Fluorescence sensing based on cation-induced conformational switching: Copper-selective modulation of the photoinduced intramolecular charge transfer of a donor-acceptor biphenyl fluorophore

Article abstract of DOI:10.1016/j.tet.2004.08.056

The fluorescence emission energy of donor-acceptor substituted biphenyls is highly sensitive towards conformational changes of the interannular twist angle. By integrating 4-dimethylamino-4′-cyano-biphenyl into the ligand backbone of a thioether-rich metal receptor we designed a fluorescence sensor that exhibits high selectivity towards copper. Upon metal binding the ligand undergoes a significant conformational change, which induces a strong hypsochromic shift of the photoinduced charge-transfer emission. Steady-state absorption and fluorescence spectroscopy revealed a high affinity towards Cu(I) with a well-defined 1:1 metal-ligand complex stoichiometry. The nature of the conformational changes upon Cu(I) coordination were analyzed in detail by ¹H NMR and 2D NOESY experiments. The spectroscopic data provide a coherent picture, which is consistent with a Boltzmann ground-state distribution of several rotamers that are locked into a more flattened geometry upon coordination of Cu(I).

Full text of DOI:10.1016/j.tet.2004.08.056

Tetrahedron 60 (2004) 11099–11107
Fluorescence sensing based on cation-induced conformational
switching: copper-selective modulation of the photoinduced
intramolecular charge transfer of a donor–acceptor
biphenyl fluorophore
*
John Cody and Christoph J. Fahrni
School of Chemistry and Biochemistry, Georgia Institute of Technology, 770 State Street, Atlanta, GA 30332, USA
Received 2 February 2004; revised 27 July 2004; accepted 19 August 2004
Available online 15 September 2004
Abstract—The fluorescence emission energy of donor–accepto0r substituted biphenyls is highly sensitive towards conformational changes of
the interannular twist angle. By integrating 4-dimethylamino-4 -cyano-biphenyl into the ligand backbone of a thioether-rich metal receptor
we designed a fluorescence sensor that exhibits high selectivity towards copper. Upon metal binding the ligand undergoes a significant
conformational change, which induces a strong hypsochromic shift of the photoinduced charge-transfer emission. Steady-state absorption
and fluorescence spectroscopy revealed a high affinity towards Cu(I) with a well-defined 1:1 metal–ligand complex stoichiometry. The nature
of the conformational changes upon Cu(I) coordination were analyzed in detail by 1H NMR and 2D NOESY experiments. The spectroscopic
data provide a coherent picture, which is consistent with a Boltzmann ground-state distribution of several rotamers that are locked into a more
flattened geometry upon coordination of Cu(I).
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
developed, indicating the considerable challenges in the
probe design.4
Cation-specific fluorescence sensors are powerful tools for
the measurement of metal ion concentrations in environ-
mental and biological samples. They typically combine high
optical sensitivity with excellent cation selectivity, and are
therefore particularly well suited for the non-invasive
visualization of labile metal pools in a biological environ-
ment.1 Perhaps the largest class of fluorescence sensors
function as simple cation-responsive switches.2 The linear
relationship between intensity and cation concentration
allows for quantitative measurements; however, the
emission intensity depends also on the sensor concentration,
which is often not known with sufficient accuracy in
biological samples. Fluorescence sensors which undergo a
spectral shift upon binding of the cation inherently provide
concentration information of the metal-free sensor and are
principally suitable for accurate quantitative measurements
via ratiometric fluorescence imaging.3 Despite their useful-
ness, only a handful of ratiometric sensors have been
Upon excitation into a charge transfer state the electronic
structure of the fluorophore is significantly altered, which
typically gives rise to considerable structural changes such
as alteration of bond angles and distances, or twisting of
conformationally flexible bonds.5 In particular the latter
relaxation mode could be effectively utilized to achieve a
spectral shift, either via cation-induced changes of the
ground-state geometry or through interference with an
excited-state conformational relaxation pathway. Despite
being a promising approach for the design of ratiometric
probes, there are currently only few sensors which are based
on a cation-induced conformational switching mechanism.6
Among suitable fluorophore platforms 4-dimethylamino-40-
cyanobiphenyl derivatives stand out due to their spectral
sensitivity towards conformational changes of the inter-
annular C–C bond (Chart 1).7,8 For example, the methyl
substituents in DMDCB enforce a near perpendicular
ground-state geometry, which results in a large bath-
ochromic shift compared to the more flattened unsubstituted
fluorophore DCB.7 To take advantage of this conformational
sensitivity we thought to integrate the DCB fluorophore
structure into a ligand backbone, such that cation binding
Keywords: Fluorescence sensor; Copper; Photoinduced charge transfer;
Ligand design; Donor–acceptor fluorophores.
* Corresponding author. Tel.: C1-404-385-1164; fax: C1-404-894-2295;
e-mail: fahrni@chemistry.gatech.edu
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.08.056

11100
J. Cody, C. J. Fahrni / Tetrahedron 60 (2004) 11099–11107
2. Results and discussion
2.1. Synthesis
While symmetrically substituted biphenyl derivatives are
accessible from a single precursor via reductive coupling,9
the synthesis of unsymmetrically substituted donor–accep-
tor biphenyls such as 1 requires two different building
blocks and is therefore significantly more laborious. To
achieve sufficient synthetic flexibility, we were looking for a
suitable precursor that could be functionalized with a wide
range of metal-coordinating motifs. Key intermediate 9
fulfills these requirements and is readily obtained via
palladium-catalyzed Suzuki-coupling of the corresponding
reaction partners 5 and 8 (Scheme 2). While the aldehyde
group is stable towards Suzuki-coupling conditions,10 it is
readily functionalized via reductive amination or conden-
sation with amines to yield the corresponding Schiff bases.
Furthermore, upon reduction and conversion to the
corresponding halide or tosylate, it can serve as a versatile
precursor for substitution with a wide range of nitrogen,
sulfur or oxygen nucleophiles. For the synthesis of ligand 1
Chart 1.
would induce a significant conformational change of the
interannular twist angle (Scheme 1). Because our current
interest is focusing on the development of copper-selective
fluorescence sensors, we used a thioether-rich ligand
environment as metal-receptor for the design of chemosen-
sor 1. However, this approach could be readily adapted for
selective fluorescence sensing of other metal cations by
attaching different coordinating motifs.
Scheme 1.
Scheme 2.

J. Cody, C. J. Fahrni / Tetrahedron 60 (2004) 11099–11107
11101
Table 1. Spectroscopic data for 1, 1-Cu(I), DCB,a and DMDCBa in anhydrous EtOH at 298 K
d
Absorption
lmax (nm),
Emission
lmax (nm)
Quantum
yieldc Ff
Stokes’ shift
(cmK1
HWem
(cmK1
)
)
3 (MK1 cmK1 b
)
Ligand 1
319 (CT), 4630
239 (A)
326 (CT), 5770
240 (A)
345 (CT)
512
473
0.08
11,800
9550
5000
4300
1C2 equiv [Cu(CH3CN)4]PF6
0.026
DCBa
456
535
0.81
0.04
7100
13,400
3400
6200
DMDCBa
312 (CT)
a
From Ref. 7.
Molar extinction coefficient.
b
c
Aerated solution, quinine sulfate in 1 N H2SO4 as quantum yield standard.12
Fluorescence emission band half-width.
d
Figure 1. Absorption (left) and fluorescence emission (right, excitation at isosbestic point) spectra for the titration of ligand 1 (100 mM in EtOH, 298 K) with
0.1 molar equiv aliquots of [Cu(CH3CN)4]PF6. For clarity the absorption spectra are only shown for 0.2 molar equiv aliquots.
the aldehyde groups were reduced with sodium borohy-
dride, chlorinated with thionyl chloride, and finally
substituted with thiol 11. The donor building block 5 was
synthesized in four steps starting from commercially
available 3-(dimethylamino)-benzoic acid 2. Aldehyde 3
was obtained in 77% overall yield via reduction with lithium
aluminum hydride followed by oxidation with manganese
dioxide in anhydrous dichloromethane. The regioselective
ortho-bromination of aldehyde 3 was accomplished in 87%
yield using 1,3-dibromo-5,5-dimethylhydantoin as reagent.
Finally, palladium-catalyzed borylation11 of aryl-bromide 4
provided the boronate 5 in 35% yield. For the synthesis of
the acceptor moiety 8 commercially available 4-bromo-3-
methyl-benzonitrile was converted via radical bromination
to the dibromomethyl derivative 7, which was then
hydrolyzed under mild conditions to give 8 in 60% overall
yield. Although the current study is focusing on thioether
ligand 1, the synthetic strategy outlined in Scheme 2 should
be suitable to provide access to a wide range of other donor–
acceptor substituted biphenyl ligands.
strongly dependent on the interannular twist angle F, the
published photophysical data7 for DCB (FgasZ398) and
DMDCB (FgasZ788) provide a valuable reference for the
interpretation of the photophysical properties of ligand 1.
The absorption spectrum of the free ligand in absolute
ethanol shows two broad feature-less bands centered around
239 and 319 nm (Fig. 1, left). Using DCB and DMDCB as
reference compounds, the bands can be unambiguously
assigned to a locally excited state of the 1La type (A), and a
charge-transfer state (1CT), which involves p-electron
2.2. Complexation studies with [Cu(I)(CH3CN)4]PF6
2.2.1. Steady state absorption and emission spectra. A
compilation of the photophysical data for ligand 1 and its
Cu(I) complex are given in Table 1. Because the absorption
and emission properties of D–A substituted biphenyls are
Figure 2. Mol-ratio plot of the UV–vis absorbance at 326 nm (B) and the
fluorescence intensity at 512 nm (C) for the titration shown in Figure 1.

11102
J. Cody, C. J. Fahrni / Tetrahedron 60 (2004) 11099–11107
significantly higher energy by 39 nm (1600 cmK1) and
decreases in intensity (Fig. 1). The UV–vis absorption traces
form a set of sharp isosbestic points throughout the entire
titration range, indicating a stoichiometrically well-defined
equilibrium which involves only two UV–vis active species,
the free ligand and its Cu(I)-coordinated form.
The mol-ratio plot of the absorbance at 326 and the
fluorescence emission at 510 nm reveals for both saturation
after addition of 1 molar equiv of Cu(I), which is consistent
with a 1:1 complex stoichiometry (Fig. 2). Furthermore, the
linearity of the absorbance increase and emission decrease
up to 1 molar equiv of Cu(I) suggests a high binding affinity
with near-quantitative fractional saturation at each copper
concentration. Because reliable binding constants can be
only obtained from titration data with a fractional saturation
of less than 80%,13 the mol-ratio plot is not suitable to
extract an accurate binding affinity. A comparison of the
fluorescence emission band half-width shows a significant
decrease by 700 cmK1 for the Cu(I)-bound ligand (Table 1)
and directly reflects the reduced conformational flexibility
upon complexation. The greater band half-width of the free
ligand is presumably due to the presence of several
energetically similar rotamer conformations, which com-
prise a dynamic ground-state Boltzmann distribution. This
interpretation is further corroborated by the observation that
the peak emission position depends strongly on the
excitation wavelength in the case of the free but not the
Cu(I)-bound ligand (Fig. 3). The free ligand shows a broad
distribution of the peak emission wavelength, which
undergoes a hypsochromic shift by more than 30 nm when
excited at increasingly lower energy. In contrast, the
fluorescence emission of the Cu(I)-bound ligand is essen-
tially independent of the excitation energy, thus suggesting
conformational ‘locking’ upon Cu(I) coordination.
Figure 3. Normalized fluorescence emission spectra of 1 and 1-Cu(I) as a
function of the excitation wavelength (300–380 nm; 100 mM 1 in EtOH,
298 K). (a) Free ligand; (b) ligand 1 in the presence of 2 molar equiv of
[Cu(CH3CN)4]PF6.
delocalization from the dimethylamino to the cyano group.
The relative intensities and positions of the two bands
resembles strongly the published absorption spectrum for
DMDCB,7 suggesting a similar twist angle of approxi-
mately 808 for the ground state equilibrium geometry. The
highly Stokes’ shifted fluorescence emission of the free
ligand exhibits a peak wavelength of 512 nm (Fig. 1, right),
which is only slightly higher in energy compared to the
published value of 535 nm for the peak emission of
DMDCB in ethanol.7 The Stokes’ shift of the fluorescence
emission of 1 is by 1600 cmK1 smaller compared to the
value for DMDCB, suggesting a slightly smaller degree of
To gain further insights into the energetics of the ground-
state rotamer equilibrium, we performed density functional
calculations at the B3LYP/6-31G* level of theory with
model compound 12 (Fig. 4). The gas-phase ground-state
potential curve for rotation about the inter-annular C–C
bond reveals indeed a very shallow well with a minimum at
FZ82.08. Although compared to DMDCB the ortho-
substituted methyl groups in 12 are expected to generate a
1
geometrical reorganization in the emissive CT state.
Upon addition of [Cu(I)(CH3CN)4]PF6 the CT absorption
band of ligand 1 undergoes a bathochromic shift by 7 nm
(670 cmK1), whereas the peak emission moves to
Figure 4. Unrelaxed gas-phase ground-state potential energy curve for 12 as a function of the biphenyl dihedral angle F (B3LYP/6-31G*). The potentials
represent single point energies of the corresponding unrelaxed molecular geometries, which were obtained from the geometry-optimized structure (FZ82.08)
by variation of F. The vertical dotted line indicates the dihedral angle of the lowest-energy rotamer.

J. Cody, C. J. Fahrni / Tetrahedron 60 (2004) 11099–11107
11103
1
Figure 5. Aromatic region of the H NMR spectra for the titration of ligand 1 (20 mM in CD3OD) with [Cu(CH3CN)4]PF6 at 298 K. The concentration of
added Cu(I) is indicated as molar equivalents of total ligand present. The proton numbering scheme is provided with the structure to the right.
Figure 6. NOESY spectra of (a) ligand 1 and (b) ligand 1 in the presence of 2 molar equiv of [Cu(CH3CN)4]PF6 in CD3OD at 298 K. For clarity the graph is
reduced to the spectrum section which shows the cross-correlation signals between the benzylic (H2C-2/20), the dimethylamino (N(CH3)2), and the aromatic
ring protons (H-6/60).
similar energy barrier for rotation about the interannular
C–C bond, the potential curve for 12 is not symmetrical. As
illustrated in Figure 4 rotation by 128 from the equilibrium
geometry towards a more acute dihedral angle increases the
potential energy by 5.3 kJ molK1, whereas a rotation of 368
is necessary to create an isoenergetic potential in the
opposite direction. Nevertheless, the calculated dihedral
angle of the lowest-energy conformation is very similar
compared to DMDCB, for which a lowest energy
conformation with FZ788 has been reported based on
semi-empirical AM1 calculations.7 Hence, the spectro-
scopic data combined with the computational study suggests
that the ground-state equilibrium is dominated by rotamers
with a near perpendicular dihedral angle. At the same time,
the observed hypsochromic shift of the peak emission is
consistent with the presence of a subpopulation of rotamers
with a more flattened geometry (FO908). With increasing
flattening of the biphenyl ring-system, the CT absorption
energy moves gradually towards lower energy. Upon
excitation at the red-edge of the UV–vis CT band, the
contribution of the flattened rotamers to the emission
Figure 7. Emission intensity of ligand 1 at 510 nm (excitation at 320 nm) in
response to various metal cations (100 mM in EtOH, 2 molar equiv metal,
298 K).

11104
J. Cody, C. J. Fahrni / Tetrahedron 60 (2004) 11099–11107
spectrum becomes increasingly important due to their
increased cross-section at lower energy.
3. Conclusions
The absorption and emission spectroscopic data combined
with the H NMR and 2D NOESY experiments provide a
1
2.2.2. NMR studies. To explore the ground-state structural
changes of the ligand backbone upon coordination of Cu(I),
coherent picture of the conformational changes which occur
in the ground-state equilibrium upon coordination of Cu(I)
to ligand 1 at room temperature. While the free ligand shows
an equilibrium distribution of rotamers in the ground-state,
Cu(I) complexation imposes conformational locking and
results in a flattened geometry with respect to the two aryl
moieties. Detailed studies by Maus et al. unequivocally
established that the photoinduced charge transfer state of
DMDCB undergoes flattening distortion in non-polar
solvents, but adopts a near perpendicular geometry in
polar solvents such as acetonitrile or ethanol.7,8,14 The
similarities of the emission quantum yield and the spectro-
scopic data of 1 compared to DMDCB suggests, that the
free ligand 1 might also adopt a near-perpendicular
geometry in the charge transfer state. However, it is
important to keep in mind that the different positioning of
the ortho-substituents on the biphenyl ring system results in
an unsymmetrical potential energy surface for ligand 1, thus
providing a better opportunity for relaxation of the charge
transfer state towards a more open dihedral angle. In either
case, conformational locking by Cu(I) would inhibit this
rotational relaxation pathway and result in reduced
geometrical distortion, which is consistent with the smaller
Stokes’ shift and higher emission energy for the Cu(I)-
complex. The reduced quantum yield of the Cu(I) bound
form might be due to the presence of an additional non-
emissive excited state (1ET), which could be accessed via
reductive electron transfer from Cu(I) to the biphenyl
fluorophore. At present, conclusions regarding the excited
state conformational dynamics and non-radiative deactiva-
tion pathways remain rather speculative and require more
detailed photophysical studies.
1
we performed a series of NMR experiments. The H NMR
spectra for titration of ligand 1 with [Cu(I)(CH3CN)4]PF6 in
CD3OD revealed significant changes in the chemical shift of
the aromatic proton resonances of the biphenyl ring system
(Fig. 5). Throughout the entire titration range a single set of
sharp resonances was observed, indicating fast metal
exchange kinetics compared to the NMR time scale.
Therefore, the chemical shifts reflect directly the fractional
contribution of free and Cu(I)-bound ligand. As already
observed for the UV–vis and fluorescence titrations the
resonances shift linearly up to 1 molar equiv of Cu(I) and
undergo no further changes at higher concentrations. It is
noteworthy that the slope of the chemical shift changes is
essentially identical for the corresponding ortho-(H-6/60)
and meta-proton (H-3/30 and H-5/50) resonances, thus
suggesting a symmetrical coordination of Cu(I) to both
thioether branches and no involvement of the dimethyl-
amino group in metal coordination.
2.3. NOESY (Nuclear Overhauser Enhancement
Spectroscopy) experiment
The NOESY experiment is the two-dimensional equivalent
of a NOE difference experiment and yields correlation
signals which are caused by dipolar cross-relaxation
between nuclei in a close spatial relationship. Because the
intensities of the cross-peaks are proportional to the sixth
power of the proton–proton distances, this technique is well
suited to investigate structural differences between the free
and Cu(I)-bound ligand. As shown in Figure 6 the
conformational differences of the biphenyl backbone are
best reflected in the changes of the cross-correlation signal
intensities of the benzylic (H2C-2/20) and ortho-protons
(H-6/60). If the integrated intensities are normalized for each
spectra using the correlation signals between the H-5/H-3
and dimethylamino protons, qualitative information of the
distance changes associated with Cu(I) coordination can be
directly extracted from the spectra. Hence, upon addition of
excess Cu(I) the cross correlation signals for H2C-2/H-60
and H2C-20/H-6 increase by a factor of 2, whereas the signal
for the cross-relaxation of H-6/H-60 decreases by a factor of
10 (not included in Fig. 6). These data unambiguously
indicate that upon Cu(I) coordination the dihedral angle of
the biphenyl backbone increases (FO908) to yield a more
flattened conformation.
In summary, this study demonstrates that cation-induced
conformational locking of the ground or excited state can
yield substantial shifts of the emission energy and represents
therefore an attractive strategy for the design of ratiometric
cation-responsive fluorescence sensors.
4. Experimental
4.1. Materials and reagents
All starting materials, reagents and solvents were purchased
from Aldrich (Milwaukee, WI) or Fisher Scientific
(Pittsburgh, PA) and used without further purification.
1
NMR: d in ppm vs. SiMe4 (0 ppm, H, 400 MHz). MS:
selected peaks; m/z. Melting points are uncorrected. Flash
chromatography: Merck silica gel (240–400 mesh). TLC:
0.25 mm, Merck silica gel 60 F254, visualizing at 254 nm or
with 2% KMnO4 solution.
2.3.1. Selectivity. To evaluate the selectivity of ligand 1 we
measured its fluorescence response in the presence of
various metal cations (Fig. 7). Among the tested mono- and
divalent metal cations only Cu(I) and Cu(II) induced a
significant change of the emission intensity. Although
Cd(II) and Hg(II) are both members of the ‘copper triangle’,
neither metal resulted in a change of the emission intensity.
Similarly, the UV–vis spectra displayed no changes upon
addition of either cation (data not shown), suggesting a
surprisingly weak interaction with the thioether ligand.
4.2. Synthesis
4.2.1. 2-Bromo-5-(dimethylamino)-benzaldehyde (4).
Solid lithium aluminum hydride (5.75 g, 152 mmol) was
added in portions to a stirring solution of acid 2 (10.0 g,
61 mmol) in anhydrous THF at 0 8C. The reaction mixture

J. Cody, C. J. Fahrni / Tetrahedron 60 (2004) 11099–11107
11105
was stirred for 1 h at room temperature and then quenched
by addition of H2O (4 equiv relative to LAH). The
precipitate was filtered off, washed with THF, and the
combined filtrates were concentrated under reduced
pressure to yield 3-(dimethylamino)-benzylalcohol as a
(18), 217.2 (100), 176.1 (16). EI HRMS m/e calcd for (MC
H) C15H22BNO3 275.1693, found 275.1759.
4.2.3. 4-Bromo-3-formyl-benzonitrile (8). A solution of
4-bromo-3-methylbenzonitrile
6
(4.0 g, 20 mmol),
1
yellow oil (8.22 g, 90%). Rf 0.15 (hexane/EtOAc 4:1); H
N-bromosuccinimide (7.63 g, 43 mmol), and benzoyl per-
oxide (250 mg, 1.0 mmol) was heated at reflux for 4 h. The
reaction mixture was diluted with H2O and extracted with
EtOAc. The organic layers were dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure.
The product was purified on silica gel (hexane/Et2O 20:1) to
yield 5.85 g of dibromide 7 as a white powder (17 mmol,
81%). Mp 149–151 8C; Rf 0.17 (20:1 hexane/Et2O); 1H
NMR (CDCl3, 400 MHz) d 7.00 (s, 1H), 7.44 (dd, JZ8.2,
1.6 Hz, 1H), 7.66 (d, JZ8.2 Hz, 1H), 8.31 (d, JZ1.6 Hz,
1H); MS (70 eV) 354.8 (MC, 9), 273.9 (100), 114.0 (46). EI
HRMS m/e calcd for (MCH) C8H4Br3N 350.7894, found
350.7892.
NMR (CDCl3, 400 MHz) d 2.62 (s, 1H broad), 2.90 (s, 6H),
4.55 (s, 2H), 6.62–6.71 (m, 3H), 7.19 (t, JZ7.7 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) d 40.6, 65.5, 111.2, 111.9, 115.2,
129.0, 141.8, 150.7; MS (70 eV) 150.1 (MC, 25), 148.1
(100), 77 (25); FAB HRMS m/e calcd for (MCH) C9H11NO
149.0841, found 149.0827.
A mixture of the crude alcohol (5.15 g, 34 mmol) and
activated MnO2 (35.5 g, 409 mmol) in anhydrous CH2Cl2
was stirred for 4 h at room temperature. The solid was
filtered off (Celite) and washed with CH2Cl2. The combined
filtrates were extracted with aq NaHCO3, dried over
anhydrous MgSO4, and concentrated under reduced
pressure to yield aldehyde 3 as a yellow-green oil (4.32 g,
85%). Rf 0.34 (8:1 hexane/EtOAc); 1H NMR (CDCl3,
400 MHz) d 2.99 (s, 6H), 6.94 (dd, JZ2.2, 1.6 Hz, 1H), 6.97
(dd, JZ2.7, 1.1 Hz, 1H), 7.17 (d, JZ1.6 Hz, 1H), 7.37 (t,
JZ7.7 Hz, 1H), 9.94 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d 40.3, 111.5, 118.2, 118.8, 129.5, 137.1, 150.7, 193.2; MS
(70 eV) 149.1 (MC, 97), 148.1 (100), 77 (25). FAB HRMS
m/e calcd for (MCH) C9H11NO 149.0841, found 149.0827.
The crude bromide 7 (5.85 g, 17 mmol) was added to a
solution of sodium acetate (5.98 g, 73 mmol), calcium
carbonate (3.70 g, 37 mmol), and tetrabutyl ammonium
bromide (1.26 g, 3.9 mmol) in 500 mL of H2O. The reaction
mixture was heated under reflux for 12 h, cooled to room
temperature, and extracted with CH2Cl2. The organic layers
were dried with MgSO4, filtered, and concentrated under
reduced pressure. The product was purified on silica gel to
yield aldehyde 8 as a white powder (2.57 g, 12 mmol, 74%)
with an overall yield from 6 of 60%. Mp 124–126 8C; Rf
To the crude benzaldehyde (4.36 g, 29 mmol) in CH2Cl2
was added 1,3-dibromo-5,5-dimethylhydantoin (3.77 g,
13 mmol). The mixture was then allowed to reflux overnight
and subsequently diluted with H2O and extracted with
CH2Cl2. The organic layers were then dried with MgSO4,
filtered and concentrated under reduced pressure. The
reaction mixture was purified on silica gel (8:1 hexanes/
EtOAc) to yield 4 as a bright yellow solid (5.80 g, 87%)
with an overall yield from 2 of 67%. Mp 55–58 8C; Rf 0.39
1
0.12 (hexane/Et2O 20:1); H NMR (CDCl3, 400 MHz) d
7.72 (dd, JZ8.2, 2.2 Hz, 1H), 7.84 (d, JZ8.2 Hz, 1H), 8.18
(d, JZ2.2 Hz, 1H), 10.35 (s, 1H); 13C NMR (CDCl3,
100 MHz) d 112.6, 116.8, 131.5, 133.3, 134.2, 135.1, 137.2,
189.5; MS (70 eV) 210 (MC, 100), 129.1 (30), 101 (42), 50
(38). EI HRMS m/e calcd for (MCH) C8H4BrNO 208.9476,
found 208.9476.
1
4.2.4. 4-Dimethylamino-2,20-diformyl-40-cyano-1,10-
biphenyl (9). 4-Bromo-3-formylbenzonitrile 8 (161 mg,
0.77 mmol) was added to a Schlenck tube containing a
solution of boronate ester 5 (209 mg, 0.76 mmol), CsF
(281 mg, 1.9 mmol), and Pd(PPh3)4 (5 mol%) in 8 mL of
dimethoxyethane. The mixture was heated under reflux
overnight at which point a bright orange precipitate was
formed. The reaction mixture was diluted with H2O and
extracted with CH2Cl2. The organic layers were dried over
anhydrous MgSO4, filtered, and concentrated under reduced
pressure. The crude product was purified on silica gel
(CH2Cl2/MeOH 4:1) to yield 135 mg of the coupling
product 9 as an orange crystalline solid (0.49 mmol, 64%).
(8:1 hexanes/EtOAc); H NMR (CDCl3, 400 MHz) d 2.98
(s, 6H), 6.80 (dd, JZ8.8, 3.3 Hz, 1H), 7.19 (d, JZ3.3 Hz,
1H), 7.43 (d, JZ8.8 Hz, 1H), 10.31 (s, 1H); 13C NMR
(CDCl3, 100 MHz) d 40.4, 112.1, 113.0, 119.0, 119.2,
133.2, 133.9, 192.7, MS (70 eV) 228 (MC, 98), 227 (100),
156 (24). EI HRMS m/e calcd for (MCH) C9H10BrNO
226.9946, found 226.9950.
4.2.2. 5-Dimethylamino-2-(4,4,5,5-tetramethyl-[1,3,2]-
dioxaborolan-2-yl)-benzaldehyde (5). 4,4,5,5-Tetra-
methyl-[1,3,2]-dioxaborolane (950 mL, 6.55 mmol) was
added to a Schlenck tube containing a solution of bromide
4 (0.5 g, 2.18 mmol) in dioxane. Triethylamine (950 mL,
6.82 mmol) and PdCl2(PPh3)2 (46 mg, 3 mol%) were added
and the solution was flushed with a light stream of argon.
The tube was then heated for 3 h at 100 8C. The reaction
mixture was then diluted with H2O and extracted with
EtOAc. The organic layers were dried with MgSO4, filtered,
and concentrated under reduced pressure. The product was
purified on silica gel (hexane/EtOAc 10:1) to yield boronate
ester 5 as a yellow oil (209 mg, 35%). Rf 0.35 (8:1 hexane/
EtOAc); 1H NMR (CDCl3, 400 MHz) d 1.35 (s, 12H), 3.03
(s, 6H), 6.86 (dd, JZ8.8, 2.7 Hz, 1H), 7.28 (d, JZ2.7 Hz,
1H), 7.80 (d, JZ8.8 Hz, 1H), 10.68 (s, 1H); 13C NMR
(CDCl3, 100 MHz) d 24.9, 40.1, 83.6, 108.9, 115.7, 137.7,
142.7, 151.8, 195.8; MS (70 eV) 275.2 (MC, 63), 261.0
1
Mp 189–191 8C; Rf 0.33 (hexane/EtOAc 2:1); H NMR
(CDCl3, 400 MHz) d 3.11 (s, 6H), 6.97 (dd, JZ8.2, 2.7 Hz,
1H), 7.14 (d, JZ8.8 Hz, 1H), 7.28 (d, JZ2.7 Hz, 1H), 7.44
(d, JZ8.2 Hz, 1H), 7.84 (dd, JZ7.7, 1.7 Hz, 1H), 8.28 (d,
JZ1.7 Hz, 1H), 9.80 (s, 1H), 9.81 (s, 1H); 13C NMR
(CDCl3, 100 MHz) d 40.6, 112.3, 113.1, 116.6, 117.9,
125.0, 131.8, 133.0, 133.3, 135.0, 135.5, 135.6, 147.0,
150.9, 189.7, 191.4; MS (70 eV) 278.1 (MC, 88), 249.2
(100), 221.2 (25) EI HRMS m/e calcd for (MCH)
C17H14N2O2 278.1055, found 278.1058.
4.2.5. 2,20-Bis(chloromethyl)-4-(dimethylamino)-40-
cyano-1,10-biphenyl (10). A solution of the aldehyde

11106
J. Cody, C. J. Fahrni / Tetrahedron 60 (2004) 11099–11107
(55 mg, 0.20 mmol) in MeOH was added slowly to a
solution of NaBH4 (17 mg, 0.45 mmol) in MeOH. The
solution was then allowed to stir at room temperature for
1 h. The reaction mixture was then quenched with H2O and
extracted with Et2O. The organic layers were dried with
MgSO4, filtered, and concentrated under reduced pressure.
The product was purified on silica gel (2:1 hexane/EtOAc)
to yield 40-(dimethylamino)-2,20-bis(hydroxymethyl)-1,10-
biphenyl-4-carbonitrile as a yellow solid (0.16 mmol, 81%).
extracted with CH2Cl2. The organic layers were dried with
MgSO4, filtered, and concentrated under reduced pressure.
The product was purified via Chromatron (1:1 hexane/
EtOAc) to yield 1 as a viscous yellow oil (0.11 mmol, 65%).
Rf 0.22 (1:1 hexane/EtOAc); 1H NMR (CD3CN, 400 MHz)
d 1.20–1.30 (m, 6H), 2.47–2.61 (m, 12H), 3.02 (s, 6H),
3.36–3.63 (m, 4H), 6.67 (dd, JZ8.8, 2.7 Hz, 1H), 6.76 (d,
JZ2.7 Hz, 1H), 7.00 (d, JZ8.2 Hz, 1H), 7.35 (d, JZ
7.7 Hz, 1H), 7.55 (dd, JZ7.7, 1.7 Hz, 1H), 7.83 (d, JZ
1.7 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d 14.7, 25.9,
30.2, 30.9, 31.2, 31.3, 32.0, 33.5, 34.7, 38.5, 40.3, 110.9,
111.1, 113.1, 118.7, 125.8, 129.9, 130.3, 131.8, 132.9,
136.0, 138.8, 145.6, 150.2; MS (70 eV) 490.2 (MC, 76),
247.2 (100), 89.1 (87). EI HRMS m/e calcd for (MCH)
C25H34N2S4 490.1605, found 490.1605.
1
Mp 132–135 8C; Rf 0.21 (2:1 hexane/EtOAc); H NMR
(CDCl3, 400 MHz) d 3.09 (s, 6H), 4.98 (s, 2H broad), 6.71
(d, JZ2.7 Hz, 1H), 6.82 (dd, JZ8.2, 2.7 Hz, 1H), 6.98 (dd,
JZ8.8, 2.7 Hz, 1H), 7.44 (d, JZ8.8 Hz, 1H), 7.79 (dd, JZ
8.2, 1.7 Hz), 8.20 (d, JZ1.7 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d 40.8, 68.8, 70.6, 112.0, 114.9, 116.6, 118.9,
130.3, 130.5, 131.9, 132.3, 133.7, 135.2, 136.7, 142.6,
151.1; MS (70 eV) 282.2 (MC, 100), 263.2 (23), 233.1 (33),
190.1 (17). EI HRMS m/e calcd for (MCH) C17H18N2O2
282.1368, found 282.1378.
4.3. Steady state absorption and fluorescence
spectroscopy
All sample stock solutions and buffer solutions were filtered
through 0.2 mm Nylon membrane filters to remove inter-
fering dust particles or fibers. UV–vis absorption spectra
were recorded at 25 8C using a Varian Cary Bio50 UV–vis
spectrometer with constant-temperature accessory. Steady-
state emission and excitation spectra were recorded with a
PTI fluorimeter and FELIX software. Throughout the
titration the sample solution was stirred with a magnetic
stirring device. For all titrations the path length was 1 cm
with a cell volume of 3.0 mL. All fluorescence spectra have
been corrected for the spectral response of the detection
system (emission correction file provided by instrument
manufacturer) and for the spectral irradiance of the
excitation channel (via calibrated photodiode). Quantum
yields were determined in aerated, anhydrous ethanol using
quinine sulfate dihydrate in 1.0 N H2SO4 (FfZ0.507G0.03
as fluorescence standard.12
Thionyl chloride (31 mL, 0.42 mmol) was added cautiously
to a solution of the alcohol (30 mg, 0.11 mmol) in CH2Cl2.
The reaction was then allowed to stir for 1.5 h at room
temperature. The reaction mixture was then diluted with
CH2Cl2 and washed with sat. NaHCO3. The organic layers
were dried with MgSO4, filtered, and concentrated under
reduced pressure. No further purification was necessary and
the chloride 10 was isolated as a pale yellow solid
(0.07 mmol, 68%). Mp 115–117 8C; Rf 0.25 (1:1 hexane/
EtOAc); 1H NMR (CDCl3, 400 MHz) d 2.97 (s, 6H), 4.12–
4.36 (m, 4H), 6.67 (dd, JZ8.2, 2.7 Hz, 1H), 6.75 (d, JZ
2.7 Hz, 1H), 6.98 (d, JZ8.8 Hz, 1H), 7.32 (d, JZ7.7 Hz,
1H), 7.57 (dd, JZ7.7, 1.7 Hz, 1H), 7.80 (d, JZ1.7 Hz, 1H);
13C NMR (CDCl3, 100 MHz) d 40.2, 43.1, 44.8, 112.0,
112.2, 113.3, 118.3, 124.9, 130.3, 131.3, 131.9, 133.5,
135.6, 138.1, 144.8, 150.7; MS (70 eV) 318.1 (MC, 100),
234.2 (48), 190.1 (19). EI HRMS m/e calcd for (MCH)
C17H16Cl2N2 318.0691, found 318.0704.
4.4. NMR spectroscopy
1
4.2.6. 2-Mercaptoethylethyl sulfide (11). A mixture of
thiourea (2.0 g, 26 mmol) and 2-chloroethylethyl sulfide
(3.2 g, 26 mmol) was heated at 100 8C under N2 atmo-
sphere. After all thiourea had dissolved a very viscous fluid
was formed at which point the reaction was allowed to cool
to room temperature. The mixture was hydrolyzed by
addition of 5 mL of 5 M NaOH and then stirred overnight.
The mixture was acidified with 6 M HCl to pH 3 and
extracted with ether. The organic extract was dried over
anhydrous MgSO4 and concentrated in vacuo yielding
2.14 g of thiol 11 as a colorless liquid (17.5 mmol, 68%). 1H
NMR (CDCl3, 400 MHz) d 1.27 (t, JZ7.1 Hz, 3H), 2.53–
2.62 (m, 2H), 2.71–2.78 (m, 4H), 5.31 (s, 1H); 13C NMR
(CDCl3, 100 MHz) d 14.7, 24.6, 25.7, 35.6; MS (EI) 122
(MC, 90), 75 (100), 61 (54), 47 (50). EI HRMS m/e
calculated for (MC) C4H10S2 122.0224, found 122.0225.
All NMR spectra (1H, 13C, H–1H NOESY) were recorded
on either a Bruker DRX 500 or Mercury VX 400. For all
titrations the total sample volume was 700 mL containing
20 mM ligand solution in CD3OD. The titrations were
performed by adding 0.1 molar equiv aliquots of a solution
of [Cu(I)(CH3CN)4]PF6 in CD3CN to a solution of ligand 1
in CD3OD.
4.5. Quantum chemical calculations
The geometry of the ground-state structure for 12 was
optimized by the density functional method using the
B3LYP hybrid functional with the split-valence polarized
6-31G* (6-31G(d)) basis set as implemented in the
Q-Chem15 electronic structure calculation suite of pro-
grams. The calculation of the ground-state potential curve is
based on unrelaxed geometries using the corresponding
dihedral angles for rotation around the interannular C–C
bond.
4.2.7. 4-Dimethylamino-2,20-bis-(2-ethylsulfanyl-ethyl-
sulfanylmethyl)-40-cyano-1,10-biphenyl (1). A solution of
the chloride 10 (52 mg, 0.16 mmol) in EtOH was added
dropwise to a solution of the thiol 11 (58 mg, 0.48 mmol) in
EtOH containing NaOH (20 mg, 0.50 mmol). The reaction
mixture was then allowed to stir at room temperature for
1 h. The reaction mixture was then diluted with H2O and
Acknowledgements
Financial support from the National Institutes of Health

J. Cody, C. J. Fahrni / Tetrahedron 60 (2004) 11099–11107
11107
2002, 31, 245. Henary, M. M.; Fahrni, C. J. J. Phys. Chem. A,
5210. Taki, M.; Wolford, J. L.; O’Halloran, T. V. J. Am. Chem.
Soc. 2004, 126, 712. Chang, C. J.; Jaworski, J.; Nolan,
E. M.; Sheng, M.; Lippard, S. J. Proc. Natl Acad. Sci. U.S.A.
2004, 101, 1129.
(R01GM67169) and the Georgia Institute of Technology is
gratefully acknowledged. Computations were supported by
the Center for Computational Molecular Science and
Technology at the Georgia Institute of Technology and
partially funded through a Shared University Research
(SUR) grant from IBM and the Georgia Institute of
Technology. We also thank D. Bostwick for mass spectral
data and Dr. L. Gelbaum for performing the NOESY
experiments.
5. Rettig, W.; Maus, M. Conformational Changes Accompanying
Intramolecular Excited State Electron Transfer; Waluk, J.,
Ed.; Wiley: New York, 2000; p 1.
6. McFarland, S. A.; Finney, N. S. J. Am. Chem. Soc. 2001, 123,
1260. Mello, J. V.; Finney, N. S. Angew. Chem. Int. Ed. Engl.
2001, 40, 1536. Morozumi, T.; Anada, T.; Nakamura, H.
J. Phys. Chem. B 2001, 105, 2923. Costero, A. M.; Andreu, R.;
Monrabal, E.; Martinez-Manez, R.; Sancenon, F.; Soto, J.
J. Chem. Soc. Dalton Trans. 2002, 1769. Lee, D. H.; Im, J. H.;
Lee, J. H.; Hong, J. I. Tetrahedron Lett. 2002, 43, 9637.
McFarland, S. A.; Finney, N. S. J. Am. Chem. Soc. 2002, 124,
1178. McFarland, S. A.; Finney, N. S. Chem. Commun. 2003,
388.
References and notes
1. Mason, W. T. Fluorescent and Luminescent Probes for
Biological Activity, 2nd ed.; Academic: San Diego, 1999.
For recent reports on the visualization of labile Zn(II) in
mammalian cells see for example: Zalewski, P. D.; Millard,
S. H.; Forbes, I. J.; Kapaniris, O.; Slavotinek, A.; Betts,
W. H.; Ward, A. D.; Lincoln, S. F.; Mahadevan, I. J.
Histochem. Cytochem. 1994, 42, 877. Budde, T.; Minta, A.;
White, J. A.; Kay, A. R. Neuroscience 1997, 79, 347. Nasir,
M. S.; Fahrni, C. J.; Suhy, D. A.; Kolodsick, K. J.; Singer,
C. P.; O’Halloran, T. V. J. Biol. Inorg. Chem. 1999, 4, 775.
Pearce, D. A.; Jotterand, N.; Carrico, I. S.; Imperiali, B. J. Am.
Chem. Soc. 2001, 123, 5160. Hirano, T.; Kikuchi, K.; Urano,
Y.; Nagano, T. J. Am. Chem. Soc. 2002, 124, 6555. Burdette,
S. C.; Frederickson, C. J.; Bu, W.; Lippard, S. J. J. Am. Chem.
Soc. 2003, 125, 1778. Kimura, E.; Aoki, S.; Kikuta, E.; Koike,
T. Proc. Natl Acad. Sci. U.S.A. 2003, 100, 3731.
7. Maus, M.; Rettig, W.; Bonafoux, D.; Lapouyade, R. J. Phys.
Chem. A 1999, 103, 3388.
8. Maus, M.; Rettig, W. Chem. Phys. 2000, 261, 323.
9. Hassan, J.; Sevignon, M.; Gozzi, C.; Schulz, E.; Lemaire, M.
Chem. Rev. 2002, 102, 1359.
10. Kumar, S. J. Chem. Soc. Perkin Trans. 1 2001, 1018.
11. Murata, M.; Oyama, T.; Watanabe, S.; Masuda, Y. J. Org.
Chem. 2000, 65, 164.
12. Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991.
13. Deranleau, D. A. J. Am. Chem. Soc. 1969, 91, 4044.
14. Maus, M.; Rettig, W. J. Phys. Chem. A 2002, 106, 2104. Maus,
M.; Rettig, W. Phys. Chem. Chem. Phys. 2001, 3, 5430. Maus,
M.; Rettig, W. Chem. Phys. Lett. 2000, 324, 57. Maus, M.;
Rettig, W. Chem. Phys. 1997, 218, 151.
2. DeSilva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.;
Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice,
T. E. Chem. Rev. 1997, 97, 1515.
15. Kong, J.; White, C. A.; Krylov, A. I.; Sherrill, C. D.; Adamson,
R. D.; Furlani, T. R.; Lee, M. S.; Lee, A. M.; Gwaltney, S. R.;
Adams, T. R.; Ochsenfeld, C.; Gilbert, A. T. B.; Kedziora, G.
S.; Rassolov, V. A.; Maurice, D. R.; Nair, N.; Shao, Y.; Besley,
N. A.; Maslen, P. E.; Dombroski, J. P.; Dachsel, H.; Zhang, W.
M.; Korambath, P. P.; Baker, J.; Byrd, E. F. C.; Voorhis, T. V.;
Oumi, M.; Hirata, S.; Hsu, C. P.; Ishikawa, N.; Florian, J.;
Warshel, A.; Johnson, B. G.; Gill, P. M. W.; Head-Gordon, M.;
Pople, J. A. Q-Chem; 2.0 ed.; Q-Chem Inc.: PA, 2000.
3. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985,
260, 3440.
4. Haugland, R. P. Handbook of Fluorescent Probes and
Research Products; Molecular Probes: Eugene, OR, 2002.
Recent developments for ratiometric measurement of Zn(II):
Maruyama, S.; Kikuchi, K.; Hirano, T.; Urano, Y.; Nagano, T.
J. Am. Chem. Soc. 2002, 124, 10650. Gee, K. R.; Zhou,
Z. L.; Ton-That, D.; Sensi, S. L.; Weiss, J. H. Cell Calcium