1,3,5-Triaryl-2-pyrazoline Fluorophores
A R T I C L E S
slow evaporation of the solvent over a period of several days. The X-ray
data were collected on a Siemens SMART 1K CCD diffractometer with
graphite-monochromated Mo KR radiation (λ ) 0.71073 Å). The
programs SADABS (Sheldrick)30 and SAINT 6.22 (Bruker)31 were used
for absorption corrections. The structure was solved by direct methods
and refined by least-squares calculations with the SHELXTL 5.10
software package.31 The hydrogen atoms were added using ideal
geometries with a fixed C-H bond distance of 0.96 Å. A summary of
the crystallographic parameters and data is included with the Supporting
Information (Tables S1 and S3).
and correlated the theoretical data with the experimental results.
In addition, we have characterized the properties of carboxylic
acid derivative (2e) in aqueous solution and evaluated the
fluorophore in vivo as a potential intracellular pH probe. The
major impetus for this work was to gain detailed insights into
the photophysics of this compound class and, hence, to provide
a basis for the rational design of cation specific fluorescent
probes in a biological environment.
Experimental Section
Computational Methods. All calculations were carried out with
the Q-Chem electronic structure calculation suite of programs.32 The
geometries of ground-state structures were optimized by the density
functional method using Becke’s gradient corrected three-parameter
exchange functional33 with the correlation functional of Lee, Yang, and
Parr34 (B3LYP). The split-valence polarized 6-31G* (6-31G(d)) basis
set was used for all geometry optimizations. Single point energies were
computed at the B3LYP/6-31G* level using the optimized geometry
(B3LYP/6-31G*//B3LYP/6-31G*). To obtain estimates of the vertical
electronic excitation energies which include some account of electron
correlation, time-dependent density functional theory (TD-DFT)35
calculations with the B3LYP functional and 6-31G* basis set were
performed. The calculation of ground-state potential curves is based
on relaxed geometries with the corresponding constraints of dihedral
angles. Molecular orbitals were visualized with the software MOLE-
KEL36 using the Q-Chem plot output data. Details for all computations
including the coordinates of the geometry-optimized structures are
provided with the Supporting Information (Tables S4 and S5).
Cell Culture Experiments. HeLa cells were cultivated in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 5% calf serum
and 200 mM L-glutamine. For the uptake experiments, cells were
coincubated with 50 nM Lysotracker Red (Molecular Probes) and 5
µM pyrazoline 2c for 60 min at 37 °C, washed 3 times with PBS, and
fixed with 3.7% paraformaldehyde for 30 min before mounting on slides
with Fluoromount13 (Molecular Probes). To equilibrate intracellular pH
gradients, cells were incubated with 1 µM nigericine (Sigma) for 30
min prior to incubation with the dyes. The cells were analyzed using
an Olympus microscope (BX40, plan apo 60×, 1.40) equipped with a
standard fluorescence excitation filter set. For quantization of the
fluorescence intensity over a large cell population (10 000 counts), cells
were analyzed with a Becton-Dickinson LSR flow cytometer.
Preparation of Liposomes. The liposomal experiment was per-
formed at a lipid concentration of 1 mg/mL PIPES buffer solution (10
mM PIPES, 0.1 KNO3). The solution was prepared by evaporation of
1.0 mL of lipid solution (20 mg/mL 1,2-dioleoyl-sn-glycero-3-phos-
phocholine in chloroform). After addition of 2.0 mL of PIPES buffer
solution, the mixture was allowed to hydrate for 1 h. The mixture was
then ultrasonicated until the solution clarified and the final concentration
was adjusted to 1 mg/mL.
Synthesis. All compounds were prepared using synthetic protocols
developed for 1,3,5-triaryl-2-pyrazoline derivatives.16 The crude prod-
ucts were purified by flash chromatography, and the purity of the final
products was tested by reversed-phase HPLC (Varian ProStar system
with UV detector, acetonitrile-water, gradient 20% f 2% water). The
chemical structures of the synthesized compounds were confirmed by
1H NMR, MS, and high-resolution mass spectrometry (HRMS).
Description of the syntheses and detailed analytical data are provided
with the Supporting Information.
Steady-State Absorption and Fluorescence Spectroscopy. All
sample solutions were filtered through 0.45 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 spectro-
meter with a constant-temperature accessory. Steady-state emission and
excitation spectra were recorded with a PTI fluorimeter and FELIX
software. 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 calibrated photodiode). Quantum yields
were determined using quinine sulfate dihydrate in 1.0 N H2SO4 as a
fluorescence standard (Φf ) 0.54 ( 0.05).26
Electrode Calibration in Aqueous Solution. Measurements were
performed with an Orion combination glass microelectrode (model
9802BN). For the determination of pK values in aqueous solution, the
electrode was calibrated for -log[H3O+] by titration of a standardized
HCl solution (Aldrich, 0.1 N volumetric standard) with KOH (Aldrich,
0.1 N volumetric standard) at 25 °C and 0.1 M ionic strength (KCl).
The end point, E°, and slope were determined using Gran’s method27
as implemented in the software GLEE.28 The electrode potential was
measured with the Corning pH/Ion Analyzer 355, and the emf
measurements were reproducible with (0.1 mV accuracy.
Determination of pK Values. The fluorescence emission spectra
of the fluorophore were monitored for a series of solutions in which
-log[H3O+] was varied between 5 and 9. The emf of each solution
was directly measured in the fluorescence quartz cell (electrode diameter
3 mm) and converted to -log[H3O+] using E° and slope as obtained
from the electrode calibration procedure described previously. The raw
spectral and emf data were processed via nonlinear least-squares fit
analysis using the SPECFIT software package.29
Results and Discussion
Cyclic Voltammetry. The cyclic voltammograms were acquired in
acetonitrile (freshly distilled over calcium hydride) containing 0.1 M
Bu4NPF6 as electrolyte using a CH-Instruments potentiostat (model
600A). The samples were measured under inert gas at a concentration
of 3 mM in a single compartment cell with a Pt working and counter
electrode and an Ag/AgCl reference electrode. The half-wave potentials
were referenced to ferrocene as the internal standard, and the measure-
ments were typically performed with 500 mV s-1 scan rate.
1. Structural Studies. Pyrazoline derivatives 1-6 were
synthesized from p-diethylamino-benzaldehyde or benzaldehyde
(30) Blessing, R. H. Acta Crystallogr., Sect. A 1995, 51, 33.
(31) SHELXTL 5.10; Bruker: Madison, WI, 1998.
(32) 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.: Export, PA, 2000.
X-ray Structure Analysis. Crystals of 1b, 2a-d, and 3b suitable
for X-ray structural analysis were grown from acetonitrile-water by
(33) Becke, A. D. J. Chem. Phys. 1993, 98, 5648.
(26) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991.
(27) Gran, G. Analyst (London) 1951, 77, 661.
(28) Gans, P.; O’Sullivan, B. Talanta 2000, 51, 33.
(29) Binstead, R. A.; Zuberbu¨hler, A. D. SPECFIT Global Analysis System,
3.0.27 ed.; Spectrum Software Associates: Marlborough, MA, 01752, 2001.
(34) Lee, C. T.; Yang, W. T.; Parr, R. G. Phys. ReV. B 1988, 37, 785.
(35) Stratmann, R. E.; Scuseria, G. E.; Frisch, M. J. J. Chem. Phys. 1998, 109,
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(36) Flu¨kiger, P.; Lu¨thi, H. P.; Portmann, S.; Weber, J. MOLEKEL 4.1, 4.1 ed.;
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