Long-wavelength analogue of PRODAN: Synthesis and properties of Anthradan, a fluorophore with a 2,6-donor-acceptor anthracene structure

Article abstract of DOI:10.1021/jo0616660

We have synthesized the environment-sensitive fluorophores 2-cyano-6-dihexylaminoanthracene and 2-propionyl-6-dihexylaminoanthracene (Anthradan) starting from 2,6-diaminoanthraquinone. Anthradan is the benzologue of the well-known family of naphthalene 2-propionyl-6-dimethylaminonaphthalene (PRODAN) fluorophores. The additional spectral red shift of the anthracene avoids the autofluorescence of many biological systems and provides for more favorable excitation wavelengths for fluorescence applications. Furthermore, Anthradan exhibits polarity-sensitive emission comparable to that of PRODAN and displays high quantum yields in a range of solvents. Single molecules of these anthracene-containing fluorophores have been imaged in polymer hosts as a proof-of-principle.

Full text of DOI:10.1021/jo0616660

Long-Wavelength Analogue of PRODAN: Synthesis and Properties
of Anthradan, a Fluorophore with a 2,6-Donor-Acceptor
Anthracene Structure
Zhikuan Lu,† Samuel J. Lord,‡ Hui Wang,† W. E. Moerner,‡ and Robert J. Twieg*,†
Department of Chemistry, Kent State UniVersity, Kent, Ohio 44242-0001, and Department of Chemistry,
Stanford UniVersity, Stanford, California 94305-5080
rtwieg@lci.kent.edu
ReceiVed August 10, 2006
We have synthesized the environment-sensitive fluorophores 2-cyano-6-dihexylaminoanthracene and
2-propionyl-6-dihexylaminoanthracene (Anthradan) starting from 2,6-diaminoanthraquinone. Anthradan
is the benzologue of the well-known family of naphthalene 2-propionyl-6-dimethylaminonaphthalene
(PRODAN) fluorophores. The additional spectral red shift of the anthracene avoids the autofluorescence
of many biological systems and provides for more favorable excitation wavelengths for fluorescence
applications. Furthermore, Anthradan exhibits polarity-sensitive emission comparable to that of PRODAN
and displays high quantum yields in a range of solvents. Single molecules of these anthracene-containing
fluorophores have been imaged in polymer hosts as a proof-of-principle.
Introduction
naphthalene (LAURDAN), 2′-(N,N-dimethylamino)-6-naph-
thoyl-4-trans-cyclohexanoic acid (DANCA), and other related
fluorophores have been introduced.3 However, one drawback
of these dyes is that they all absorb in the blue, thus stimulating
cellular autofluorescence4 that can diminish the signal/back-
ground ratio, thereby making ultrasensitive measurements more
difficult in future cell applications.
Our continued interest in fluorescent dyes for single-molecule
fluorescence spectroscopy5 in biologically relevant systems led
us to explore the anthracene analogues of these naphthalene
dyes. We anticipated that the push-pull structure of a 2,6-
substituted anthracene would retain the useful properties found
in PRODAN (i.e., polarity-induced shift of emission and
sensitivity to local nanoenvironments) while shifting the
absorption further to the red for better results in cellular imaging.
No amine-donor anthracene chromophores have been reported
Fluorescence probes are widely used in photobiology, and
the enhancement of function of these materials is actively
sought.1 Especially attractive are molecules that exhibit a
reporter function arising from some observable that reflects
features or changes in the local environment. One of the best
known among polarity-sensitive fluorophores is 2-propionyl-
6-dimethylaminonaphthalene (PRODAN), which was originally
introduced by Weber and Farris.2 This specific dye and many
of its derivatives have subsequently been extensively explored
as fluorescence probes for studying nanoenvironments in various
chemical and biological systems. PRODAN is a push-pull
charge-transfer chromophore with an electron-donating di-
methylamino group and an electron-withdrawing propionyl
group located at the 2,6-positions of naphthalene. Numerous
modifications of PRODAN such as 6-acryloyl-2-dimethylami-
nonaphthalene (ACRYLODAN), 6-lauroyl-2-dimethylamino-
(3) (a) Samanta, A.; Fessenden, R. W. J. Phys. Chem. A 2000, 104,
8972-8975. (b) Davis, B. N.; Abelt, C. J. J. Phys. Chem. A 2005, 109,
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Xiao, Y.; Liu, F. Y.; Qian, X. H.; Cui, J. N. Chem. Commun. 2005, 239-
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† Kent State University.
‡ Stanford University.
(1) For review: (a) Valeur, B. Molecular Fluorescence, Principles and
Applications; Wiley-VCH: New York, 2002. (b) Lakowicz, J. R. Principles
of fluorescence spectroscopy, 2nd ed.; Kluwer Academic/Plenum: New
York, 1999. (c) Willets, K. A.; Nishimura, S. Y.; Schuck, P. J.; Twieg, R.
J.; Moerner, W. E. Acc. Chem. Res. 2005, 38, 549-556. (d) de Silva, A.
P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C.
P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515-1566.
(2) Weber, G.; Farris, F. J. Biochemistry 1979, 18, 3075-3078.
10.1021/jo0616660 CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/23/2006
J. Org. Chem. 2006, 71, 9651-9657
9651

Lu et al.
SCHEME 1. Unsuccessful Approaches to Prepare Asymmetric 2,6-Disubstituted Anthraquinonesa,b
a In all cases, the asymmetrical product (4, 6, 8, 9) was not produced in a useful yield. b(a) Zn, NH4OH; (b) 1 equiv of t-BuONO, 1 equiv of CuBr2,
CH3CN; (c) 2.2 equiv of t-BuONO, 2.2 equiv of CuBr2, CH3CN; (d) dihexylamine, K2CO3, DMSO, 125-140 °C; (e) CuI, NH4OH.
with a donor-acceptor pair located only at the 2,6-positions.
Here, we report the synthesis and physical properties of 2,6-
substituted anthracene push-pull molecules that possess a
modest change in solvent-dependent absorption but a large
change in solvent-dependent fluorescence. The potential utility
of these dyes is illustrated by a demonstration of direct single-
molecule imaging.
Simple anthracene derivatives have been extensively used as
fluorescent probes in biology, chemical sensors, and optoelec-
tronic materials.6 For instance, Tocanne and co-workers studied
methyl 8-(2-anthroyl)octanoate as a solvatochromic lipophilic
probe.7 Coleman and Mortensen synthesized 1- and 2-anthran-
cenyl â-C2′-deoxyribosides to study the dynamic properties of
DNA using ultrafast photophysical techniques.8 Sakamoto and
co-workers synthesized anthracene-linked polythiazaalkane and
polythiaalkane derivatives as silver-ion-selective fluoroiono-
phores.9 The absorption and fluorescence of these simple
anthracene derivatives is typically in the range of 350-400 nm.
However, red-shifted 2,6-donor-acceptor-substituted anthracenes,
in which the two groups are located at maximum distance across
the acene, are rare. Only very recently, Ihmels and co-workers
reported a 2,6-donor-acceptor-substituted anthracene with a
moderate-strength methoxy donor group and a benzoxazole or
oxazoline acceptor, which were derived from a carboxylic acid.10
These molecules exhibit moderate solvatochromism and were
explored as acid-sensitive fluorescence probes. An anthracene
with a 6-amino donor and a 2,3-dicarboxylic acid acceptor pair
was prepared by building the anthracene core in several steps.11
Sun and Desper’s recent modification involved protection of
2,3-dimethylanthracene and subsequent nitration and reduction.
However, the starting material was not commercially available
and it had to be prepared in several steps.12 These methods are
not useful for the 2,6-donor-acceptor-substituted anthracenes
with an amino donor.
Results and Discussion
The synthetic approaches we pursued to prepare 2,6-donor-
acceptor-substituted anthracenes all began with commercially
available 2,6-diaminoanthraquinone 1 (Scheme 1). There are
two key issues concerning the application of this precursor: (1)
breaking the symmetry of the molecule which involves retaining
one amine and converting the other amine to a different
functional group (e.g., dialkylating one amine and converting
the other to a bromide) and (2) reduction of the 9,10-
anthraquinone system to anthracene. The second issue had
already been dealt with in a variety of other substituted
anthraquinones using either sodium borohydride13 or zinc in
(4) (a) Campbell, R. E.; Tour, O.; Palmer, A. E.; Steinbach, P. A.; Baird,
G. S.; Zacharias, D. A.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 7877-7882. (b) Baird, G. S.; Zacharias, D. A.; Tsien, R. Y. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 11984-11989.
(5) (a) Willets, K. A.; Ostroverkhova, O.; He, M.; Twieg, R. J.; Moerner,
W. E. J. Am. Chem. Soc. 2003, 125, 1174-1175. (b) Nishimura, S. Y.;
Lord, S. J.; Klein, L. O.; Willets, K. A.; He, M.; Lu, Z.; Twieg, R. J.;
Moerner, W. E. J. Phys. Chem. B 2006, 110, 8151-8157.
(6) (a) Lee, S. K.; Yang, W. J.; Choi, J. J.; Kim, C. H.; Jeon, S. J.; Cho,
B. R. Org. Lett. 2005, 7, 323-326. (b) Quesada, E.; Ardhammar, M.;
Norden, B.; Miesch, M.; Duportail, G.; Bonzi-Coulibaly, Y.; Nakatani, Y.;
Ourisson, G. HelV. Chim. Acta 2000, 83, 2464-2476. (c) Mathauer, K.;
Vahlenkamp, T.; Frank, C. W.; Wegner, G. Langmuir 1993, 9, 1582-1586.
(7) Perochon, E.; Lopez, A.; Tocanne, J. F. Chem. Phys. Lipids 1991,
59, 17-28.
(10) (a) Ihmels, H.; Meiswinkel, A.; Mohrschladt, C. J.; Otto, D.;
Waidelich, M.; Towler, M.; White, R.; Albrecht, M.; Schnurpfeil, A. J.
Org. Chem. 2005, 70, 3929-3938. (b) Ihmels, H. Eur. J. Org. Chem. 1999,
1595-1600. (c) Ihmels, H.; Meiswinkel, A.; Mohrschladt, C. J. Org. Lett.
2000, 2, 2865-2867. (d) Bohne, C.; Ihmels, H.; Waidelich, M.; Yihwa, C.
J. Am. Chem. Soc. 2005, 127, 17158-17159.
(11) Gundermann, K. D.; Klockenbring, G.; Roker, C.; Brinkmeyer, H.
Liebigs Ann. Chem. 1976, 1873-1888.
(12) Sun, S.; Desper, J. Tetrahedron 1998, 54, 411-422.
(8) Coleman, R. S.; Mortensen, M. A. Tetrahedron Lett. 2003, 44, 1215-
1219.
(9) Ishikawa, J.; Sakamoto, H.; Nakao, S.; Wada, H. J. Org. Chem. 1999,
64, 1913-1921.
9652 J. Org. Chem., Vol. 71, No. 26, 2006

Analogue of PRODAN: Synthesis of Anthradan
SCHEME 2 Overall Synthesis of the Asymmetric Anthracenes 11-13 Starting from 2,6-Diaminoanthraquinone 1a
a (a) t-BuONO, CuBr2, CH3CN; (b) CsF, DMSO, 110 °C; (c) R ) hexyl, R2NH, DMSO, K2CO3, 70 °C (18% yield, b and c, two steps); (d) (1) NaBH4,
i-PrOH, (2) HCl aq, (3) NaBH4, i-PrOH, (4) HCl aq (53%); (e) CuCN, DMF, reflux 12 h (83%); (f) CH3CH2MgCl, CuCl (cat.), reflux 24 h (77%).
ammonia.14 Using either of these procedures, we successfully
reduced 2,6-diamino-9,10-anthraquinone to 2,6-diamino-
anthracene (2). However, all attempts to selectively modify only
one of the two amines were unsuccessful. An attempt to carry
out a selective Sandmeyer reaction (conversion of amine to
bromide) on only one amine group failed at least in part due to
solubility problems, resulting mainly in dibromide 3, and no
monobromide 4 was isolated.
We noticed that the commercially available 2,6-diamino-
anthraquinone (1) had already been successfully converted to
2,6-dibromoanthraquinone (5) using the Sandmeyer reaction.15
Using the method described for this double Sandmeyer reaction,
an effort to convert only one of the amino groups to a bromide
by using 1 equiv or less of tert-butyl nitrite and cupric bromide
resulted in only a mixture of 5 and the starting 1. Direct
nucleophilic aromatic substitution of 5 with a secondary amine
was not efficient and usually afforded an inseparable mixture.
For example, reaction of 5 with dihexylamine in the presence
of K2CO3 in DMSO at 125-140 °C afforded a mixture
containing less than 5% of the desired 2-bromo-6-dihexylami-
noanthraquinone (8). Other products included 2,6-bisdihexyl-
aminoanthraquinone (7), but the mixture was too difficult to
separate for any practical application. Amination of 5 with a
CuI catalyst and an excess of ammonium hydroxide under
pressure afforded only the starting amine 1.16 These failed
attempts to obtain asymmetrically 2,6-disubstituted anthracenes
and anthraquinones are summarized in Scheme 1.
reported that 5,6-dibromo-1,2-acenaphthenequinone was suc-
cessfully converted to 5,6-difluoro-1,2-acenaphthenequinone by
reaction with CsF in anhydrous DMSO,17 and we felt this
approach might also be useful in the case of the 2,6-dibromo-
anthraquinone system at hand (Scheme 2). With 1.0 equiv of
CsF in DMSO at 110 °C, the result was encouraging in that
about 45% of the starting dibromide (5) was converted to
2-bromo-6-fluoroanthraquinone (10) accompanied by about 10%
of 2,6-difluoroanthraquinone. An increase of the amount of CsF
to 2 equiv, under the same conditions, consumed all the
dibromide starting material but now gave about a 1:2 mixture
of monofluoride and difluoride. We finally found that treatment
of 5 with 1.3 equiv of CsF in anhydrous DMSO at 110 °C gave
about a 2:1 ratio of 10 and 2,6-difluoroanthraquinone as detected
by GC-MS (60% 2-bromo-6-fluoroanthraquinone, 30% 2,6-
difluoroanthraquinone, and the remainder was the starting 5).
No attempt was made to separate this mixture. Instead, the
halogen exchange reaction mixture in DMSO was directly
treated with N,N-dihexylamine and potassium carbonate at 70
°C to give, after chromatography, 2-bromo-6-dihexylamino-
anthraquinone (8) in overall 18% yield for the two-step, one-
pot reaction.
Reduction of the 2-bromo-6-dihexylaminoanthraquinone was
performed in 53% yield using the method of Klanderman
involving successive sodium borohydride reduction and dehy-
dration.13 The product, 2-bromo-6-dihexylaminoanthracene (11),
was treated with cuprous cyanide in DMF to afford 2-cyano-
6-(dihexylamino)anthracene, 12.18 Finally, 6-propionyl-2-(di-
hexylamino)anthracene (Anthradan) 13 was prepared by reaction
of this cyano compound with ethylmagnesium chloride catalyzed
with cuprous chloride followed by hydrolysis of the imine
intermediate.
One option to break the symmetry of the 2,6-disubstituted
anthraquinone involves finding an intermediate that has similar
or less solubility or reactivity than the starting substrate, so as
to avoid preferential reactivity for the monofunctionalized
product. In such a circumstance, at least a statistical product
distribution might be obtained. We assume 2-bromo-6-fluoro-
anthraquinone (10) has solubility comparable to 5 and that the
fluorine could be preferentially replaced by the dialkylamino
functionality via aromatic nucleophilic substitution. It was
Absorption and Emission Properties. The absorption
spectra of 11, 12, and Anthradan 13 in THF are all shown in
Figure 1. The spectra are significantly different from those for
anthroyl derivatives that have no push-pull substitution. The
maximum long-wavelength absorption band of a 2-anthroyl
chromophore is around 375-400 nm, and this band was
(13) Criswell, T. R.; Klanderman, B. H. J. Org. Chem. 1974, 39, 770-
774.
(14) Yanagimoto, T.; Takimiya, K.; Otsubo, T.; Ogura, F. J. Chem. Soc.,
Chem. Commun. 1993, 519-520.
(17) Mallory, F. B.; Mallory, C. W.; Butler, K. E.; Lewis, M. B.; Xia,
A. Q.; Luzik, E. D.; Fredenburgh, L. E.; Ramanjulu, M. M.; Van, Q. N.;
Francl, M. M.; Freed, D. A.; Wray, C. C.; Hann, C.; Nerz-Stormes, M.;
Carroll, P. J.; Chirlian, L. E. J. Am. Chem. Soc. 2000, 122, 4108-4016.
(18) Friedman, L.; Shechter, H. J. Org. Chem. 1961, 26, 2522-2524.
(15) Hodge, P.; Power, G. A.; Rabjohns, M. A. Chem. Commun. 1997,
73-74.
(16) Quick, A. J. J. Am. Chem. Soc. 1920, 42, 1033-1042.
J. Org. Chem, Vol. 71, No. 26, 2006 9653

Lu et al.
TABLE 1. Absorption and Emission of PRODAN, 12, and 13 in
Solvents with a Range of ∆f (Orientational Polarizability)a
fluorophore 12
PRODANb
solvent
hexane
13
∆f
λabs
λem
390c
λabs
λem
λabs
λem
0.0001 341c
445 483 439 483
toluene
0.0134 349d 416d 451 507 447 507
chloroform
THF
acetonitrile
DMF
DMSO
1-propanol
ethanol
methanol
1,2-propanediol 0.2708 370
ethylene glycol 0.2745 375
0.1482 357
0.2096 348c
0.3053 350
0.2745 355
440
431c
462
461
459 525 458 536
455 533 450 529
460 551 455 557
464 557 458 557
0.2640 358d 464d 467 566 460 569
0.2493 360d 480d 456 537 456 585
0.2886 360
0.3084 362
496
505
510
515
454 545 452 601
457 546 456 604
462 546 462 598
464 554 464 617
a The concentration for absorption measurement was 10-5 M. The
concentration for emission measurement was 10-7 M, and the excitation
wavelength was at the absorption λmax.
b Unless otherwise specified, all λabs
and λem data for PRODAN are from ref 2. c Ref 23. d Ref 3d (in i-PrOH).
FIGURE 1. Comparison of the absorption spectra of 11, 12, and 13
in THF. All of these three anthracene derivatives have a common
2-dihexylamine donor but differ in the 6-substituent (Br, CN, and
COCH2CH3, respectively).
assigned as the longitudinally polarized band with vibrational
structure.19 Although the absorption of molecules 12 and 13
exhibits the same absorption with vibrational detail at the same
wavelength, the most notable absorption feature of 12 and 13
is the appearance of a new broad band around 440-460 nm
that is assigned as an intramolecular charge-transfer band. The
longitudinally polarized band with vibrational features in the
anthroyl derivative is diminished substantially. Bromide 11,
which is the precursor of 12 and has the same 2,6-substitution
pattern, has much less absorption intensity for the intramolecular
charge-transfer band due to the lack of a strong electron-
withdrawing group. The maximum charge-transfer molar extinc-
tion coefficients of 12 and 13 in THF are 1.03 × 104 and 1.21
× 104 L cm-1 mol-1, respectively.
The absorption and emission maxima of PRODAN, 12, and
Anthradan 13 in a variety of solvents spanning a wide range of
polarity including nonpolar, polar, and polar aprotic solvents
are found in Table 1. Examination of structure 13 reveals that
it is quite similar to PRODAN, with the exception of the
extension of the conjugation from naphthalene in PRODAN to
anthracene in 13 and the different dialkyl groups on the amine
donor. The dihexylamine group was employed with Anthradan
to enhance solubility in ordinary organic solvents and will not
significantly influence the absorption and emissions relative to
the dimethylamino group as found in PRODAN. Similarly,
structure 12 is an anthracene analogue of the classic 4-di-
methylaminobenzonitrile (DMABN) which was the first ex-
ample of the twisted intramolecular charge-transfer (TICT)
state;20 thus, we anticipated that the emission of 12 and 13 might
strongly depend on solvent polarity. The influence of selected
solvents on the absorption of 12 and 13 is shown in Figures 2
and 3, respectively. The absorptions of both 12 and 13 were
found to not be very sensitive to changes in the polarity of the
solvent, and only moderate absorption solvatochromism was
FIGURE 2. Normalized absorption of 12 in hexane (1), toluene (2),
chloroform (3), DMSO (4), methanol (5), and ethylene glycol (6).
observed. In the low-polarity solvents hexane and toluene, a
charge-transfer band with some structure features was observed,
whereas in all other higher-polarity solvents, only a single broad
charge-transfer band was observed. The absorption maximum
of 12 changes from 445 (hexane) to 467 nm (DMSO). Polar
protic solvents have no specific effect on the absorption of 12.
The absorption maximum of 13 ranges from 439 (hexane) to
464 nm (ethylene glycol). Anthradan (13) and PRODAN are
quite similar in their response to solvents, except Anthradan
absorbs at approximately 100-nm longer wavelengths.
The fluorescence quantum yields of 12 and 13 in toluene,
ethanol, and acetone are tabulated in Table 2. These solvents
were selected to represent both a range of polarity and the ability
to form hydrogen bonds. The quantum yields in these three
solvents are comparable and show no obvious trends.
The influence of selected solvents on the emission of 12 and
13 is shown in Figures 4 and 5, respectively. In contrast to the
absorption, the solvent polarity has a dramatic effect on the
emission wavelength. The emission maximum of 12 ranges from
483 (hexane) to 566 nm (DMSO), and the emission maximum
(19) Tamaki, T. Bull. Chem. Soc. Jpn. 1978, 51, 2817-2821.
(20) Rotkiewicz, K.; Grellmann, K. H.; Grabowski, Z. R. Chem. Phys.
Lett. 1973, 19, 315-318. For a review, see: Rettig, W. In Top. Curr. Chem.;
Mattay, J., Ed.; Springer-Verlag: New York, 1994; Vol. 169, pp 253-299.
9654 J. Org. Chem., Vol. 71, No. 26, 2006

Analogue of PRODAN: Synthesis of Anthradan
Stokes shift and orientational polarizability shown in Figure 6A
reveals only a dipolar solvent effect here. On the other hand, in
Figure 6B, all the protic solvents are located on the top and
right side of the plot indicative of a specific solvent effect most
likely due to hydrogen bonding.
Fluorophore 13 is structurally related to PRODAN, and
comparisons of the Stokes shift vs ∆f in various solvents are
shown in Figure 6B. Although the slope for PRODAN is slightly
higher, it is clear that the sensitivity to solvent polarity is quite
similar for PRODAN and Anthradan (13) which both contain
the propionyl acceptor group. Fluorophore 12 with a nitrile
acceptor group has no specific solvent effect and is slightly less
sensitive to the solvent polarity compared with both PRODAN
and Anthradan (13).
Single-Molecule Properties. A strong test of the quality of
a fluorophore is its ability to be imaged at the single-molecule
level, which requires bright fluorescence, weak coupling with
dark states, and photostability. Our single-molecule analysis
reveals that single copies of Anthradan molecules in poly(methyl
methacrylate) (PMMA) are visible in an epifluorescence image
of a typical sample, as shown in Figure 7. However, the
photostability of these molecules needs further development.
To characterize a single-molecule emitter using one simple
parameter, we recorded the distribution of the number of photons
emitted from single fluorophores before photobleaching. After
recording single-molecule distributions, which were exponential
in shape as expected, a fit to the exponential yields an average
number of detected photons, Ntot,d ∼ 20 000 per molecule. The
photon collection efficiency21 of our setup is D ) 0.10.
Correcting the Ntot,d value for the collection efficiency yields a
value for the total number of photons emitted, Ntot,e ∼ 2.0 ×
105 photons emitted per molecule. This value is an order of
magnitude lower than that for DCDHF-6 or R6G (2.4 × 106
and 1.9 × 106 photons emitted per molecule, respectively),22
both of which are good single-molecule fluorophores. Neverthe-
less, we demonstrated that single copies of Anthradan are
observable, which gives us hope in being able to further
synthetically develop this fluorophore for enhanced photophysi-
cal properties required for future single-biomolecule imaging.
FIGURE 3. Normalized absorption of 13 in hexane (1), toluene (2),
chloroform (3), DMSO (4), methanol (5), and ethylene glycol (6).
of 13 ranges from 483 (hexane) to 617 nm (ethylene glycol).
Examination of Table 1 shows that the emission wavelengths
for both 12 and 13 are quite comparable to each other in
nonpolar and polar aprotic solvents. One of the distinct
differences between these two fluorophores is their response to
polar protic solvents. In polar protic solvents, emission of 13
has moved to a longer wavelength relative to 12. The Stokes
shift of 13 in a series of alcohols (1-propanol, methanol, 1,2-
propanediol, and ethylene glycol) is approximately 40 nm greater
than that of 12.
To better explore the solvatochromic behavior of these
fluorophores, we used a standard dipolar model for solvent
stabilization. When a fluorophore is excited from the ground
to excited state, the solvent dipole can reorient around the
excited-state dipole moment. This solvent relaxation can lower
the energy of the excited state. This effect becomes larger as
the solvent polarity is increased, thus the emission will be red
shifted. More precisely, this solvent effect is determined by the
index of refraction and the relative dielectric constant of the
solvent, described by the Lippert equation in which the
fluorophore is considered to be a dipole located in a cavity with
a radius of a in a continuous solvent-dipole environment (eq
1).1b
Conclusions
New 2,6-dipolar anthracene fluorophores have been synthe-
sized which have bathochromically shifted absorption and
emission relative to their well-known naphthalene analogues.
These new fluorophores retain the same photophysical properties
that have made the naphthalene compounds so useful, namely
a pronounced environmental sensitivity for the emission. The
anthracene compound Anthradan has been successfully imaged
at the single-molecule level, but the number of photons emitted
and detected is low relative to other high-performance single-
molecule chromophores, perhaps reflecting instability problems
intrinsic to chromophores containing larger acene substructures.
(µE - µG)2
2
νjA - νjF ) ∆f
hc
ꢀ - 1
2ꢀ + 1
n2 - 1
+ c and ∆f )
-
a3
2n2 + 1
(1)
In the equation, νjA and νjF are the wavenumbers of the
absorption and emission, n is the refractive index, ꢀ is the
relative low-frequency dielectric constant of the solvent, h is
Planck’s constant, c is the speed of light, and ∆f is called the
orientation polarizability. The linear correlation between the
Stokes shifts and ∆f predicted by the Lippert equation does not
hold for a specific interaction between the fluorophore and
solvent molecules such as hydrogen bonding.
Experimental Section
Sample Preparation. Solutions of fluorophores were prepared
in toluene, and bulk absorption and emission spectra were taken
on conventional spectrometers. Quantum yields were measured
(21) Moerner, W. E.; Fromm, D. P. ReV. Sci. Instrum. 2003, 74, 3597-
3619.
(22) Soper, S. A.; Nutter, H. L.; Keller, R. A.; Davis, L. M.; Shera, E.
B. Photochem. Photobiol. 1993, 57, 972-977.
Plots of the Stokes shift as a function of the solvent
orientational polarizability of 12 and 13 are shown in Figure
6A and B, respectively. The linear correlation between the
J. Org. Chem, Vol. 71, No. 26, 2006 9655

Lu et al.
TABLE 2. Fluorescence Quantum Yields (QY), λabs,max, and λem,max of 12 and 13 in Selected Solvents
PRODAN
12
Anthradan (13)
solvent
QY
λabs,max
λem,max
QY
λabs,max
λem,max
QY
λabs,max
λem,max
toluene
ethanol
acetone
349b
360c
350c
416b
496c
452c
0.51
0.51
0.44
452
454
458
509
545
551
0.58
0.41
0.51
446
452
452
507
601
557
0.71a ( 0.03
a Ref 24. b Ref 3d. c Ref 2.
FIGURE 6. Lippert plots. Circles denote aprotic solvents, and triangles
denote protic solvents. (A) Lippert plot for fluorophore 12. The slope
of the fit to all solvents is 5223 cm-1 (R2 ) 0.9015). (B) Lippert plots
for Anthradan 13 (closed symbols) and PRODAN (open symbols). For
Anthradan, the slope for the fit to aprotic solvents is 5921 cm-1 (R2 )
0.9233) and for protic solvents is 10 656 cm-1 (R2 ) 0.6845). The
Lippert plot for PRODAN yields a slope for the fit to aprotic solvents
of 10 162 cm-1 (R2 ) 0.9933) and that for protic solvents of 12 803
cm-1 (R2 ) 0.5627).
FIGURE 4. Normalized emissions of 12 in hexane (1), toluene (2),
chloroform (3), DMSO (4), methanol (5), and ethylene glycol (6).
using a continuous-wave 488 nm laser; the intensity at the sample
was approximately 0.5 kW/cm2. The emission was collected through
a 100×, 1.4 N.A. microscope objective, filtered to remove scattered
excitation light, and imaged onto a back-illuminated frame-transfer
Si CCD camera with an integration time of 100 ms/frame.
2-Bromo-6-dihexylaminoanthraquinone (8). A mixture of 2,6-
dibromo-9,10-anthraquinone (3.66 g, 10 mmol), anhydrous CsF
(1.62 g, 11 mmol), and 200 mL of anhydrous DMSO was heated
under nitrogen at 140-150 °C for 5 h with magnetic stirring. The
black reaction mixture was then cooled to 70 °C. Then, dihexyl-
amine (3.71 g, 20 mmol) and potassium carbonate (2.76 g, 20
mmol) were added to the flask, and the mixture was stirred at 70
°C for 36 h. The resulting red-brown mixture was poured into
1000 mL of water and extracted with dichloromethane (200 mL ×
3). The combined organic layers were washed with water and brine
and dried over anhydrous MgSO4. After removal of the dichlo-
romethane, the residue was separated by flash chromatography with
hexane and a hexane/dichloromethane (4:1) mixture as eluent. The
2-bromo-6-dihexylaminoanthraquinone was obtained as a red solid
FIGURE 5. Normalized emissions of 13 in hexane (1), toluene (2),
chloroform (3), DMSO (4), methanol (5), and ethylene glycol (6).
1
(0.84 g, 18% yield). Mp 114.2-115.4 °C (CH2Cl2/hexane). H
NMR (400 MHz, CDCl3) δ 8.39 (d, J ) 2.0 Hz, 1H), 8.12 (d, J )
9.0 Hz, 1H), 8.09 (d, J ) 8.2 Hz, 1H), 7.81 (dd, J ) 8.2, 2.0 Hz,
1H), 7.39 (d, J ) 2.7 Hz, 1H), 6.88 (dd, J ) 9.0, 2.7 Hz, 1H), 3.43
(t, J ) 7.7 Hz, 4H), 1.70-1.61(m, 4H), 1.42-1.31 (m, 12H), 0.93
against DCDHF-6 in toluene and were corrected for differences in
optical density and solvent refractive index.1b
Single-molecule samples were prepared by spin-casting 1% (by
mass) solutions of PMMA in toluene, doped with nanomolar
fluorophore concentrations, onto clean glass coverslips. After
drying, these samples were studied using an inverted microscope
in an epifluorescence configuration.21 Samples were illuminated
(23) Bunker, C. E.; Bowen, T. L.; Sun, Y. P. Photochem. Photobiol.
1993, 58, 499-505.
(24) Davis, B. N.; Abelt, C. J. J. Phys. Chem. A 2005, 109, 1295-1298.
9656 J. Org. Chem., Vol. 71, No. 26, 2006

Analogue of PRODAN: Synthesis of Anthradan
2-Cyano-2-(dihexylamino)anthracene (12). A mixture of 2-bro-
mo-6-dihexylaminoanthracene (220 mg, 0.5 mmol) and cuprous
cyanide (269 mg, 3 mmol) in 6 mL of anhydrous DMF was stirred
and refluxed under a nitrogen atmosphere for 12 h. The reaction
mixture was then cooled and treated with 1 mL of ethylenediamine
and 10 mL of water at 50 °C for a half hour. Water (50 mL) was
added, and the resulting mixture was extracted with dichloromethane
(60 mL × 2). The combined organic layer was washed with water
(50 mL × 2), dried (MgSO4), concentrated, and loaded on silica
gel. Purification by silica gel flash chromatography with hexane/
dichloromethane elution afforded the product as a yellow solid (160
1
mg, 83% yield). Mp 81.9-82.5 °C (hexane). H NMR (CDCl3) δ
8.29 (d, J ) 1.5 Hz, 1H), 8.27 (s, 1H), 8.09 (s, 1H), 7.91-7.87
(m, 2H), 7.4 (dd, J ) 8.8, 1.5 Hz, 1H), 7.27 (dd, J ) 9.5, 2.5 Hz,
1H), 6.87 (d, J ) 2.5 Hz, 1H), 3.45 (t, J ) 7.7 Hz, 4H), 1.75-1.65
(m, 4H), 1.47-1.33 (m, 12H), 0.95 (t, J ) 7.0 Hz, 3H). 13C NMR
(CDCl3) δ 146.7, 136.0, 135.8, 132.4, 129.7, 128.5, 127.5, 127.1,
126.9, 124.3, 122.2, 120.2, 118.6, 105.8, 102.0, 51.2, 31.7, 27.5,
26.9, 22.7, 14.1. UV-vis (THF) λ1 ) 253 nm, ꢀ1 ) 5.20 × 104 L
cm-1 mol-1, λ2 ) 318 nm, ꢀ2 ) 6.38 × 104 L cm-1 mol-1, λ3 )
456 nm, ꢀ3 ) 1.03 × 104 L cm-1 mol-1. IR (cm-1) 2926, 2215,
1626. Anal. Calcd for C27H34N2: C, 83.89; H, 8.87; N, 7.25.
Found: C, 83.85; H, 9.15; N, 7.61. HRMS (m/z): [M + H] calcd
for C27H35BrN2, 387.2800; found, 387.2797.
6-Propionyl-2-(dihexylamino)anthracene (Anthradan, 13). A
mixture of 2-cyano-6-dihexylaminoanthracene (157 mg, 0.406
mmol) was dissolved in anhydrous THF (10 mL). Ethyl magnesium
chloride (0.18 mL, 25% weight in THF, 0.49 mmol) and a trace of
cuprous chloride were added, and the mixture was stirred and heated
to reflux under a nitrogen atmosphere for 24 h. The reaction flask
was covered by aluminum foil to avoid possible photoreactions.
The reaction mixture was then cooled, and hydrochloric acid (5
mL, 1:1) was added. The mixture was refluxed for another 12 h.
Then the reaction was cooled, and the solution was neutralized with
NaHCO3. Water (100 mL) was added, and the resulting mixture
was extracted with dichloromethane (60 mL × 3). The combined
organic layers were washed with water (50 mL × 2), dried
(MgSO4), concentrated, and loaded on silica gel. Purification by
silica gel flash chromatography with hexane/dichloromethane (up
to 1:2) as eluent afforded the product as yellow solid (130 mg,
77% yield). Mp 66.2-66.9 °C (hexane). 1H NMR (CDCl3) δ 8.57
(s, 1H), 8.36 (s, 1H), 8.09 (s, 1H), 7.94 (dd, J ) 9.0, 1.6 Hz, 1H),
7.91-7.86 (m, 2H), 7.27 (dd, J ) 9.4, 2.5 Hz, 1H), 6.89 (d, J )
2.5 Hz, 1H), 3.44 (t, J ) 7.7 Hz, 4H), 3.16 (q, J ) 7.3 Hz, 2H),
1.75-1.65 (m, 4H), 1.46-1.36 (m, 12H), 1.33 (t, J ) 7.3 Hz, 3H),
0.95 (t, J ) 6.9 Hz, 3H). 13C NMR (CDCl3) δ 200.6, 146.4, 135.8,
133.7, 131.7, 131.4, 129.6, 128.7, 127.7, 126.6, 122.7, 121.9, 118.0,
102.3, 51.2, 31.8, 31.5, 27.5, 26.9, 22.7, 14.1, 8.7. UV-vis (THF)
λ1 ) 257 nm, ꢀ1 ) 4.46 × 104 L cm-1 mol-1, λ2 ) 323 nm, ꢀ2 )
5.31 × 104 L cm-1 mol-1, λ3 ) 449 nm, ꢀ3 ) 1.21 × 104 L cm-1
mol-1. IR (cm-1) 2929, 1682, 1622. Anal. Calcd for C29H39NO:
C, 83.40; H, 9.41; N, 3.35. Found: C, 83.11; H, 9.67; N, 3.59.
HRMS (m/z): [M + H] calcd for C29H40NO, 418.3110; found,
418.3110.
FIGURE 7. Epifluorescence images of single copies of Anthradan in
a PMMA film imaged with 488 nm excitation.
(t, J ) 7.0 Hz, 6H). 13C NMR (100 Hz, CDCl3) δ 183.6, 179.8,
152.3, 135.8, 135.7, 134.9, 132.3, 130.1, 129.9, 129.5, 128.6, 120.9,
115.6, 108.3, 51.2, 31.6, 27.2, 26.7, 22.6, 14.0. Anal. Calcd for
C26H32BrNO2: C, 66.38; H, 6.86; N, 2.98. Found: C, 66.42; H,
7.09; N, 3.27. HRMS (m/z): [M + Na] calcd for C26H32BrNO2Na,
492.1514; found, 492.1517.
2-Bromo-6-dihexylaminoanthracene (11). A suspension of
2-bromo-6-dihexylaminoanthraquinone (0.84 g, 1.79 mmol) and
NaBH4 (3.0 g, 79.3 mmol) in i-PrOH (100 mL) was stirred for 12
h at room temperature and then heated under reflux for 10 h. After
cooling to room temperature, the solvent was removed by rotary
evaporation and the residue was treated with water. The mixture
was neutralized with 6 M HCl and heated under reflux for 4 h.
After cooling to room temperature, the mixture was extracted with
dichloromethane and the organic layers were dried over MgSO4.
After the solvent was removed, the residue was added to i-PrOH
(50 mL) and treated with another portion of NaBH4 (2.0 g, 52.2
mmol) with subsequent refluxing for 12 h. After cooling, i-PrOH
was removed and aqueous 3 M HCl (10 mL) was added carefully
until bubbling ceased. The mixture was refluxed for 2 h under
nitrogen protection. The mixture was cooled down to room
temperature and extracted with dichloromethane. The combined
organic layers were washed with water and brine and dried over
anhydrous MgSO4. The solvent was removed, and the residue was
purified by flash chromatography with hexane as eluent to afford
2-bromo-6-dihexylaminoanthracene as a glassy yellow solid (420
mg, 53% yield). 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 8.04
(s, 1H), 8.02 (d, J ) 2.0 Hz, 1H), 7.81 (d, J ) 9.4 Hz, 1H), 7.70
(d, J ) 9.1 Hz, 1H), 7.37 (dd, J ) 9.1, 2.0 Hz, 1H), 7.18 (dd, J )
9.4, 2.4 Hz, 1H), 6.83 (d, J ) 2.4 Hz), 3.38 (t, J ) 7.7 Hz, 4H),
1.69-1.61 (m, 4H), 1.40-1.31 (m, 12H), 0.92 (t, J ) 7.0 Hz, 6H).
13C NMR (100 Hz, CDCl3) δ 145.7, 134.1, 130.6, 129.91, 129.87,
129.14, 129.11, 128.5, 126.8, 124.9, 122.4, 118.5, 116.7, 102.5,
51.2, 31.8, 29.7, 27.5, 26.9, 22.7, 14.1. Anal. Calcd for C26H34-
BrN: C, 70.90; H, 7.78; N, 3.18. Found: C, 70.71; H, 7.84; N,
3.47. HRMS (m/z): [M + H] calcd for C26H35BrN, 440.1953; found,
440.1955.
Acknowledgment. This work was supported in part by the
Department of Energy, Grant No. DE FG02-04ER63777, and
by the National Institutes of Health, Grant No. 5P20-HG003638-
02.
Supporting Information Available: Hard copies of H1 and C13
NMR spectra for compounds 8 and 11-13 are provided. This
material is available free of charge via the Internet at http://
pubs.acs.org.
JO0616660
J. Org. Chem, Vol. 71, No. 26, 2006 9657