Kinetically controlled photoinduced electron transfer switching in Cu(I)-responsive fluorescent probes

Article abstract of DOI:10.1021/ja908326z

Copper(I)-responsive fluorescent probes based on photoinduced electron transfer (PET) switching consistently display incomplete recovery of emission upon Cu(I) binding compared to the corresponding isolated fluorophores, raising the question of whether Cu(I) might engage in adverse quenching pathways. To address this question, we performed detailed photophysical studies on a series of Cu(I)-responsive fluorescent probes that are based on a 16-membered thiazacrown receptor ([16]aneNS₃) tethered to 1,3,5- triarylpyrazoline-fluorophores. The fluorescence enhancement upon Cu(I) binding, which is mainly governed by changes in the photoinduced electron transfer (PET) driving force between the ligand and fluorophore, was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding a maximum 29-fold fluorescence enhancement upon saturation with Cu(I) in methanol and a greater than 500-fold enhancement upon protonation with trifluoroacetic acid. Time-resolved fluorescence decay data for the Cu(I)-saturated probe indicated the presence of three distinct emissive species in methanol. Contrary to the notion that Cu(I) might engage in reductive electron transfer quenching, femtosecond time-resolved pump-probe experiments provided no evidence for formation of a transient Cu(II) species upon photoexcitation. Variable temperature ¹H NMR experiments Revealed a dynamic equilibrium between the tetradentate NS₃-coordinated Cu(I) complex and a ternary complex involving coordination of a solvent molecule, an observation that was further supported by quantum chemical calculations. The combined photophysical, electrochemical, and solution chemistry experiments demonstrate that electron transfer from Cu(I) does not compete with radiative deactivation of the excited fluorophore, and, hence, that the Cu(I)-induced fluorescence switching is kinetically controlled.

Full text of DOI:10.1021/ja908326z

Published on Web 12/18/2009
Kinetically Controlled Photoinduced Electron Transfer
Switching in Cu(I)-Responsive Fluorescent Probes
Aneese F. Chaudhry, Manjusha Verma, M. Thomas Morgan, Maged M. Henary,
Nisan Siegel, Joel M. Hales, Joseph W. Perry,* and Christoph J. Fahrni*
School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience,
Georgia Institute of Technology, 901 Atlantic DriVe, Atlanta, Georgia 30332
Received September 30, 2009; E-mail: fahrni@chemistry.gatech.edu; perry@chemistry.gatech.edu
Abstract: Copper(I)-responsive fluorescent probes based on photoinduced electron transfer (PET) switching
consistently display incomplete recovery of emission upon Cu(I) binding compared to the corresponding
isolated fluorophores, raising the question of whether Cu(I) might engage in adverse quenching pathways.
To address this question, we performed detailed photophysical studies on a series of Cu(I)-responsive
fluorescent probes that are based on a 16-membered thiazacrown receptor ([16]aneNS3) tethered to 1,3,5-
triarylpyrazoline-fluorophores. The fluorescence enhancement upon Cu(I) binding, which is mainly governed
by changes in the photoinduced electron transfer (PET) driving force between the ligand and fluorophore,
was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding
a maximum 29-fold fluorescence enhancement upon saturation with Cu(I) in methanol and a greater than
500-fold enhancement upon protonation with trifluoroacetic acid. Time-resolved fluorescence decay data
for the Cu(I)-saturated probe indicated the presence of three distinct emissive species in methanol. Contrary
to the notion that Cu(I) might engage in reductive electron transfer quenching, femtosecond time-resolved
pump-probe experiments provided no evidence for formation of a transient Cu(II) species upon
1
photoexcitation. Variable temperature H NMR experiments revealed a dynamic equilibrium between the
tetradentate NS3-coordinated Cu(I) complex and a ternary complex involving coordination of a solvent
molecule, an observation that was further supported by quantum chemical calculations. The combined
photophysical, electrochemical, and solution chemistry experiments demonstrate that electron transfer from
Cu(I) does not compete with radiative deactivation of the excited fluorophore, and, hence, that the Cu(I)-
induced fluorescence switching is kinetically controlled.
Introduction
undesired quenching pathways can be reduced or in some cases
even eliminated through the use of electronically decoupled
The development of synthetic fluorescent probes for the
detection of transition metal cations represents a very active
and steadily growing research field.1 An important class of
cation-responsive probes is based on a photoinduced electron
transfer (PET) switching mechanism, which translates the metal-
binding event into a fluorescence emission increase.2 While
numerous PET probes have been reported for the detection of
closed-shell metal cations, including Zn(II),3 Cd(II),4 and
Hg(II),5,6 the detection of redox-active transition metals such
as Cu(I/II)6–8 and Fe(II/III)9 has received less attention. The
fluorescence-based detection of these cations is particularly
challenging due to interfering metal-mediated quenching path-
ways, for example through enhanced spin-orbit coupling or
competing energy and electron transfer processes. While these
metal receptor moieties,10 reports on Cu(I)-responsive PET
(5) Prodi, L.; Bargossi, C.; Montalti, M.; Zaccheroni, N.; Su, N.; Bradshaw,
J. S.; Izatt, R. M.; Savage, P. B. J. Am. Chem. Soc. 2000, 122, 6769.
Rurack, K.; Resch-Genger, U.; Bricks, J. L.; Spieles, M. Chem.
Commun. 2000, 2103. Nolan, E. M.; Lippard, S. J. J. Am. Chem. Soc.
2003, 125, 14270. Yoon, S.; Albers, A. E.; Wong, A. P.; Chang, C. J.
J. Am. Chem. Soc. 2005, 127, 16030. Nolan, E. M.; Racine, M. E.;
Lippard, S. J. Inorg. Chem. 2006, 45, 2742. Meng, X. M.; Liu, L.;
Hu, H. Y.; Zhu, M. Z.; Wang, M. X.; Shi, J.; Guo, Q. X. Tetrahedron
Lett. 2006, 47, 7961. Nolan, E. M.; Lippard, S. J. J. Am. Chem. Soc.
2007, 129, 5910. Yoon, S.; Miller, E. W.; He, Q.; Do, P. H.; Chang,
C. J. Angew. Chem., Int. Ed. 2007, 46, 6658. Wang, J.; Qian, X. Org.
Lett. 2006, 8, 3721. For a comprehensive review see also: Nolan, E. M.;
Lippard, S. J. Chem. ReV. 2008, 108, 3443.
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Chem. Soc. 2000, 122, 968.
(7) (a) Yang, L.; McRae, R.; Henary, M. M.; Patel, R.; Lai, B.; Vogt, S.;
Fahrni, C. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11179. (b)
Verma, M.; Chaudry, A. F.; Morgan, M. T.; Fahrni, C. J. Org. Biomol.
Chem. 2010, 8, 363.
(1) Demchenko, A. P. Introduction to Fluorescence Sensing; Springer:
New York, 2008.
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A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV.
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10.1021/ja908326z  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 737–747 737

A R T I C L E S
Chaudhry et al.
for a given donor potential E(D+/D) as expressed by the
Rehm-Weller equation, eq 1.14
Scheme 1
∆Get ) E(D+/D) - E(A/A-) - ∆E00 + wp
(1)
The term wp refers to the Coulombic stabilization energy of
the radical ion pair formed upon PET. In polar solvents, this
value is typically small due to dielectric screening.
The fluorophore π-system in 1,3,5-triarylpyrazolines is com-
posed of the central pyrazoline core and the two aryl rings in
the 1- and 3-positions, which are coupled through the sp2-
hybridized nitrogen and carbon atoms of the heterocycle.
Electron withdrawing substituents R2 on the aryl ring in the
1-position of the pyrazoline system yield a large increase of
∆E00 but only a small change of E(A/A-). Conversely, substit-
uents R1 on the 3-aryl ring influence E(A/A-) more strongly
than ∆E00. This property can be exploited to gradually adjust
the PET driving force for any given receptor unit and thus to
optimize fluorescence enhancement upon analyte binding.12 The
aryl ring in the 5-position is electronically decoupled from the
fluorophore π-system, thus offering an opportunity to attach a
metal-receptor R3 for PET switching.7 To bind Cu(I) with high
affinity and sufficient redox stability,15 we chose a 16-membered
trithiazacyclohexadecane ([16]aneNS3) macrocycle as the recep-
tor moiety (Chart 1b). Furthermore, previous studies showed
that the donor potential of N,N-dialkylanilines is well matched
with the acceptor potential of pyrazolines carrying a p-cyano
substituent on the 3-aryl ring.12 On the basis of these consid-
erations, we synthesized a series of pyrazoline derivatives 1a-e
with varying PET driving forces and evaluated their photo-
physical properties. To gauge the degree of fluorescence
recovery upon saturation with Cu(I), we also characterized each
derivative in acidic solution. Because the protonated anilinium
cation is electrochemically inactive, acidic conditions should
lead to a full recovery of the fluorescence emission.12 Finally,
we performed detailed time-resolved fluorescence and transient
absorption experiments to elucidate the role of the proposed
excited-state pathways.
probes have consistently shown incomplete recoveries of the
fluorescence quantum yields compared to the corresponding
isolated fluorophores,7 thus raising the question of whether Cu(I)
might be directly involved in adverse quenching pathways. Here
we report on detailed photophysical studies that were aimed at
clarifying the role of Cu(I) in mediating fluorescence quenching
in PET probes.
The molecular architecture of PET probes is based on a
donor-spacer-acceptor (D-A) framework that combines a
cation receptor with a suitable fluorophore. In the absence of
the analyte, the fluorescence emission is strongly quenched due
to a fast intramolecular photoinduced electron transfer reaction
from the electron-rich cation receptor (D) to the excited
fluorophore (A*) acting as the electron acceptor (Scheme 1, left).
The formed radical ion pair (D+-A-) undergoes nonradiative
charge recombination back to the initial ground state. Binding
of the metal cation to the receptor moiety results in a less
favorable donor potential, which slows down the kinetics for
PET quenching and thus increases the emission quantum yield.
Because redox-active cations such as Cu(I) may also act as
electron donors, two additional nonradiative deactivation path-
ways A and B must be considered (Scheme 1, right). In pathway
A, an analogous radical ion pair (D+-A-) is formed first, albeit
with a slower rate due to an increase in donor potential in the
presence of Cu(I), followed by an electron transfer from Cu(I)
to the radical cation (D•+). Back-electron transfer to the
intermediately formed Cu(II) species then regenerates the initial
ground state. Because electron donation from Cu(I) is preceded
by formation of the radical ion pair, only the rate constant ket
of the first ET reaction will affect the emission response. Hence,
Cu(I) would appear to act as a Lewis acid, and in principle, it
should be possible to fully recover the fluoresence emission of
the unquenched fluorophore. In contrast, pathway B directly
competes with radiative deactivation to form the same Cu(II)-
intermediate (Scheme 1, right). Depending on the rate constant
k′′et, this pathway might severely compromise the fluorescence
recovery, even if ket is much slower than kr in the presence of
Cu(I).
Experimental Section
Synthesis. Procedures for the synthesis of derivatives 1a-e and
all intermediates are provided in the Supporting Information.
Steady-State Absorption and Fluorescence Spectroscopy. All
sample solutions were filtered through 0.2 µm Teflon membrane
filters to remove interfering dust particles or fibers. UV-vis
absorption spectra were recorded at 25 °C using a Varian Cary
Bio50 UV-vis spectrometer with constant-temperature accessory.
Steady-state emission and excitation spectra were recorded with a
PTI fluorimeter. For all measurements the path length was 1 cm
with a cell volume of 3.0 mL. The 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 a calibrated
photodiode). Fluorescence titrations with Cu(I) were carried out at
a fluorophore concentration of 2.3 µM in MeOH by adding 0.1
molar equiv from a 1 mM stock solution of [Cu(I)(CH3CN)4]PF6
in MeOH containing 10% CH3CN (v/v). Because the pKa of trifluo-
roacetic acid (TFA) is approximately 5 pKa units higher in methanol
compared to aqueous solution,16 a large excess of TFA (180 mM)
was required to ensure quantitative protonation of the aniline
nitrogen as determined by saturation experiments. Quantum yields
While any Cu(I)-responsive PET probe with D-A architec-
ture would be suitable to explore the importance of these
pathways, we decided to take advantage of the synthetic
accessibility and photophysical tunability of 1,3,5-triarylpyra-
zoline-based probes (Chart 1a).11-13 In these fluorophores, two
of the key PET parameters, the excited-state equilibrium energy
∆E00 and the acceptor potential E(A/A-), can be rationally tuned
to adjust the change in free energy of the PET reaction (∆Get)
(11) Verma, M.; Chaudhry, A. F.; Fahrni, C. J. Org. Biomol. Chem. 2009,
7, 1536.
(12) Cody, J.; Mandal, S.; Yang, L.; Fahrni, C. J. J. Am. Chem. Soc. 2008,
130, 13023.
(14) Rehm, D.; Weller, A. Isr. J. Chem. 1970, 8, 259.
(13) Fahrni, C. J.; Yang, L. C.; VanDerveer, D. G. J. Am. Chem. Soc. 2003,
125, 3799.
(15) Addison, A. W. Inorg. Chim. Acta 1989, 162, 217.
(16) Rived, F.; Rose´s, M.; Bosch, E. Anal. Chim. Acta 1998, 374, 309.
9
738 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010

Cu(I)-Responsive Fluorescent Probes
A R T I C L E S
Chart 1
were determined using quinine sulfate dihydrate in 1.0 N H2SO4
as the fluorescence standard (Φf ) 0.54 ( 0.05).17
recommended by IUPAC.19 Measurements were typically per-
formed with a scan rate of 100 mV s-1 or as indicated in the text.
Computational Methods. All calculations were carried out with
the Q-Chem electronic structure calculation suite of programs.20
Ground-state geometries were energy minimized by DFT with the
B3LYP hybrid functional21 and Pople’s 6-31G(d) split valence basis
set with added polarization functions. The starting geometry for
the Cu(I) complex of ligand 3 (N-phenyl-1,5,9-trithia-13-azacyclo-
hexadecane) was constructed from the crystal structure coordinates
of [1,5-dithia-9,13-diazacyclohexadecane-copper(I)]tetrafluoroborate
(Cambridge crystallographic database, code VEMWAL).22 To
ensure a stationary point on the ground-state potential surface, all
geometry optimized structures were verified by a vibrational
frequency analysis. For the calculation of gas phase reaction
enthalpies, single point energies were calculated at the B3LYP/6-
31+G(d,p) level of theory and corrected with the respective scaled
harmonic zero-point vibrational energies (ZPVE) obtained from the
frequency analysis at the B3LYP/6-31G(d) level (scaling factor
0.9806).23 Molecular orbitals were visualized with the software
VMD24 using the Q-Chem plot output data. Details of the
computational results including the coordinates of the geometry-
optimized structures are provided in the Supporting Information.
Picosecond Fluorescence Spectroscopy. Time-resolved fluo-
rescence decay data were acquired at an emission wavelength of
480 nm with a sample concentration of 0.8 µM using a single
photon counting spectrometer (Edinburgh Instruments, LifeSpec
Series) equipped with a pulsed laser diode as the excitation source
(372 nm, fwhm ) 80 ps, 10 MHz repetition rate, 1024 channel
resolution). Sample solutions of the fluorophore saturated with
excess Cu(I) were prepared on the basis of steady-state fluorescence
titrations as described above. For measurements under acidic
conditions, trifluoroacetic acid was directly added to the sample
solution in MeOH (final concentration 180 mM). The time decay
data were analyzed by nonlinear least-squares fitting with decon-
volution of the instrumental response function using the FluoFit
software package.18
Femtosecond Transient Absorption Spectroscopy. Spectra
were acquired using a commercial transient absorption spectroscopy
system (Newport, Helios). This system accepts two input laser
beams, one of variable wavelength used as the pump beam and
one of a fixed wavelength (800 nm) used to generate the probe
beam in a proprietary nonlinear optical crystal. For the experimental
pump beam, the light source was an ultrafast optical parametric
amplifier (Newport, TOPAS) running at a repetition rate of 1 kHz,
pumped by a Ti:Sapphire regenerative amplifier (Newport, Spitfire).
All experiments were performed using the TOPAS output at 350
nm with a pulse width of approximately 120 fs. Approximately
5% of the Spitfire fundamental at 800 nm was used for white light
generation (420-800 nm) in the Helios crystal to provide the
experimental probe beam. At these specifications, the instrument
response function is approximately 200 fs. The time window that
can be studied is 3200 ps; the transient spectra were acquired
starting at a delay of -1 ps and continued to a delay of 3000 ps.
At each time point, data were averaged for 1.5 s. The Helios pump
beam was chopped at 500 Hz to obtain pumped (signal) and
nonpumped (reference) absorption spectra of the sample. A
correction factor for chirp in the probe beam was generated using
the ultrafast response of CS2, and was applied to all data sets. The
data were stored as 3-D wavelength-time-absorbance matrices
that were exported for use with the fitting software. The solutions
studied had an OD350 in 2 mm path-length cuvettes of 0.07-0.45,
and were stirred continually throughout the data acquisition.
Cyclic Voltammetry. The cyclic voltammograms were acquired
in methanol or acetonitrile containing 0.1 M Bu4NPF6 as the
electrolyte using a CH-Instruments potentiostat (model 600A). The
samples were measured under inert gas at concentrations ranging
from 50 µM to 3 mM in a single compartment cell with a glassy
carbon working electrode and a Pt counter electrode. An aqueous
Ag/AgCl reference electrode (3 M KCl) was used in methanol, and
a nonaqueous Ag/AgNO3 (10 mM in 0.1 M Bu4NPF6/CH3CN)
reference electrode in acetonitrile. The half-wave potentials were
referenced to ferrocene as the internal or external standard as
Results and Discussion
Contrast Optimization. Following the previously delineated
strategy,11,12 we optimized the contrast ratio upon Cu(I)-binding
by progressively increasing the electron withdrawing ability of
the 1-aryl ring through fluoro-substitution. All pertinent pho-
tophysical data including estimations of the PET driving force
for each derivative are provided in Table 1. With increasing
number of fluoro substituents, the absorption and emission
spectra shift to higher energies while maintaining a uniform
band shape across all derivatives (Figure 1). The resulting
increase of the corresponding zero-zero transition energies
∆E00, estimated as the average between the absorption and
emission maxima, directly parallels the increase in the PET
driving force (-∆G00), which in turn is responsible for the
progressively decreasing quantum yields in neutral MeOH
(Table 1). For derivative 1e the emission intensities and the
associated signal/noise ratios were too low for accurate
quantum yield determinations. Upon addition of excess
[Cu(I)(CH3CN)4]PF6, all derivatives exhibited substantial fluo-
rescence emission increases. Following the trend of the previous
model study,12 the emission enhancement factors fe, defined as
(19) Gritzner, G.; Kuta, J. Pure Appl. Chem. 1982, 54, 1527.
(20) Shao, Y. E. A.; et al. Phys. Chem. Chem. Phys. 2006, 8, 3172.
(21) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (a) Lee, C. T.; Yang,
W. T.; Parr, R. G. Phys. ReV. B 1988, 37, 785.
(22) Balakrishnan, K. P.; Riesen, A.; Zuberbu¨hler, A. D.; Kaden, T. A.
Acta Crystallogr., Sect. C: Cryst. Soc. Commun. 1990, 46, 1236.
(23) Scott, A. P.; Radom, L. J. Phys. Chem. 1996, 100, 16502.
(24) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14,
33.
(17) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991.
(18) Enderlein, J.; Erdmann, R. Opt. Commun. 1997, 134, 371.
9
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A R T I C L E S
Chaudhry et al.
Table 1. Photophysical Data of Pyrazoline Derivatives 1a-e in Methanol at 298 K
c
ΦF
fed
compd
abs λmax (nm)
em λmax (nm)
Stokes shift (cm-1
)
∆E00 (eV)a
∆Get (eV)b
neutrale
acidic f
Cu(I)g
acidic f
Cu(I)g
1a
1b
1c
1d
1e
394
381
373
356
350
487
480
464
445
448
4850
5410
5260
5620
6250
2.85
2.92
3.00
3.13
3.15
-0.22
-0.31
-0.39
-0.47
-0.54
0.0072
0.0033
0.0024
0.0010
n.d.h
0.53
0.54
0.60
0.55
0.46
0.15
74
21
29
20
0.095
0.048
0.020
0.020
164
250
550
n.d.h
20
n.d.h
a Zero-zero transition energy, estimated on the basis of ∆E00 ) (Eabs(max) + Eem(max))/2. b PET free energy change calculated according to eq 1
with wp ) -0.045 eV;12 the experimental donor and acceptor potentials are provided in the Supporting Information. c Fluorescence quantum yield,
quinine sulfate as reference. d Fluorescence enhancement factor fe ) ΦF/Φneutral e Probe in methanol alone. f 180 mM TFA in methanol. g 10 µM
.
[Cu(I)(CH3CN)4]PF6 in methanol (0.1% acetonitrile). h Signal/noise ratio insufficient for accurate determination.
Figure 1. Normalized absorption and emission spectra of pyrazoline
Figure 2. Fluorescence titration of pyrazoline 1a (2.3 µM) with
[Cu(I)(CH3CN)4]PF6 in MeOH (298 K, excitation at 345 nm). Inset shows
a molar-ratio plot for the fluorescence intensity change at 490 nm.
derivatives 1a-e in methanol (298 K). Absorption traces are depicted with
dotted lines (left) and emission spectra with solid lines (right). The shaded
area indicates the tunable range of the excited-state energy ∆E00 within
each compound series.
tion species that might compromise the quantum yield and lower
the fluorescence enhancement factors compared to the acidic
solution.
the ratio of the quantum yields in the presence and absence of
Cu(I), first increased before decreasing with increasing PET
driving force, reaching a maximum of 29-fold enhancement for
the difluoro-substituted derivative 1b. In contrast, protonation
of the aniline nitrogen under acidic conditions resulted in
substantially greater enhancement factors across all derivatives
(Table 1), raising the question of whether Cu(I) might be
involved in mediating fluorescence quenching. To address this
issue, we systematically investigated both the solution chemistry
as well as the excited-state dynamics of this system.
Copper Redox Chemistry. To study the electrochemical
properties of the Cu(II/I) couple coordinated to the thiazacrown
receptor without interference from the pyrazoline fluorophore,
we utilized the truncated ligand 3 as a model compound. The
formal potential of the Cu(II/I) couple coordinated to the ligand
can be readily measured by cyclic voltammetry of the Cu(II)
complex.25 To determine the amount of Cu(II) needed to saturate
ligand 3, we first performed a molar-ratio titration with Cu(OTf)2
in methanol. Only at a large excess of Cu(II) were we able to
discern changes in the UV-vis absorption spectrum, indicating
a very low affinity of 3 toward Cu(II). In the presence of 10
mM Cu(OTf)2, we observed formation of a new band with a
maximum at 478 nm (Figure S3a, Supporting Information).
Unlike the broad shape expected for a charge-transfer band, the
absorption trace showed distinct vibronic structure that re-
sembled the spectrum of the N,N,N′,N′-tetramethylbenzidine
radical cation,26 apparently indicating oxidative dimerization of
the ligand. Treatment of the isolated dimer (6, see Supporting
Information) with Cu(OTf)2 yielded the same spectral signature
at 478 nm, thus independently confirming the formation of the
dimer radical cation (Figure S3b, Supporting Information). In
contrast, no changes in the UV-vis spectrum were observed
for compound 1a under the same conditions (data not shown),
indicating that the pyrazoline substituent in the para position
Solution Equilibrium with [Cu(I)(CH3CN)4]PF6. The presence
of multiple coordination equilibria could complicate the fluo-
rescence response of the probes upon addition of Cu(I). Of
particular concern was the possibility of Cu(I)-mediated forma-
tion of dimers or higher oligomers, which might undergo self-
quenching and thus lower the quantum yield and contrast ratio
compared with the acidic solution. To investigate the ligand-Cu(I)
binding stoichiometry in methanol, we first performed a molar-
ratio titration with fluorophore 1a and [Cu(I)(CH3CN)]4PF6. As
illustrated in Figure 2, the fluorescence emission of 1a increased
linearly and was saturated at 1 molar equiv. Excess Cu(I) did
not lead to further changes of the emission intensity. In agree-
1
ment with this observation, a H NMR titration of the isolated
ligand N-phenyl-1,5,9-trithia-13-azacyclohexadecane (3) with
[Cu(I)(CH3CN)4]PF6 also indicated saturation at 1 molar equiv
of Cu(I) (Figure S1, Supporting Information). At substoichio-
metric concentrations of Cu(I), only single resonances for each
set of equivalent protons were observed, indicating rapid metal
exchange kinetics exceeding the fast exchange limit on the NMR
time scale. The solution chemistry studies thus confirmed the
presence of a defined 1:1 ligand-Cu(I) equilibrium and provided
no evidence for formation of aggregates or oligomeric coordina-
(25) Kakos, S. H.; Dressel, L. T.; Bushendorf, J. D.; Kotarba, C. P.;
Wijetunge, P.; Kulatilleke, C. P.; McGillivary, M. P.; Chaka, G.; Heeg,
M. J.; Ochrymowycz, L. A.; Rorabacher, D. B. Inorg. Chem. 2006,
45, 923.
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Amsterdam, 1988.
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Cu(I)-Responsive Fluorescent Probes
A R T I C L E S
Repeated HPLC purification of 1a resulted in a decrease of the
long-lifetime component, indicating the presence of a difficult
to remove impurity (<0.1%). Despite the small fractional
contribution of the long-lifetime component, the triexponential
fit yielded greatly improved residuals compared to a biexpo-
nential decay model (ꢀ2 ) 2.56).
Upon addition of trifluoroacetic acid (TFA), monoexponential
decay kinetics with a lifetime of 3.76 ns were observed. For
comparison, we also measured the decay kinetics of compound
2, which contains the same pyrazoline fluorophore but lacks
the thiazacrown moiety. In neutral methanol, 2 displayed
monoexponential decay kinetics with a lifetime of 3.76 ns.
Consistent with effective inhibition of the PET quenching
process, under acidic conditions compound 1a yielded indis-
tinguishable decay kinetics compared with 2 in neutral methanol.
Saturation of 1a with Cu(I) in methanol resulted in surpris-
ingly complex decay kinetics. Nonlinear least-squares fitting
revealed three distinct decay components with similar fractional
amplitudes but distinctly different lifetimes of 84 ps, 0.77 ns,
and 3.23 ns (Table 2), suggesting the presence of three distinct
emissive species emitting with different decay kinetics. It is
noteworthy that the long-lifetime component decays with
kinetics comparable to the acidic conditions. The species
responsible for this component must therefore invoke similar
reduction in the PET quenching kinetics compared to the
protonated form. In agreement with weak Cu(II)-binding,
addition of 10 µM Cu(II)(OTf)2 to a solution of 1a in methanol
had no effect on the fluorescence decay kinetics.
Steady-State Fluorescence Emission Profiles. To investigate
whether divergent nonequilibrating excited-state pathways might
be responsible for the complex decay kinetics, we acquired the
fluorescence emission spectrum of 1a saturated with Cu(I) and
also under acidic conditions over a broad excitation energy range
of 22 000-33 000 cm-1 (Figure 4). Contour plots of the
emission spectra revealed symmetrical profiles with a single
emission maximum centered at 487 nm (20 530 cm-1) for both
conditions. Given the nearly indistinguishable 2D profiles of
the protonated versus Cu(I)-saturated probe, we can conclude
that the nature of the emissive state is very similar for both
conditions. Excited-state processes involving multiple non-
equilibrated deactivation pathways can therefore be ruled out
in explaining the observed triexponential decay kinetics in the
presence of Cu(I).
Transient Absorption Spectroscopy. While picosecond fluo-
rescence decay measurements can provide insights into the
excited-state dynamics, the method is not suitable to identify
the transient reaction products. To gain further information
regarding the excited-state intermediates formed in the presence
of Cu(I) compared to neutral or acidic conditions, we performed
a series of ultrafast pump-probe experiments with derivative
1a. Of particular interest was the detection of the charge
separated radical ion pair formed through the PET reaction.
Transient absorption spectra were recorded at different delay
times after photoexcitation at 350 nm with a femtosecond laser
pulse (120 fs fwhm). To visualize the evolution of the difference
spectra over the entire time course, we constructed a set of color-
coded contour plots (Figure 5). Nonlinear least-squares fitting
of the time-dependent absorption traces over the entire spectral
range yielded deconvoluted spectra for each transient species
(Figure 6). All pertinent numerical data including fitting results
are compiled in Table 3.
Figure 3. Time-dependent fluorescence decay profile of 1a in methanol,
and in the presence of 10 µM [Cu(I)(CH3CN)4]PF6, or 180 mM trifluoro-
acetic acid (TFA). A 1 µM solution of 1a was excited at 372 nm (80 ps
fwhm), and the emission signal was detected by single photon counting at
480 nm. Nonlinear least-squares fitted profiles are shown as solid lines (see
Table 2 for fitting data; IRF ) instrument response function).
of the aniline ligand inhibits dimerization. While Cu(II)-
mediated oxidative coupling of N,N-dialkylanilines has been
reported in the literature,27,28 the reaction required acetonitrile,
a solvent that greatly favors the Cu(I) state.29 A formal potential
of 0.40 V versus SHE has been reported for the Cu(II/I) couple
solvated in methanol.29 To compare this value with the donor
potential of ligand 3 measured against Fc+/0 as the reference,
the following conversion can be applied. According to the
manufacturer, the potential of the aqueous Ag/AgCl (3 M KCl)
reference electrode used in our measurements is 0.21 V versus
SHE. Given the half-wave potential of 0.38 V of the Fc+/0 couple
under the experimental conditions, we calculated a corrected
potential of -0.19 V versus Fc+/0 for the solvated Cu(II/I) couple
in methanol. While there remains some uncertainty in the above
conversion due to the exclusion of junction potentials, the
solvated Cu(II/I) couple is clearly a much weaker oxidant by
approximately 0.58 V compared to the radical cation of ligand
3 (E(D+/D) ) 0.39 vs Fc+/0). Because formation of the latter
species is a prerequisite for the observed oxidative coupling
reaction, we can conclude that the Cu(II) complex of 3 must
act as an oxidant. This in turn implies that the reduction potential
of this complex is similar or greater than the estimated donor
potential of 0.39 V (vs Fc+/0) of the free ligand 3. Concluding
from the electrochemical properties of the Cu(II/I) couple
coordinated to the thiazacrown receptor, a competing PET
quenching pathway mediated by Cu(I) is thermodynamically
feasible.
Fluorescence Decay Kinetics. To gain information about the
excited-state dynamics and possibly the role of Cu(I) in
fluorescence deactivation, we performed time-resolved fluores-
cence decay measurements with derivative 1a as a representative
compound. The compound was excited at 372 nm with a pulsed
laser (80 ps fwhm), and the emission decay signal was collected
by single photon counting at 480 nm. As illustrated in Figure
3, in neutral methanol the fluorescence emission decayed rapidly
involving complex kinetics. The data fit best with a triexpo-
nential decay involving two short components with 34 and 232
ps lifetimes, and a component with 3.53 ns lifetime (Table 2).
(27) Kirchgessner, M.; Sreenath, K.; Gopidas, K. R. J. Org. Chem. 2006,
71, 9849.
(28) Sumalekshmy, S.; Gopidas, K. R. Chem. Phys. Lett. 2005, 413, 294.
(29) Milazzo, G.; Caroli, S. Tables of Standard Electrode Potentials; Wiley:
New York, 1978.
In methanol alone, two distinct regions at 480 and 630 nm
(dashed circles) appeared in the difference spectra after a delay
9
J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010 741

A R T I C L E S
Chaudhry et al.
Figure 4. Two-dimensional fluorescence excitation-emission contour plots for 1a in methanol containing 10 µM [Cu(I)(CH3CN)4]PF6 (a) or 180 mM
trifluoroacetic acid (b).
Figure 5. Transient absorption spectra of pyrazoline 1a in (a) methanol, (b) 180 mM TFA in MeOH, and (c) 10 µM [Cu(I)(CH3CN)4]PF6 in MeOH. Top
row shows color-coded contour plots of the transient difference absorption traces on a logarithmic time scale. Each plot is represented on the absorption
scale shown to the right. Bottom row shows transient absorption progression at 480 nm.
Table 2. Time-Resolved Fluorescence Decay Data for Compounds 1a and 2 in Methanol at 298 K
a
d
e
cmpd
conditions
τi (ns)
aib
fic
ꢀ2
k0 (s-1
)
1a
MeOH
0.0341 ( 0.0001
0.232 ( 0.011
3.53 ( 0.05
0.967
0.032
0.001
1.000
0.347
0.359
0.294
1.000
0.740
0.167
0.093
1.000
0.023
0.221
0.756
1.000
0.851
2.9 × 1010
4.3 × 109
2.8 × 108
2.7 × 108
1.2 × 1010
1.3 × 109
3.1 × 108
2.7 × 108
1a
1a
TFA (180 mM) in MeOH
[Cu(I)(CH3CN)4]PF6 (10 µM) in MeOH
3.76 ( 0.01
1.145
0.970
0.084 ( 0.03
0.774 ( 0.007
3.23 ( 0.04
2
MeOH
3.76 ( 0.01
1.380
b
a Fluorescence lifetime. Fractional amplitudes ai ) Ri/∑ni)1Ri with ∑ni)1Ri ) 1 for the multiexponential decay function I(t) ) ∑in)1Ri exp (-t/τi).
c Fractional intensities fi ) Riτi/∑in)1Riτi. d Goodness of fit parameter. e Excited-state deactivation rate k0 ) 1/τi.
cation,28,30 the 480 nm band can be assigned to the thiazacrown
time of approximately 30 ps. The corresponding changes were
substituted 5-aryl radical cation formed during the course of
less pronounced in the presence of Cu(I) and entirely absent
under acidic conditions. Given the absorption maximum of 470
nm of the structurally related N,N-dimethylaniline radical
(30) Maroz, A.; Hermann, R.; Naumov, S.; Brede, O. J. Phys. Chem. A
2005, 109, 4690.
9
742 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010

Cu(I)-Responsive Fluorescent Probes
A R T I C L E S
formation of qualitatively similar species (Figure 6a,b). Vibra-
1
tional cooling from FC to LE again occurred with a fast time
constant of 1.1 ps. Interestingly, 1LE did not immediately decay
to the ground state S0, but was rapidly converted to two
additional intermediate species with qualitatively similar dif-
ference absorption spectra (Figure 6b). Using a simplified model
composed of a single intermediate species, an average time
constant of 90 ps was obtained; however, the fit significantly
improved with the expanded model to give two closely spaced
time constants of 71 and 110 ps. The latter agreed well with
the slightly delayed fluorescence emission already observed in
the picosecond lifetime measurements. Given the similarity
between the deconvoluted difference spectra, the two intermedi-
ate species must have a closely related electronic structure. To
explore the potential role of TFA in the delayed fluorescence
emission, we also conducted pump-probe experiments in
neutral methanol with pyrazoline 2, which lacks the thiazacrown
receptor. The deconvoluted transient absorption traces agreed
well qualitatively with the deconvoluted spectra of 1a under
acidic conditions (Figure 6c); however, the data better matched
the simpler four species model lacking the second intermediate.
While the pump-probe experiments indicate that strong acidic
conditions impose some effect on the excited-state dynamics,
there is no substantive evidence for the formation of a
structurally different intermediate. Earlier reports on the excited-
state dynamics of pyrazolines concluded that the emissive state
is not identical with the initial photoexcited singlet state.31 Gas
phase quantum chemical calculations on a broad range of
pyrazoline fluorophores uniformly identified a strongly allowed
lowest excited state S1 with π-π* character,13 making the
involvement of higher lying excited states along with slow
internal conversion processes quite unlikely. Consistent with
the broad unstructured emission of 1a reminiscent of a polarized
charge-transfer state 1CT, vertical excitation into S1 is presum-
ably followed by substantial structural reorganization combined
with solvent relaxation, both of which may contribute to the
delayed radiative deactivation.32 The polarized nature of the
emissive states is also in agreement with previous solvatochro-
mic studies employing the Lippert-Mataga formalism, which
revealed a substantially increased excited-state dipole moment
compared to the ground state with ∆µge ranging between 8 and
13 D for a broad range of substituted 1,3,5-triarylpyrazolines.13,33
The charge transfer state (1CT) decayed with a time constant
of 3.7 ns, a value that is consistent with the fluorescence lifetime
measurements.
Figure 6. Deconvoluted difference absorption spectra for the transient
excited-state species of 1a in (a) methanol and (b) methanol containing
180 mM TFA. (c) Difference absorption spectra for the transient excited-
state species of 2 lacking the PET donor. Legend: FC ) Franck-Condon
state (dashed red), 1LE ) vibrationally cooled, local excited state (solid
yellow), 1ET ) nonemissive singlet electron transfer state (solid green),
1CT ) emissive singlet charge transfer state (blue).
the PET process. The transient absorption progression at 480
nm highlights the differences in evolution of the radical cation
in neutral methanol compared to the other two conditions (Figure
5, bottom). The difference spectra fit well with a model
composed of a series of four distinct species linked by three
consecutive first-order rate constants:
k1
k2
k3
FC 98 1LE 98 1ET
98 GS
(2)
At first glance, transient difference spectra in the presence
of Cu(I) revealed a similar picture compared to that in methanol
alone; however, the bands at 480 and 630 nm characteristic of
the 1ET state appeared with substantially reduced intensity
(Figure 5c). As illustrated with the transient absorption trace at
The initially formed Franck-Condon (FC) state decayed with
a time constant of 0.6 ps to a local excited state (1LE), which
was converted to the electron-transfer state (1ET) with a time
constant of 24 ps. The characteristic 480 and 630 nm bands of
1
1
the ET state rapidly decayed with a 77 ps time constant back
480 nm, the ET state seems to evolve with similarly rapid
to the initial ground state (GS).
Consistent with efficient inhibition of PET, the transient
absorption traces of 1a in acidic methanol did not exhibit bands
at 480 and 630 nm characteristic of the ET state (Figure 5b).
The absorption traces were fit again with a model that included
five species and four consecutive first-order rate constants:
kinetics compared to the probe in methanol alone, an observation
that contradicts the strong fluorescence enhancement upon Cu(I)
coordination. Given the triexponential decay kinetics of the
fluorescence emission, it was not surprising that the transient
absorption data did not fit to a model solely based on consecutive
first-order reactions. To account for the multiexponential decay,
1
k1
k2
k3
k4
FC 98 1LE 98 1CT* 98 1CT
98 GS
(3)
(31) Blair, J. T.; Sahyun, M. R. V.; Sharma, D. K. J. Photochem. Photobiol.,
A 1994, 77, 133.
(32) Yang, G.; Wu, H. J. Photochem. Photobiol., A 1992, 66, 69.
(33) Gu¨sten, H.; Heinrich, G.; Fru¨hbeis, H. Ber. Bunsen Ges. Phys. Chem.
1977, 81, 810. Yan, Z. L.; Wu, S. K. J. Lumin. 1993, 54, 303. Rurack,
K.; Bricks, J. L.; Schulz, B.; Maus, M.; Reck, G.; Resch-Genger, U.
J. Phys. Chem. A 2000, 104, 6171.
The deconvoluted difference spectra of the first two inter-
mediates strongly resemble the spectra of the Franck-Condon
and local excited states under neutral conditions, which indicates
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J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010 743

A R T I C L E S
Chaudhry et al.
Table 3. Pump-Probe Transient Absorption Data for Compounds 1a and 2 in Methanol (298 K)
cmpd
conditions
k1 (τ1)
k2 (τ2)
k3 (τ3)
k4 (τ4)
1a
1a
1a
MeOH
TFA (180 mM) in MeOH
[Cu(I)(CH3CN)4]PF6 (10 µM) in MeOH
1.6 × 1012 (0.6 ps)
9.1 × 1011 (1.1 ps)
1.5 × 1012 (0.7 ps)
4.2 × 1010 (24 ps)
1.4 × 1010 (71 ps)
1.2 × 1010 (83 ps)
1.3 × 109 (0.77 ns)
9.4 × 109 (0.11 ns)
1.5 × 1011 (6.6 ps)
1.3 × 1010 (77 ps)
9.4 × 109 (0.11 ns)
1.9 × 1010 (53 ps)
2.7 × 108 (3.7 ns)
3.1 × 108 (3.2 ns)
2
MeOH
1.3 × 1012 (0.8 ps)
1.3 × 1010 (77 ps)
2.6 × 108 (3.8 ns)
we assumed a model that involves three coordination species,
A, B, and C, with distinct PET dynamics:
sistent with the steady-state fluorescence emission profiles, the
nature of all involved transient species remains qualitatively
unchanged; only the kinetics of their interconversion differs as
a function of Cu(I) or proton concentration.
k1
k2a
k3a
98 GSA
FCA 98 1LEA 98 1ETA
Variable Temperature 1H NMR Spectra. Although the
fluorescence molar-ratio titrations firmly established a 1:1
stoichiometry for the interaction of Cu(I) with 1a or ligand 3,
the fluorescence lifetime and pump-probe data unequivocally
support the presence of three distinct species. Furthermore, given
the uniform nature of the observed transient reaction products,
we concluded that these three species must somehow differ with
regard to their coordination mode only, for example through
binding of a counterion or solvent molecule in the first
coordination sphere. To find supporting evidence for this
hypothesis, we conducted variable temperature 1H NMR experi-
ments. The initial molar-ratio titration of 3 with Cu(I) at room
temperature indicated a fast exchange equilibrium with regard
to the NMR time scale. If there are multiple equilibria present
involving several distinct coordination species, changes in
temperature should give rise to nonuniform chemical shift
k1
k2b
k3b
98 GSB
FCB 98 1LEB 98 1ETB
(4)
k1
k2c
k4
FCC 98 1LEC 98 1CTC
98 GSC
If equilibration of the three species is slow with regard to
the nanosecond time scale of the experiment, each deactivation
pathway can be modeled with an independent set of differential
equations (see Supporting Information). Species A and B
undergo PET quenching with rates k2a and k2b, corresponding
to the two fast decay components of the fluorescence emission.
Both species are assumed to form the vibrationally relaxed
1
excited state LE from the Franck-Condon state FC with the
same rate constant k1. Furthermore, the two charge recombina-
tion rate constants k3a and k3b are also assumed to be identical.
Species C corresponds to the slow component that involves
formation of the radiative charge transfer state 1CT. The
vibrational cooling rate constant k1 is again assumed to be
identical with the other two species. The rate constant k2c was
obtained from the delayed fluorescence emission rate, and k4
from the slow component of the fluorescence decay. Because
neither the thiazacrown nor its Cu(I) complex absorb within
the wavelength range of the acquired transients, we can assume
that the extinction coefficients for the corresponding intermedi-
ates for each species remain identical. Furthermore, the triex-
ponential decay profile implies that the three species equilibrate
substantially slower than the time scale of radiative deactivation.
Therefore, the concentration of the three species at time zero
corresponds to the fractional amplitudes obtained from the
fluorescence lifetime analysis (Table 2). Because the rate
constants k2a, k2b, k2c, and k4 are given by the corresponding
inverse lifetimes of the individual components, only k1, k3, and
the four molar extinction coefficients were used as variables
for nonlinear least-squares fitting. Despite the considerable
constraints imposed by the fixed rate constants, the model fit
very well with the data as illustrated with the single time trace
at 480 nm (Figure 5c). The inverse rate constant for conversion
of FC to LE was found to be 0.7 ps and compared well with
the data in neutral and acidic methanol. Similarly, the charge
recombination time constant of 56 ps agreed well with the
previous fits. The deconvoluted transient absorption traces at
480 nm for each of the three species are shown as dotted lines
in Figure 5c. In agreement with the fluorescence lifetime data,
the pump-probe experiments indicate the presence of three
species with distinct PET quenching kinetics. Most importantly,
in the presence of Cu(I) we found no evidence for the formation
of an additional transient reaction product that was not also
formed in methanol alone and under acidic conditions. Con-
1
changes. Figure 7 shows representative H NMR traces of the
aromatic and aliphatic region of ligand 3 in the presence of 1
molar equiv of [Cu(I)(CH3CN)4]PF6 in CD3OD at three tem-
peratures. Compared to the uncoordinated ligand, which is
shown at the bottom, all resonances are shifted downfield. When
the temperature is decreased from 20 to -20 °C, the aromatic
resonances of the Ha/Ha′, Hb/Hb′, and Hc protons gradually drift
back toward the respective positions of the free ligand (Figure
7a, left). While the chemical shift itself, even in the absence of
a dynamic equilibrium, is sensitive to temperature variations,
the observed changes are larger than expected. This is in fact
directly confirmed by the insignificant shift of the thioether
methylene resonances in the aliphatic region (Figure 7a, right).
A plot of the chemical shift changes as a function of temperature
best illustrates the dramatically different behavior of the two
sets of proton resonances (Figure 7b). While the binding affinity
of chelate complexes is expected to decrease with decreasing
temperature, we would anticipate a gradual shift of all reso-
nances upon decomplexation. The temperature insensitivity of
the chemical shift for the thioether methylene protons therefore
suggests that Cu(I) remains coordinated. At the same time,
weakening of the nitrogen-Cu(I) interaction at lower temper-
atures would be expected to enhance the π-donation ability of
the aniline nitrogen due to an increasingly flattened geometry
with enhanced sp2 character. As a consequence, the proton
resonances are expected to undergo an upfield shift, as was
indeed observed. Even at the lowest accessible temperature in
CD3OD, the equilibrium system did not approach the slow
exchange regime or coalescence point, which indicates a fast
dynamic exchange between these species. It is important to note
that the time scale of NMR is vastly different compared to
fluorescence spectroscopy; therefore, the fast exchange dynamics
does not contradict the triexponential decay profile observed in
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744 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010

Cu(I)-Responsive Fluorescent Probes
A R T I C L E S
[3-Cu(I)]+ + solv h [3-Cu(I)(solv)]+ ∆Haux
(5)
the lifetime measurements. The observed temperature depen-
dence of the chemical shift changes is consistent with an
equilibrium that involves at least two types of Cu(I) coordination
species. Besides the expected 4-coordinate complex with the
thiazamacrocycle, an additional species containing an auxiliary
ligand, either a solvent molecule or possibly the counterion,
must be present. As the temperature is lowered, the equilibrium
is shifted in favor of the latter species due to the decreasing
contribution of the entropy term (T∆S). This in turn implies
that the mixed coordination species is enthalpically more
favorable compared to the tetradentate macrocyclic complex.
Table 4 shows all pertinent data obtained from these
calculations. The lowest energy structure of complex [3-Cu(I)]+
assumes an all-trans t,t,t configuration,22,34 in which all of the
6-membered chelate rings adopt a chair conformation (Figure
8, left; see also Supporting Information for more detailed
structural data). Replacement of the aniline nitrogen donor with
methanol yielded the ternary complex t,t-[3-Cu(I)(MeOH)]+,
which revealed a hydrogen bonding interaction between the OH
group of methanol and the aniline nitrogen (Figure 8, middle;
Supporting Information). The gas phase enthalpy for formation
of t,t-[3-Cu(I)(MeOH)]+ as defined by the equilibrium in eq 5
is exothermic with ∆HMeOH ) -12 kcal/mol (Table 4), a result
that is consistent with the proposed coordination of methanol
at low temperature in the NMR study. If the configuration of
the complex is switched from trans,trans to trans,cis the aniline
nitrogen is pushed further away from the copper center and is
no longer accessible for H-bonding (Figure 8, right). The
geometry optimized structure of this complex c,t-[3-Cu(I)-
(MeOH)]+ was found to be slightly higher in energy than the
Quantum Chemical Calculations. Due to the rapid exchange
dynamics, the variable temperature NMR experiments prevented
us from identifying the individual components of the equilibrium
system. To gain further insights into the nature of possible
coordination species containing an auxiliary ligand, we per-
formed a series of quantum chemical calculations. To assess
the stability of the Cu(I) complex of ligand 3 coordinated to an
additional solvent ligand, we used density functional theory to
calculate the gas phase enthalpy ∆Haux of the following
equilibrium (eq 5):
1
Figure 7. Variable temperature H NMR experiment of ligand 3 in the presence of 1 molar equiv of [Cu(I)(CH3CN)4]PF6 in CD3OD.
Table 4. Computational Data for the Geometry Optimized Cu(I) Complexes of Ligand 3 and Methanol as an Auxiliary Liganda
c
species
Σb
∆Haux (kcal/mol)
HOMO energy (eV)
donor obital energyd (eV)
t,t,t-[3-Cu(I)]+
326.7
336.7
351.0
0
-8.239
-8.306
-7.824
-9.746
-9.001
-7.824
t,t-[3-Cu(I)(MeOH)]+
c,t-[3-Cu(I)(MeOH)]+
-12.4
-10.2
a B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level of theory. b Sum of bond angles at aniline nitrogen N1 (C7-N1-C8, C8-N1-C11, C7-N1-C11; the atom
numbering scheme is provided in the Supporting Information). c Association enthalpy as defined by eq 5. d homologous to aniline HOMO with B2 symmetry.
9
J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010 745

A R T I C L E S
Chaudhry et al.
Figure 8. Effect of solvent coordination on the orbital energy levels of Cu(I) complexes with ligand 3. The depicted orbital diagrams refer to the corresponding
energy levels marked in blue (see text for details).
H-bonded structure; however, the gas phase association enthalpy
remained exothermic with ∆HMeOH ) -10 kcal/mol.
with Cu(I) indeed fit best a monoexponential rate law with a
long lifetime of τ ) 4.03 ns (Figure S4d, Supporting Informa-
tion), thus confirming the absence of a Cu(I)-promoted quench-
ing channel.
Coordination of methanol to [3-Cu(I)]+ imposes significant
structural changes that directly affect the PET thermodynamics.
Judging from the sum of the bond angles at the aniline nitrogen
(Σ, Table 4), solvent coordination results in flattening of the
macrocycle geometry at this position. In the parent complex
[3-Cu(I)]+, the aniline nitrogen is sp3 hybridized and adopts a
near ideal trigonal pyramidal geometry with Σ ) 331°. While
in the H-bonded complex t,t-[3-Cu(I)(MeOH)]+ the aniline
nitrogen is still somewhat pyramidalized, the increased Σ of
337° implies some flattening and therefore increased p-character
of the nitrogen lone pair. In the cis,trans isomer c,t-[3-
Cu(I)(MeOH)]+, the aniline nitrogen is engaged in neither metal
coordination nor H-bonding, and assumes a near trigonal planar
geometry with Σ ) 351°. With the change from a trigonal
pyramidal to a trigonal planar geometry, the aniline nitrogen is
transformed from a strong σ-acceptor into a π-donor. Previous
quantum chemical calculations showed that the aniline molecular
orbital with B2 symmetry serves as the electron donor in the
PET process.13 As illustrated in Figure 8, the energy of this
orbital is strongly affected by the structural changes imposed
on the aniline nitrogen. As the aniline nitrogen approaches a
more flattened geometry, its π-donor strength increases, which
in turn results in an increased MO energy and therefore more
favorable electron transfer thermodynamics. At the same time,
the energy of the highest occupied metal-centered d-orbital
remains largely unchanged, a result that strongly contrasts the
widely different deactivation rates extracted from the fluores-
cence lifetime data of 1a in the presence of Cu(I) (Table 2).
The experimental data are therefore best reconciled with a model
that entails three distinct coordination species with varying
ability to allow PET from the aniline B2 MO as the electron
donor. To further support this hypothesis, we repeated the above
time-resolved fluorescence decay measurements in dichlo-
romethane, a noncoordinating solvent in which no solvent-
associated species should form. The decay profile of 1a saturated
In summary, Figure 9 illustrates the relevant excited-state
processes and the associated dynamics found for the various
solution conditions. In neutral methanol, the initially formed
Franck-Condon state (FC) undergoes vibrational cooling to a
1
local excited singlet state LE, which is rapidly converted to
the electron transfer state 1ET followed by charge recombination
back to the initial ground state GS (Figure 9, center). Under
strongly acidic conditions, the aniline nitrogen is protonated
which effectively inhibits the PET process (Figure 9, left). The
1
initially formed local excited state LE is now converted to a
more polarized (pyrazoline centered) charge-transfer state, which
undergoes radiative deactivation back to the ground state.
Finally, saturation of 1a with Cu(I) in neutral methanol leads
to the formation of three coordination species with distinct
deactivation pathways (Figure 9, right).
While according to the derived model all species form a
1
similar local excited state LE, the subsequent deactivation
pathways differ depending on the energy level of the ligand-
centered MO acting as the electron donor in PET. In the NS3-
coordinated, all-trans configured complex t,t,t-[1a-Cu(I)]+ (A),
the energetics for PET are unfavorable and the initially formed
1LE state decays through 1CT with fluorescence emission back
to the ground state. This complex is in equilibrium with two
additional solvent coordinated species, assigned as t,t-[1a-
Cu(I)(MeOH)]+ (B) and c,t-[1a-Cu(I)(MeOH)]+ (C), which
based on the gas phase quantum chemical calculations are
expected to have a more favorable PET driving force. Although
all three complexes are engaged in a dynamic exchange
equilibrium that exceeds the NMR time scale, the fluorescence
decay and pump-probe data can be modeled as a superimposi-
tion of the individual nonequilibrating decay pathways.
Contrary to the notion that Cu(I) might engage in reductive
electron transfer quenching, we found no evidence for formation
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746 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010

Cu(I)-Responsive Fluorescent Probes
A R T I C L E S
Figure 9. Jablonski diagram for the excited-state deactivation pathways of probe 1a in neutral methanol (center), under acidic conditions (left), and in the
presence of [Cu(I)(CH3CN)4]PF6 (right). The indicated lifetime data are based on the pump-probe experiments (GS ) ground state, FC ) Franck-Condon
1
1
1
excited state, LE ) local excited state, ET ) electron transfer state, CT ) emissive charge transfer excited state).
of a transient Cu(II) species upon photoexcitation of Cu(I)-
saturated 1a. Such a quenching pathway would require electron
transfer kinetics comparable to the observed excited-state
deactivation rate of k0 ) 2.6 × 108 s-1. While many efforts
have been made to generate Cu(II/I) complexes with large self-
successfully optimized the contrast ratio of a Cu(I)-responsive
fluorescent probe. While we achieved a strong 29-fold fluores-
cence enhancement upon saturation with Cu(I) in methanol, the
contrast ratio remained significantly lower compared to acidic
conditions. Detailed time-resolved spectroscopic studies revealed
that the contrast ratio for Cu(I)-binding is not compromised by
reductive electron transfer from Cu(I) but by the formation of
several ternary coordination species with varying PET driving
forces. Most importantly, the combined photophysical, electro-
chemical, and solution chemistry experiments suggest that
electron transfer from Cu(I) does not compete with radiative
deactivation of the excited fluorophore, and hence, that the
Cu(I)-induced fluorescence switching is kinetically controlled.
The results of this study highlight the challenges associated with
designing high-contrast ratio probes for Cu(I), which not only
require careful tuning of the PET thermodynamics but also
careful control of the Cu(I) solution chemistry. The conclusions
derived from this study are not limited to pyrazoline-based
probes but are generally applicable to the design of Cu(I)-
selective probes composed of a rigid, electronically decoupled
receptor unit attached to any other fluorophore platform.
exchange electron transfer rate constants, only a few systems
35
were described that exceeded 105 M-1 s-1
.
The structurally
related copper complex of 1,5,9,13-tetrathiacyclohexadecane
([16]aneS4), which has the same ring size as ligand 3, exhibited
a comparatively slow self-exchange rate with k11 ) 3.7 × 103
35,36
M-1 s-1
.
The reorganizational energy barriers for Cu(II/I)
systems are typically high due to large changes brought about
the inner-coordination sphere of copper, often involving bond
breakage or formation. Furthermore, the large distance between
the Cu(I) center and the fluorophore also renders PET from Cu(I)
less favorable. As a result, PET quenching is kinetically
controlled and determined by the energetics of the aniline donor
but not the Cu(I) center. Most importantly, the redox stability
of the Cu(II/I) couple is not an impediment for the design of
Cu(I)-selective turn-on sensors. If the solution equilibria were
devoid of ternary solvent adducts and included only the binary
complex t,t,t-[1a-Cu(I)]+ (A), the fluorescence enhancement of
1a would increase from 21- to 90-fold as judged by the ratio of
the excited-state deactivation rate constants of the free ligand
and the slowest component in the presence of Cu(I).
Acknowledgment. Financial support from the National Institutes
of Health (R01GM067169) is gratefully acknowledged. Computa-
tions were supported by the Center for Computational Molecular
Science and Technology at the Georgia Institute of Technology
and under National Science Foundation CRIF Award CHE-
0443564. We thank David Bostwick for mass spectral data.
Conclusions
Following the previously reported approach for tuning of the
PET thermodynamics in triarylpyrazoline-based probes,12 we
Supporting Information Available: Synthesis and spectro-
scopic characterization of derivatives 1-6 and additional
information as mentioned in the text. This material is available
free of charge via the Internet at http://pubs.acs.org.
(34) McCrindle, R.; Ferguson, G.; McAlees, A. J.; Parvez, M.; Ruhl, B. L.;
Stephenson, D. K.; Wieckowski, T. Dalton Trans. 1986, 2351.
(35) Rorabacher, D. B. Chem. ReV. 2004, 104, 651.
(36) Martin, M. J.; Endicott, J. F.; Ochrymowycz, L. A.; Rorabacher, D. B.
Inorg. Chem. 1987, 26, 3012.
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