz, 1H), 7.71-7.42 (m, 9 H), 7.16 (t, J ) 6.0 Hz,
2H), 3.82 (s, 4H), 3.71 (s,2H), 2.40 (s,3H); 13C NMR (75 MHz,
CDCl3): δ/ppm 159.6, 155.0, 153.1, 151.6, 149.8, 149.1, 140.2,
139.3, 137.5, 136.5, 133.7, 131.8, 129.0, 122.9, 122.1, 121.4, 120.9,
120.1, 93.4, 86.5, 60.2, 58.4, 18.5; HRMS (ESI+): calcd. (C32H27N5
+ Na+) 504.2164, found 504.2161.
Compound 23. A dry DCM solution (10 mL) of 22 (515 mg,
1.50 mmol) was added dropwise of a dry DCM solution (10 mL)
of Br2 (1.50 mmol, 77 µL) at 0 °C. The reaction mixture was stirred
for another 3 h after addition was completed as the temperature of
the reaction container rose to rt. The excess of Br2 and solvent
was removed carefully on a rotary evaporator at rt with Na2S2O3
in the receiving flask to absorb Br2. The residue from the
bromination reaction was dissolved in anhydrous THF (5.0 mL)
and cooled to 0 °C. tBuOK (6.0 mmol, 673 mg) was added and the
reaction mixture was stirred for overnight. The reaction mixture
was poured into icy water and product was extracted using DCM
(50 mL × 3). The organic portions were combined and dried over
Na2SO4 followed by solvent removal under vacuum. The crude
product was analyzed by TLC (silica, EtOAc, Rf ≈ 0.8). Compound
23 was isolated using silica chromatography eluted by EtOAc in
DCM (gradient 0-15%). The yield was 25%. 1H NMR (300 MHz,
CDCl3): δ/ppm 8.79 (d, J ) 2.1 Hz, 1H), 8.52 (s, 1H), 8.37 (d, J
) 8.1 Hz, 1H), 8.31 (d, J ) 8.1 Hz, 1H), 7.92 (dd, J ) 2.1, 8.1
Hz, 1H), 7.63 (dd, J ) 2.1, 8.1 Hz, 1H), 7.59 (d, J ) 8.1 Hz, 2H),
7.49 (d, J ) 8.1 Hz, 2H), 5.84 (s,1H), 4.15-4.03 (m, 4H), 2.41 (s,
3H); 13C NMR (75 MHz, CDCl3): δ/ppm 155.2, 153.0, 151.7, 149.8,
139.4, 138.6, 137.6, 133.8, 131.8, 126.7, 123.6, 121.0, 120.1, 119.9,
103.3, 93.1, 87.1, 65.4, 18.5; HRMS (ESI+): calcd. (C22H18N2O2
+ H+) 343.1446, found 343.1431.
Acknowledgment. This work was supported by the Florida
State University through a start-up fund, a New Investigator
Research (NIR) grant from the James and Esther King Biomedi-
cal Research Program administered by the Florida Department
of Health, and National Science Foundation (CHE 0809201).
We thank Professor Kenneth Goldsby for the use of a Princeton
Applied Research potentiostat (VersaStat), and Professor Mikhail
Shatruk for helpful discussions and critical reading of the
manuscript. We also thank the Institute of Molecular Biophysics
at FSU for providing access to a VP-ITC microcalorimeter
(Microcal) and Dr. Claudius Mundoma for technical assistance.
Supporting Information Available: Syntheses and charac-
terization of new compounds, additional spectra, ITC data, and
cyclic voltammograms. This material is available free of charge
via Internet at http://pubs.acs.org.
Compound 24. Compound 23 (74 mg, 0.22 mmol) was
dissolved in a mixed solvent (14 mL) of HCl (37%)/H2O/THF
JO8015083
8330 J. Org. Chem. Vol. 73, No. 21, 2008