1,1-Dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP): A solvent polarity and viscosity sensitive fluorophore for fluorescence microscopy

Article abstract of DOI:10.1021/ja9543356

1,1-Dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (II, DDNP) has been synthesized as a fluorescent dye whose intramolecular rotational relaxation is solvent polarity and viscosity dependent. Its fluorescence emission (λ max: 470-610 nm in hydrophobic and viscous environments) is in a very favorable region for its use with visible fluorescence microscopy, without interference from cell or tissue autofluorescence. Its fluorescence emission quantum yield in an aqueous environment is very low, allowing for facile differentiation between lipid or protein bound state and aqueous media. II crystallizes into two solid isoforms containing one (red crystals) and two conformers (yellow crystals), exhibiting differing spectral properties. X-ray crystal structures reveal a planar arrangement between dimethylamino and naphthalenyl moieties, with out-of-plane arrangements between the malononitrile and naphthalene ring found in each of the three conformers.

Full text of DOI:10.1021/ja9543356

5572
J. Am. Chem. Soc. 1996, 118, 5572-5579
1,1-Dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene
(DDNP): A Solvent Polarity and Viscosity Sensitive
Fluorophore for Fluorescence Microscopy
A. Jacobson,§ A. Petric,‡ D. Hogenkamp,§ A. Sinur,† and J. R. Barrio*,§
Contribution from the Department of Molecular and Medical Pharmacology and the Laboratory
of Structural Biology and Molecular Medicine, UCLA School of Medicine,
Los Angeles, California 90095-6948, Faculty of Chemistry and Chemical Technology,
UniVersity of Ljubljana, 6100 Ljubljana, SloVenia
ReceiVed December 29, 1995X
Abstract: 1,1-Dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (II, DDNP) has been synthesized as a fluorescent
dye whose intramolecular rotational relaxation is solvent polarity and viscosity dependent. Its fluorescence emission
(λ max: 470-610 nm in hydrophobic and viscous environments) is in a very favorable region for its use with
visible fluorescence microscopy, without interference from cell or tissue autofluorescence. Its fluorescence emission
quantum yield in an aqueous environment is very low, allowing for facile differentiation between lipid or protein
bound state and aqueous media. II crystallizes into two solid isoforms containing one (red crystals) and two conformers
(yellow crystals), exhibiting differing spectral properties. X-ray crystal structures reveal a planar arrangement between
dimethylamino and naphthalenyl moieties, with out-of-plane arrangements between the malononitrile and naphthalene
ring found in each of the three conformers.
Introduction
Chart 1
Environmentally sensitive fluorescence, by virtue of varying
responses to solvent media, provides a wealth of information
on the physical and chemical properties of molecular microen-
vironments. Fluorophores that are electronically interactive with
solvents or other solute molecules are fluorescent chemosen-
sors,1 sensitive to an array of factors, such as ions,2 environ-
mental rigidity,3 or electric polarization in cell membranes.4 The
range of experiments in which environmentally sensitive fluo-
rophores can be used, however, is limited by the wavelength
ranges in which useful environmental sensitivity can be found.
Although a number of these fluorophores are used in biological
assays (i.e., 2-acyl-6-dimethylaminonaphthalene derivatives, like
ADMAN (I) and PRODAN),5,6 few polarity or viscosity
sensitive probes are available with absorption in the wavelength
range around 420 nm, below which significant autofluorescence
occurs in many biological systems.
In this work we report the development of a novel fluorophore
with excitation in nonaqueous solvents or rigid media centered
at 430-440 nm and emissions at 470-610 nm, with excellent
properties for visible wavelength fluorescence microscopy using
standard argon, argon/krypton laser illumination, or conventional
lamp sources. The compound, 1,1-dicyano-2-[(6-dimethylami-
no)naphthalen-2-yl]propene (DDNP) (II) (Chart 1), demon-
strates solvent polarity dependent changes in excitation and
emission wavelengths and viscosity dependent changes in
quantum yield. Its environmental sensitivity shows character-
istics of an excited state charge transfer (CT) complex.7-12
Recently fluorophores structurally related to trans-stilbenes, 4-p-
dimethylaminostyrylpyridinium salts, were reported to be sensi-
tive to temperature and viscosity in protic solvents.13 These
derivatives are charged fluorophores with dual fluorescence in
the visible regions and a low energy twisted intramolecular
charge transfer (TICT) band sensitive to viscosity.
DDNP crystallizes into at least two solid isoforms exhibiting
markedly different absorption and fluorescence emission spectra.
X-ray crystallographic data are also presented to correlate the
putative molecular conformations of the two forms with their
spectroscopic properties. The details of this investigation are
presented herein.
Experimental Section
Heptadecane and mineral oils were obtained from Aldrich (Milwau-
kee, WI). Poly(ethylene)glycols were purchased from Serva (Paramus,
NJ). Deionized water was used for the preparation of solutions
(Millipore RO system). Bovine serum albumin (BSA) was procured
* To whom correspondence should be addressed: Department of
Molecular and Medical Pharmacology, B2-086A Center for the Health
Sciences, Los Angeles, CA 90095-6948.
(7) Loutfy, R. O.; Law, K. Y. J. Phys. Chem. 1980, 84, 2803-2808.
(8) Rollinson, A. M.; Drickamer, H. G. J. Chem. Phys. 1980, 73, 5981-
5996.
† University of Ljubljana.
‡ On leave from the University of Ljubljana, Ljubljana, Slovenia.
§ UCLA School of Medicine.
(9) Nowak, W.; Adamczak, P.; Balter, A.; Sygula, A. J. Mol. Struct.
(Theochem.) 1986, 139, 13-23.
Key words: fluorescence/environmental sensitivity/conformers.
X Abstract published in AdVance ACS Abstracts, June 1, 1996.
(1) Czarnik, A. W. Acc. Chem. Res. 1994, 27, 302-308.
(2) Tsien, R. Y. Ann. ReV. Neurosci. 1989, 12, 227-253.
(3) Loutfy, R. O. Macromolecules 1981, 14, 270-275.
(4) Loew, L. M.; Simpson, L. L. Biophys. J. 1981, 34, 353.
(5) Weber, G.; Farris, F. J. Biochemistry 1979, 18, 3075-3078.
(6) Macgregor, R. B.; Weber, G. Nature 1986, 319, 70-73.
(10) Rettig, W. Angew. Chem., Int. Ed. Engl. 1986, 25, 971-988.
(11) Heisel, F.; Miehe, J. A.; Szemik, A. W. Chem. Phys. Lett. 1987,
138, 321-326.
(12) Balter, A.; Nowak, W.; Pawelkiewicz, W.; Kowalczyk, A. Chem.
Phys. Lett. 1988, 143, 565-570.
(13) Wandelt, B.; Turkewitsch, P.; Stranix, B. R.; Darling, G. D. J. Chem.
Soc., Faraday Trans. 1995, 91, 4199-4205.
S0002-7863(95)04335-6 CCC: $12.00 © 1996 American Chemical Society

DDNP: A Fluorophore for Fluorescence Microscopy
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5573
from Sigma (St. Louis, MO). Other solvents used were spectroscopic
grade or better and were obtained from Fisher (Tustin, CA). All
solvents were degassed and sparged with argon prior to use. Absorption
spectra were determined at concentrations around 2 × 10-5 M with a
Beckman DU-50 spectrophotometer. NMR and IR spectra were
recorded on a Bruker AM-360WB spectrometer and a Perkin-Elmer
710B IR spectrophotometer, respectively. Fluorescence spectra were
determined on SLM 4800C (ADMAN, DDNP) and Perkin-Elmer LS-
50B (DDNP) fluorescence spectrophotometers, with slit widths routinely
set at 4-5 nm. For DDNP measurements, both standard and red
sensitive (R-928) photomultiplier tubes were used. Fluorescence was
referenced to solvent blank using matched cells (Spectrocell, Inc), with
emission corrected for photomultiplier wavelength response. Quantum
yields (Q) were determined relative to quinine sulfate in 0.1 N H2SO4
(Regis Chemicals, Morton Grove, IL) (Q ) 0.55, excitation ) 365
nm)14,15 or Rhodamine 101 (Kodak, Rochester NY) (Q ) 0.97-1;
excitation ) 393-460 nm).16-18 Melting points were determined on
an electrothermal melting point apparatus and are uncorrected. HPLC
conditions for analysis of II were as follows: Reverse phase Econosil
C-18 column 5 µm, 250 × 4.6 mm. Solvent: H2O:CH3CN, 7:13, with
detection by UV absorption at 360 nm. Elemental analyses were
performed by Galbraith Laboratories, Inc., Knoxville, TN.
Organic extracts were combined, dried, and evaporated to give a yellow
solid. Recrystallization from ethanol afforded 3.67 g (64%) of a yellow
solid, melting at 153.5-155 °C: 1H NMR (CDCl3, TMS) δ 2.67 (s,
3H, COCH3), 3.15 (s, 6H, N(CH3)2), 6.87 (d, 1H, H-5), 7.17 (dd, 1H,
H-7), 7.63 (d, 1H, H-4), 7.80 (d, 1H, H-8), 7.92 (dd, 1H, H-3), 8.32
(bs, 1H, H-1). J1,3 ) 2.3 Hz, J3,4 ) 8.7 Hz, J5,7 ) 2.4 Hz, J7,8 ) 9.3
Hz. MS (M+) 213; found: 213. Anal. Calcd for C14H15NO: C, 78.84;
H, 7.09; N, 6.57. Found: C, 78.96; H, 7.10; N, 6.45.
1,1-Dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP,
II). A mixture of malononitrile (436 mg, 6.6 mmol) and 2-acetyl-6-
(dimethylamino)naphthalene (ADMAN, I) (1.278 g, 6 mmol) was
heated to 110 °C in 20 mL of pyridine for 19 h. After cooling, the
remaining red solid was dissolved in 100 mL of methylene chloride,
adsorbed onto 10 g of flash silica gel (230-400 mesh) and chromato-
graphed with toluene. Appropriate fractions were combined and
evaporated to give 1.12 g (72%) of II. Recrystallization from benzene-
hexane gave red needles melting at 154.5-155 °C: 1H NMR (CDCl3,
TMS) δ 2.69 (s, 3H, CH3), 3.11 (s, 6H, N(CH3)2), 6.85 (d, 1H, H-5),
7.18 (dd, 1H, H-7), 7.56 (dd, 1H, H-3), 7.66 (d, 1H, H-4), 7.76 (d, 1H,
H-8), 8.02 (d, 1H, H-1). J1,3 ) 2.04 Hz, J3,4 ) 9.13 Hz, J5,7 ) 2.5 Hz,
J7,8 ) 9.11 Hz. IR (CHCl3) 2250 cm-1 (CN stretching). MS (M+):
261; found: 261. Anal. Calcd for C17H15N3: C, 78.13; H, 5.79; N,
18.08. Found: C, 78.17; H, 5.68; N, 17.91.
X-ray Structure Determination. X-ray crystallographic data were
obtained on the two solid isoforms of DDNP, A (red needles) and B
(yellow plates). Crystals of both isoforms were grown from benzene
solution under petroleum ether (30-60°) vapor. For isoform (A):
monoclinic, P21/n, no. 14, a ) 4.031(1), b ) 13.269 (3), c ) 26.030
(7) Å, â ) 93.63(2)°, V ) 1389.5(6) Å3, Z ) 4, Dx ) 1.249 Mg/m3,
Dm ) 1.24(2) Mg/m3, Mo KR radiation, λ ) 0.71069 Å, µ ) 0.07075
mm-1, T ) 293 (2) K, crystal shape: needle, crystal size: 0.08 mm ×
0.20 mm × 0.44 mm. For isoform (B): monoclinic, P21/n, no. 14, a
) 17.668(5), b ) 7.627(1), c ) 20.948(5) Å, â ) 90.16(2)°, V )
2823(1) Å3, Z ) 8, Dx ) 1.230 Mg/m3, Dm ) 1.22(2) Mg/m3, Mo KR
radiation, λ ) 0.71069 Å, µ ) 0.06965 mm-1, T ) 293(2) K, crystal
shape: irregular plate, crystal size: 0.20 mm × 0.56 mm × 0.72 mm.
The unit cell dimensions were determined by least squares with 25
centered reflections for the red isoform (A) (6.0 e θ e 10.9°) and 75
reflections for the yellow isoform (B) (6.1° e θ e 14.4°) using graphite
monochromated Mo KR radiation. Both structures were solved by
direct methods using MULTAN88 programs.19 Atomic scattering
factors for neutral atoms and dispersion corrections were used. The
Xtal13.220 system of crystallographic programs was used for the
correlation and reduction of data, structure refinement, and interpreta-
tion. ORTEPII21 was used to produce molecular graphics, with
calculations performed on VAX 8550 computers. Stereo pairs and
space filling models were also generated with the Insight II V2.3.5
(Biosym Technologies, San Diego, CA) on a Silicon Graphics Extreme
work station.
Results
Chemistry. Standard conditions described in the literature
for the Knoevenagel condensation of malononitrile with sub-
stituted acetophenones23 were not successful with II. Recently
described conditions employing TiCl4 as a catalyst could not
be used with acetophenone because of side reactions.24 Reaction
in benzene or toluene with a piperidinium acetate catalyst and
azeotropic removal of water gave only low conversion to the
desired adduct after 12 h at reflux. Pyridine has long been
known to catalyze the dehydration of the initial addition product
in the Knoevenagel condensation and has been used in conjunc-
tion with secondary amines to accelerate the reaction. It was
found that reaction of I with 1.05 equiv of malononitrile in
pyridine at reflux gave a 66-85% yield of the adduct, II. The
product was easily identified by TLC as a visible orange spot
of higher Rf than the starting ketone (solvent, 100% benzene,
silica gel). The compound appeared as a red solid after flash
chromatography. The structure of II is consistent with its
spectral properties.
Analytical samples of DDNP are shelf-stable at room tem-
perature. Methanolic solutions of II were found to be stable
for several weeks, as determined by their single emission peak
at 610 nm upon excitation at either 340 or 420 nm. On the
other hand, II slowly decomposed in other solvents (i.e., CH3-
CN and ether containing solvents) giving rise to a second pair
of excitation and emission bands at shorter wavelengths. The
observed second fluorescent transition was found to be solvent
polarity dependent, resembling the fluorescence properties of
the acetyl precursor, ADMAN (I) (Table 1). Verification of
this decomposition was obtained by HPLC analysis, wherein
the decomposition product eluted as a single peak when
coinjected with a purified sample of I (DDNP: Rt ) 9.02 min;
I and decomposition product of II: Rt ) 12.56 min). Using
this technique, the t1/2 of decomposition of II in CH3CN at 20
°C was determined to be approximately 36 days.
2-Acetyl-6-(dimethylamino)naphthalene (ADMAN, I).5 To a
solution of 5.26 g (117 mmol) of dimethylamine in 29 mL of freshly
distilled hexamethylphosphoric triamide (HMPT) were added 31 mL
of dry toluene and 780 mg (112 mmol) of Li in small pieces. The
mixture was stirred under argon at room temperature for 1.5 h.
2-Acetyl-6-methoxynaphthalene22 (5.57 g, 27.8 mmol) was added in
one portion and stirring continued for 20 h. The mixture was cooled
in an ice-water bath and poured into a cold water/ethyl acetate mixture
(300 mL each). After thorough mixing, the layers were separated, and
the water layer was extracted twice with 225 mL of ethyl acetate.
(14) Melhuish, W. H. J. Opt. Soc. Am. 1964, 54, 183-186.
(15) Chen, R. F. Anal. Biochem. 1967, 19, 374-387.
(16) Schnerzel, R. E.; Klosterman, N. E. NBS Special Publication 1976,
526, 3-4.
(17) Drexhage, K. N. NBS Special Publication 1977, 466, 33-40.
(18) Karstens, T.; Kobs, K. J. Phys. Chem. 1980, 84, 1871-1872.
(19) Debaerdemaeker, T.; Germain, G.; Main, P.; Refaat, L. S.; Tate,
C.; Woolfson, M. M. MULTAN88: A System of Computer Programs for
the Automatic Solutions of Crystal Structures from X-ray Diffraction Data;
University of York: England, 1988.
(20) Hall, S. R.; Stewart, J. M. The Xtal3.0 system; University of Western
Australia: Australia, and University of Maryland: U.S.A., 1990.
(21) Johnson, C. K. ORTEPII. Report ORNL-3794; Oak Ridge National
Laboratory: Oak Ridge, Tennessee, 1976.
(22) Arsenijevic, L.; Arsenijevic, V.; Horeau, A.; Jacques, J. Org. Synth.
Coll. 1988, 6, 34-36.
Electronic Absorption Spectroscopy. Substantial differ-
ences exist between the electronic absorption spectra of DDNP
(II) and those of the parent I. The ultraviolet absorption
maximum exhibited by the acetyl derivative I, centered at 360
nm (ꢀ ) 1.8 × 104), is red-shifted by 50-80 nm in the product
(23) Coligny, T. H.; Normant, C. R. Acad. Sci. Paris 1971, 272C, 1425-
1430.
(24) Lehnert, W. Tetrahedron 1972, 28, 663-666.

5574 J. Am. Chem. Soc., Vol. 118, No. 24, 1996
Jacobson et al.
Table 1. DDNP, ADMAN Electronic Absorption, and Fluorescence Dataa
absorption
maxima (nm)
excitation
maxima (nm)
emission
maxima (nm)
ꢀ(1 × 10-4
)
quantum yields
solvent
ET(30)b ADMAN DDNP ADMAN DDNP ADMAN DDNP ADMAN DDNP ADMAN DDNP τ DDNP (ps)
water
methanol
ethanol
acetonitrile
chloroform
dichloromethane
cyclohexane
hexane
63.1
55.5
51.9
46.0
41.1
39.1
31.2
30.9
355
366
363
352
358
385
426
434
428
444
438
415
412
421
422
422
c
c
360
365
365
355
360
393
428
410
428
450
456
528
506
498
492
436
529
610
600
600
548
560
470
468
468
468
468
0.22
0.48
0.63
0.70
0.17
0.015
0.013
106
210
302
306
1.8
1.8
1.8
1.9
2.0
2.0
1.9
0.053
0.028
2.0
245
343
2.1
345
394
0.03
2.5
2.5
2.6
2.6
410
0.0020
0.0032
0.0082
0.0091
0.060
heptadecane
low viscosity mineral oil
high viscosity mineral oil
γ-cyclodextrin
a Absorption spectra determined at concentrations on the order of 1-5 µM. Fluorescence spectra were measured at concentrations of less than
0.1 O.D. at the excitation maxima. b ET(30) values from ref 25. c Concentration not determined.
aqueous media are dissimilar.] Addition of protein (e.g., bovine
serum albumin, BSA) to aqueous solutions of II elicits an
increase in fluorescence intensity and a hypsochromic shift of
the fluorescence excitation and emission maxima (Figure 2),
an effect likely due to DDNP binding to hydrophobic pockets
on the protein surface. The substantial red-shift in excitation
spectra with BSA addition (Figure 2, inset) is similar to changes
observed in moving from water to more intermediate polarity
solvents (e.g., CH3CN) (Table 1). In order to assess the effect
of viscosity, DDNP fluorescence emission spectra were mea-
sured in a series of poly(ethylene)glycols (Figure 3). Fluores-
Figure 1. 1H NMR chemical shifts for naphthalene protons in DDNP
cence emission intensity and relative quantum yields were found
to increase strongly with increasing kinematic viscosity over
the range 18-140 centiStokes. Emission wavelengths were
noted to shift slightly to higher energies with increasing chain
length.
vs solvent ET(30) values.25 Proton assignments were maded from single
proton decoupling experiments.
of malononitrile addition. The absorption maximum of DDNP
appears as a broad single transition in the 380-450 nm region,
that is relatively less sensitive to solvent polarity than fluores-
cence excitation or emission. DDNP absorption maxima were
found in the range 412 (hexane) to 444 nm (chloroform), with
extinction coefficients of ∼2 × 104 (Table 1). Absorption
maxima did not follow a simple regression but instead fell
generally into an inverted “U” shaped pattern when compared
by solvent polarity ET(30).25 In investigating the ground state
As the nature of the solvent changes somewhat with increas-
ing chain length in the ethylene glycols, experiments were
conducted with a series of alkanes in order to separate the effect
of changing solvent functionality from viscosity sensitivity
(Figure 4). Even though fluorescence quantum yields increase
4.6-fold over a viscosity range of 0.45-156 centiStokes, no
change in wavelength was observed under these conditions,
confirming the absence of solvatochromatic effects with viscos-
ity as the only changing parameter. Similarly, fluorescence
intensity of the emission spectrum in glycerol at -50 °C
increased three-fold over that at room temperature in the same
solvent. As shown in Figure 5A, the fluorescence maximum
at -50 °C is at 550 nm, reducing the Stokes shift from 6.6 ×
103 to 1.1 × 102 cm-1 since no similar shift was observed in
the excitation spectrum with temperature. The temperature itself
is not an important factor in this change since the fluorescence
spectrum in methanolic solution at -50 °C was instead red-
shifted to 620 from 600 nm at room temperature. As expected,
however, the fluorescence yield proved to be very sensitive to
temperature (Figure 5B).
Solid State Properties. DDNP (II) was isolated in two
different crystalline forms from benzene solution under petro-
leum ether vapor: (A) as fine red needles, formed with fast
growth in concentrated solution, and (B) as yellow plates,
formed slowly from dilute solution. X-ray measurements
confirm that A and B are polymorphs of the same substance.
Although these isoforms produce identical 1H NMR, absorption,
and fluorescence properties when in solution, they revealed very
different fluorescent emission properties in the solid state, with
the red and yellow material showing emission maxima of 645
and 580 nm, respectively (Figure 6).
1
polarity of II, H NMR spectra were determined in various
1
solvents. Like absorption maximum, the H chemical shifts
show an inverted “U”-shaped curve when plotted against solvent
polarity (Figure 1), with higher chemical shifts at intermediate
solvent polarities.
Fluorescence Properties. Highly purified samples of II were
used for absorption and fluorescence determinations. DDNP
was found to be poorly fluorescent in organic and aqueous
solvent media. Quantum yields varied from 4 × 10-4 to 5 ×
10-2 and were considerably lower than those of I (Table 1). As
with I, however, quantum yields appear highest in solvents with
intermediate polarity, with lower yields in aqueous and nonpolar
solvents. Emission wavelengths appear to move to successively
lower energies with increasing solvent polarity, as is generally
found with excited state charge transfer (CT) complexes. In
water, fluorescence was anomalously blue-shifted with a
significant reduction in Stokes shift (Table 1), which can be
attributed to the formation of microaggregates. [The blue-shift
is coincident with the fluorescent emission spectra of I. The
fluorescence emission observed is not believed to be due to
decomposition to II in water, however, as there was no
chromatographically detectable amount of I in the sample tested.
Moreover, the excitation spectra of the two compounds in
(25) Reichardt, C. Solvent Effects in Organic Chemistry; Verlag
Chimie: Weinheim, 1979; pp 242-244.
The asymmetric unit of modification A contains only one

DDNP: A Fluorophore for Fluorescence Microscopy
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5575
Figure 2. Corrected fluorescence emission spectra of DDNP in water with increasing concentrations of bovine serum albumin (BSA) at 20 °C:
(1) water; (2) 0.5 mg/mL BSA; (3) 1 mg/mL BSA; (4) 2 mg/mL BSA; (5) 7 mg/mL BSA; (6) 11 mg/mL BSA. Higher concentration of BSA
induced no additional fluorescence changes. Excitation ) 420 nm. Inset shows DDNP fluorescence excitation spectra in water (1) and upon
addition of BSA (1 mg/mL) (2).
Figure 3. Corrected room temperature fluorescence emission spectra of DDNP in MeOH (1, 0.695 centiStokes), ethylene glycol (2, 18 centiStokes),
and neat polyethylene glycol mixture of different viscosities (3), PEG200, 58 centiStokes, (4), PEG400, 105 centiStokes, and (5), PEG 600, 140
centiStokes. Excitation ) 420 nm. Inset shows the relationship between quantum yield and viscosity in the polyethylene glycol series. Log10
curve fit R2 ) 0.982. Note: changes in fluorescence emission wavelength are probably due to changes in solvent polarities.
molecule, whereas modification B has two formula units: (a)
and (b). Most bond angles of all conformers showed remarkable
similarities, except for the orientation of substituent groups
around the C(2)-C(11) bond (Table 2). The orientation of
dihedral angles appeared to be reversed between B(a) and B(b)
in the yellow solid. The malononitrile group orients toward

5576 J. Am. Chem. Soc., Vol. 118, No. 24, 1996
Jacobson et al.
Figure 4. Corrected room temperature fluorescence emission spectra of DDNP in alkanes of different viscosities: hexane (1, 0.445 centiStokes),
heptadecane (2, 4.79 centiStokes), low viscosity mineral oil (3, 45.99 centiStokes), and high viscosity mineral oil (4, 156.10 centiStokes). Inset
shows the relationship between quantum yield and viscosity in this series. Log10 curve fit R2 ) 0.932.
Ltk- mouse fibroblast cells (Figure 9). With DDNP staining,
intense fluorescence was observed with cell membranes and
Table 2. Selected Torsion Angles (in deg) for DDNP Crystal
Isoforms (for Numbering see Figure 8)a
internal structures (488 nm illumination and emission above 525
nm). With the fluorophore applied in concentrations ranging
from 50 nM to 10 µM, with 20-30 s exposure prior to imaging,
no increase in background signal was observed. These cells
could be imaged for several hours when kept alive in growth
medium, with little or no photobleaching or other loss in signal
strength.
dihedral
isoform (A)
(B)(a)
(B)(b)
C1-C2-C11-C12
C1-C2-C11-C13
C3-C2-C11-C12
C3-C2-C11-C13
C2-C11-C13-C14
C2-C11-C13-C15
C12-C11-C13-C14
C12-C11-C13-C15
C6-C7-N3-C16
C6-C7-N3-C17
C8-C7-N3-C16
C8-C7-N3-C17
28.7
-152.3
-147.9
31.0
37.7
-141.5
-140.7
40.1
143.3
-38.0
-34.9
143.7
-179.9
-2.2
-175.1
-178.0
9.3
1.7
3.8
2.8
-1.2
-171.8
179.8
-3.7
0.6
177.1
-177.5
176.5
175.1
-5.1
Discussion
0
-173.3
-179.5
7.3
The fluorescence probe 1,1-dicyano-2-[6-(dimethylamino)-
naphthalen-2-yl]propene (II, DDNP) was developed as a neutral,
lipophilic probe for use with fluorescence spectroscopy. Be-
cause II is uncharged at physiological pH, it can cross membrane
barriers, and when linked to a molecule of biological interest,
it can be used to study intracellular in ViVo processes. When
used with an argon ion or argon/krypton laser source, a probe
for fluorescence microscopy should have significant excitation
at 488 nm and a large enough Stokes shift to filter appropriately
excitation from emission wavelengths. Indeed, II exhibits an
intense absorption band in the visible region (Table 1). The
long wavelength S0 f S1 absorption band was observed to move
to generally lower energies with increasing solvent polarity, with
the exception of water where DDNP absorption was found to
be strongly blue-shifted. Increases in absorption band-width
were also observed with increasing solvent polarity. Fluores-
cence emission spectra (S1 f S0) exhibited the same sensitivity
to solvent polarity (Table 1) demonstrating the highly polar
nature of the S1 excited state. The long wavelength fluorescence
emission of DDNP observed in more polar solvents is undoubt-
edly due to an intramolecular π* f π26 charge transfer (CT)
transition. A question can be raised, however, as to whether
DDNP fluorescence observed in solvent systems is due to
-4.9
175.0
a Both isoforms crystallize in P21/n space group, which possesses a
center of symmetry. Therefore, in both solid isoforms, 50% of the
molecules have the torsion angles listed above, and the other 50% have
the same absolute values but with opposite sign.
carbon-3 in B(a) (dihedral C(1)-C(2)-C(11)-C(13) ) 37.9°)
or carbon-1 in B(b) (dihedral C(1)-C(2)-C(11)-C(13) )
143.3°) in the naphthalene ring. The orientation of the
malononitrile group in A (red modification) is similar to that
of B(a), but with smaller C(1)-C(2)-C(11)-C(13) torsion
angle (28.7°) (Figure 7). In all three conformers, the dimethy-
lamino moiety is close to planar with the naphthalene ring ((0-
7° rotation, Table 2). Bond lengths are similar in all three forms
and correspond to the expected values. The shortest inter-
molecular contact in (A) is 3.56(1) Å between C(12) and N(2)(i)
((i): 3/2-x, 1/2+y, 1/2-z), and in (B) 3.27(1) Å between N(1b)
and N(1b)(ii) of two symmetry related molecules ((ii): -x, 1 -
y, -z). Significant aromatic ring overlap (vertical stacking)
exists in the yellow form between the two conformers (Figure
8).
Labeling Applications. A functional example of the utility
of DDNP environmental sensitivity was demonstrated in its
application as a general cell stain in confocal imaging of live
(26) Lippert, E.; Luder, W.; Boos, H. Advances in Molecular Spectros-
copy; Mangini, A., Ed.; Pergamon Press: Oxford, 1962; p 443.

DDNP: A Fluorophore for Fluorescence Microscopy
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5577
Figure 6. Corrected fluorescence emission spectra for the red (A) and
yellow (B) isoforms of DDNP.
Figure 7. Newman projections of parts of molecules of (A) and (B),
looking in the C(2)-C(11) direction.
Figure 5. A. Molecule fluorescence emission spectra of DDNP in
glycerol at room temperature (s) and glycerol at -50% (- - -); B,
in methanol at room temperature (s) and methanol at -50° (- - -).
Excitation at 460 nm.
emission from planar or “twisted” charge transfer states.10
Although we have not yet attempted to determine orientation
in solvent systems, a number of features of the fluorescent
properties of the solid state structure reflect on the importance
of the orientation of donor and acceptor substituents to the π
system.
The peak of fluorescence emission in the yellow solid (B(a)
and B(b)) is somewhat bathochromically shifted from that of
the red form (A), suggesting an increased energy of the
prominent CT transition. The lesser twisting in the longer
wavelength (red) fluorescent form of the solid would seem to
raise the possibility that a more planar, “biradical” or biradi-
caloid” CT27,28 transition is of the lowest energy in this system.
Figure 8. ORTEP representations of DDNP red (A) and yellow (B)
crystal isoforms with position and bond lengths shown.
In that the yellow crystal form reveals an extremely broad
emission, it is likely that the observed fluorescence is a
composite of emission from the two conformers (B(a) and B(b))
present in the crystal. On the other hand, while the difference
in planarity of malononitrile groups is notable, it would appear
(27) Bonacˇic´-Koutecky´, V.; Michl, J. J. Am. Chem. Soc. 1985, 107,
1765-1766.
(28) Nowak, W.; Rettig, W. J. Mol. Struct. (Theochem) 1993, 102, 1-12.

5578 J. Am. Chem. Soc., Vol. 118, No. 24, 1996
Jacobson et al.
Figure 9. Cultured Ltk-mouse fibroblast cells stained with 500 nM DDNP. Image generated on BioRad MRC 600 confocal fluorescent scanning
microscope. Excitation ) 488 nm, argon ion laser. Emission above 525 nm. Scale bar is 25 µm.
unlikely that only differences in torsion angle would account
for the approximate 80 nm red shift in fluorescence maxima
between the two crystal forms. This large wavelength change
may require much larger changes in the geometry or conjugation
of the molecule, a factor that may be provided by the
intermolecular effects of stacking in the crystal. In the yellow
form there are two conformers of DDNP stacked in close
proximity in a head to toe fashion, whereas in the red form
very little overlap exists between the molecules (Figure 8). From
the overlap and closeness of the ring systems of the yellow form,
it would appear that significant π-π stacking may occur. This
raises the possibility that, along with the out-of-plane rotation
of the malononitrile group, intermolecular electronic (excimer)
effects may participate in the broadening and bathochromic shift
of fluorescence maxima.
Clearly, our solid state fluorescence data have limited
quantitative significance but offer direct evidence that fluores-
cence emission in DDNP originates in out-of-plane conformers.
The dihedral angle (θ) between the naphthalene ring and the
malononitrile moieties in the crystal structure for all conformers
found (A, red; B(a), B(b), yellow) departs significantly from
zero. Although our findings of approximate planarity between
dimethylamino and naphthalene moieties in both crystal species
are consistent with previous reports of PRODAN crystal
structure,29 the observation of long wavelength CT emission
would appear to contrast with the general view of intramolecular
charge transfer states in highly extended donor-aryl-acceptor
systems.9-12,29-35 Modeling of electronic transitions in these
systems has generally ascribed long wavelength fluorescence
to twisted intramolecular CT, with the dialkylamino electron
donor group perpendicular to the ring system plane in the excited
state. Though dual fluorescence may occur in solution from
both planar and twisted conformations of the dimethylamino
moiety in DDNP or with interconversion between states, the
crystal structures presented here demonstrate conclusively that
a twisted donor-ring structure, with complete charge separation,
is not a necessity to produce the long wavelength emission. In
this system, the significant dihedral angle between the aromatic
ring and the electron acceptor moiety (malononitrile) may
produce effective charge separation to effect low energy
solvatochromatic emission sensitivity.
The fluorescence quantum yield of II is low in most solvents
but is found to increase with solvent viscosity (Figures 3 and
4) suggesting that the π* CT excited state is greatly depopulated
during the fluorescent lifetime by internal conversion within
the molecule. By analogy with similar systems, this S1 state
relaxation most likely occurs by photoisomerization around the
propene double bond.36 In comparison with the isomerization
and twisting of the CdC bond in trans-stilbene, wherein the
rate is estimated at ∼1 × 1010 s-1 37
, rotation around the ethylene
bond in II is expected to be much greater considering the large
difference in electronegativity of substituent groups across the
bond.38 By contrast, fluorescence deexcitation of II is unlikely
to occur by torsion of the donor or acceptor groups from the
naphthalene plane. The rotation rates in the ground state of
the malononitrile and dialkyl amino groups in [p-N,N-dialkyl-
aminobenzylidene]malononitriles have been reported to be
approximately 5 × 104 and 1 × 107 s-1, respectively.39 In the
excited state these rotations are expected to be yet slower due
to the attainment of partial double bond character and are
essentially frozen during the fluorescent lifetime.40 Rotation
of these groups thus cannot account for the low quantum yield
of II.
(29) Ilich, P.; Pendergast, F. G. J. Phys. Chem. 1989, 93, 4441-4447.
(30) Grabowski, Z. R.; Rotkiewicz, K.; Siemiarczuk, A.; Cowley, D. J.;
Baumann, W. NouV. J. Chim. 1979, 3, 443-454.
(31) Rotkiewicz, K.; Grellman, K. H.; Grabowski, Z. R. Chem. Phys.
Lett. 1973, 19, 315-318; erratum 1973, 21, 212.
(32) Rettig, W.; Bonacˇic´-Koutecky´, V. Chem. Phys. Lett. 1979, 62, 115-
120.
Taken together, these data suggest that the very low fluo-
rescence yield of II in nonviscous solvents is the result of a
fast nonradiative deactivation of the singlet excited state
(36) Safarzadeh-Amiri, A. Chem. Phys. Lett. 1986, 129, 225-230.
(37) Greene, B. I.; Hochstrasser, R. M.; Weisman, R. B. J. Chem. Phys.
1979, 71, 544-545.
(33) Lipinski, J.; Chojnacki, H.; Grabowski, Z. R.; Rotkiewicz, K. Chem.
Phys. Lett. 1980, 70, 449-453.
(34) Grabowski, Z. R.; Dobkowski, J.; Ku¨hnle, W. J. Mol. Struct. 1984,
114, 93-100.
(38) Bonacˇic´-Koutecky´, V.; Ko¨hler, J.; Michl, J. Chem. Phys. Lett. 1984,
104, 440-443.
(35) Rulliere, C.; Grabowski, Z. R.; Dobkowski, J. Chem. Phys. Lett.
1987, 137, 408-413.
(39) Safarzadeh-Amiri, A. Can. J. Chem. 1984, 62, 1895-1898.
(40) Porter, G.; Suppan, P. Trans. Faraday Soc. 1965, 61, 1664-1673.

DDNP: A Fluorophore for Fluorescence Microscopy
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5579
associated with C(11)-C(13) double bond isomerization. In-
ternal rotational relaxation of the bond that connects the
naphthalene ring and the dicyanopropylene group would produce
conformers susceptible to fluorescence deactivation36 in the
excited state. Restrictions placed on C(11)-C(13) double bond
isomerization with increased probability of intramolecular
rotation around the donor-acceptor C(2)-C(11) bond may
additionally be responsible for the environmental sensitivity of
II to protein binding. Addition of bovine serum albumin (BSA)
to DDNP in aqueous solutions results in over six-fold increase
in fluorescence intensity, a complete shift in excitation wave-
length, from 393 nm to 464 nm, and a concomitant shift in
emission wavelength, from 530 nm to 570 nm (Figure 2).
Therefore, in the presence of proteins any contribution to the
fluorescent signal from the unbound fluorophore is negligible
compared with the emission of the probe bound to the
macromolecule. This constitutes a highly desirable property
for fluorescence microscopy. Similarly, the same rotor effect39,41
can be used to explain the rigidity dependence (i.e., with
γ-cyclodextrin) and the viscosity effects3 observed when the
fluorescence emission properties are determined in glycol or
alkane series at room temperature or with glycerol at -50 °C,
where quantum yields are found to increase dramatically with
increased kinematic viscosity (Figures 3-5).
ViVo. To separate in cell labeling experiments the contributions
of viscosity and polarity to fluorescence emission would be
difficult. However, this limitation should not detract from the
use of DDNP in biological experiments. Unlike the UV
fluorescent compound PRODAN, a broad long wavelength
excitation spectra in protein and intermediate polarity solvent
media is found for DDNP, allowing its use with 488 nm
illumination from the argon ion, or argon/krypton lasers
commonly used in visible wavelength fluorescent confocal
microscopy, or conventional fluorescence microscopy using a
lamp source.
The intensity of emission at submicromolar concentrations
of DDNP, with very short ligand exposure time, demonstrates
its high efficacy for protein and membrane labeling. The rapid
labeling of internal components suggests rapid diffusion of II
through membranes. Moreover, a negligible signal from DDNP
in unbound state in the cytosol (or bathing media) adds a unique
advantage in its combination of solvent polarity and viscosity
sensitivity in the visible wavelength range with low background
signal. Conjugation of this probe to high affinity ligands for
investigating in ViVo biochemical processes (e.g., receptor
binding and modulation) increases its range of applicability. This
work will be published elsewhere.
Acknowledgment. This work was supported in part by
Department of Energy Grant DE-FC0387-ER60615 and in part
by the Ministry of Science and Technology of the Republic of
Slovenia (travel expenses for A.P.). Special thanks are given
to Professor G. Weber for valuable discussions and determi-
nation of fluorescence lifetimes.
Because of the strong visible wavelength solvatochromatism
and viscosity sensitivity, DDNP appears to be a highly useful
probe for biological assays. As DDNP is hydrophobic, and
uncharged at physiological pH, it is able to readily pass cell
membranes and, if conjugated with a pharmacological agent,
could be used to study a variety of intracellular processes in
(41) Kung, C. E.; Reed, J. K. Biochemistry 1989, 28, 6678-6686.
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