An organic white light-emitting fluorophore

Article abstract of DOI:10.1021/ja0632207

The synthesis of new benzo[a]- and [b]xanthene dye frameworks is described. A unique benzo[a]xanthene, seminaphtho[a]fluorone (SNAFR-1), is studied in a variety of media. The optimization of solution parameters and excitation wavelengths allows SNAFR-1 to display red, green, and blue emission bands of approximately equal intensities and also to produce white light. Ratiometric red (anion) and green (neutral) emissions are observed upon varying solution pH. A pH-independent violet-blue emission band is due to the addition of nucleophiles to the benzylic carbon of SNAFR-1.

Full text of DOI:10.1021/ja0632207

Published on Web 10/10/2006
An Organic White Light-Emitting Fluorophore
Youjun Yang, Mark Lowry,* Corin M. Schowalter, Sayo O. Fakayode,
Jorge O. Escobedo, Xiangyang Xu, Huating Zhang, Timothy J. Jensen,
Frank R. Fronczek, Isiah M. Warner,* and Robert M. Strongin*
Contribution from the Department of Chemistry, Louisiana State UniVersity,
Baton Rouge, Louisiana 70803
Received May 9, 2006; E-mail: rstrong@lsu.edu
Abstract: The synthesis of new benzo[a]- and [b]xanthene dye frameworks is described. A unique benzo-
[a]xanthene, seminaphtho[a]fluorone (SNAFR-1), is studied in a variety of media. The optimization of solution
parameters and excitation wavelengths allows SNAFR-1 to display red, green, and blue emission bands
of approximately equal intensities and also to produce white light. Ratiometric red (anion) and green (neutral)
emissions are observed upon varying solution pH. A pH-independent violet-blue emission band is due to
the addition of nucleophiles to the benzylic carbon of SNAFR-1.
in excitation intensity.14 Ratiometric probes have been shown
to be useful for intracellular pH,15 viscosity,13a,16 and Ca2+
Introduction
Terenin et al. reported the first example of dual fluorescence
resulting from an intermolecular excited-state proton transfer
(ESPT) in 1947.1a In 1950, Forster’s first paper on ESPT was
published.1b Since then, several compounds, such as 4-(N,N-
dimethylamino)-benzonitrile (DMABN) and its analogues,2
biaryls,3 benzo[c]xanthenes,4 3-hydroxyflavones,5 hydroxy-
camptothecin,6 6-hydroxyquinoline-N-oxides,7 aromatic dicar-
boximides,8 carotenoids,9 1,3-diphenyl-1H-pyrazolo[3,4-b]-
quinoline,10 etc., have been reported to display dual signaling
excitation and emission properties based on intermolecular or
intramolecular ESPT,11 twisted intramolecular charge transfer
(TICT),12 tautomerization,4 and other mechanisms.13
measurement.17 Additionally, they are important in applications
such as flow cytometry, confocal microscopy, emission ratio
imaging, as well as many others.4a,18 Benzo[c]xanthenes (Figure
1) are of interest as dual fluorescence ratiometric indicators
because of (i) their well-resolved emission bands, (ii) rela-
tively long wavelength absorptions and emissions, (iii) near
neutral pKa values, and (iv) clear isosbestic and isoemissive
points.4a,19
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Dual ratiometric fluorescence signaling reduces errors due
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J. AM. CHEM. SOC. 2006, 128, 14081-14092
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A R T I C L E S
Yang et al.
Traditional methods of molecular white light generation typically
involve mixing different primary colors from different emitting
materials, such as red, green, and blue fluorophores.24 Alterna-
tive and single component recent examples include (i) white
light photoluminescence from nanocrystals,25 (ii) electrolumi-
nescence from a fullerene adduct,26 (iii) electroluminescence
from a π-conjugated aromatic enyne,27 (iv) electroluminescence
from platinum-functionalized random copolymer,28 and (v)
photoluminescence from microporous metal phosphates and
silicates.29
Fluorophores with tunable emissive properties are of current
interest for multiplexing applications. In addition to serving as
a simple white light-emitting fluorophore, SNAFR-1 can be
excited anywhere within the UV to the deep red spectral regions.
It exhibits a unique emission spectrum corresponding to each
excitation wavelength. SNAFR-1 is pH sensitive, soluble in cell
culture media, stains cells, and exhibits no cytotoxicity in studies
carried out to date.
Figure 1. The xanthene dye framework and the three types of benzoxan-
thenes. Benzoxanthene types are each distinguished via the orientation of
their naphthyl moieties.
The regioisomers of benzo[c]xanthene, namely benzo[a]-
xanthene and benzo[b]xanthene, were essentially overlooked
until Wolfbeis et al. investigated their properties using semiem-
pirical molecular orbital calculations. They were predicted to
absorb and emit at longer wavelengths compared to their type
[c] analogues. Apparently because of prior limitations in
xanthene synthesis methodology, they had not been prepared
to date.19
We have recently reported a novel synthesis of xanthene
dyes.20 It involves the initial formation of methylated car-
binol intermediates, followed by demethylation and con-
comitant condensation (Scheme 1). Current studies embody an
extension of this prior work to access novel benzannulated
derivatives.
Herein, we report the synthesis of type [a] and [b] benzox-
anthenes. Importantly, we find that a new seminaphtho[a]-
fluorone (SNAFR-1) can display three emission maxima of
approximately equal intensities, in the blue, green, and red
spectral regions, respectively. When excited at ca. 300 nm, it
emits near white light, which resembles the white light from
an incandescent light bulb. White light is to date highly sought-
after owing to potential applications in (i) light emitting diodes
(LEDs),21 replacements for current illumination devices such
as incandescent bulbs and fluorescent lamps, (ii) flat panel
displays (FPDs),22 as the next generation display devices after
liquid crystal displays (LCDs), and (iii) electronic paper dis-
plays (E-PADs),23 as an electronic analogue of paper, etc.
Results and Discussion
Benzo[a]- and [b]xanthene Dyes Synthesis. Xanthene dyes
are typically synthesized via the acid-catalyzed condensation
between resorcinol and various analytes, for example, phthalic
anhydride, acid chloride, ester, or aldehyde.30 Upon replacing
resorcinol with 1,6-dihyxdroxynaphthalene, under classical acid-
catalyzed thermal conditions, known benzo[c]xanthene is formed.
It has been shown by others that when 1,6-dimethoxynaph-
thalene (1) is treated with n-BuLi and quenched with MeI, 2
and 3 are obtained in yields of 84% and 12%, respectively.31
When a solution of lithiated 1 is treated with methyl benzoate,
we obtain 4 and 5 in isolated yields of 71% and 12%,
respectively. When phthalic anhydride is used instead of
methylbenzoate, 6 is obtained in 55% yield. Upon treating
lithiated 1,6-dimethoxynaphthalene with 2,4-dimethoxyben-
zophenone, 7 and 8 are isolated in 11% and 71% yields,
respectively (Scheme 2). Additionally, trace amounts of 9 are
obtained. This is presumably because of trace amounts of
2-hydroxy-4-methoxybenzophenone in the reaction mixture from
incomplete methylation of 2,4-dihydroxybenzophenone. Single-
crystal X-ray structure analysis confirms the assigned structures
of 4-9 (Figure 2).
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An Organic White Light-Emitting Fluorophore
A R T I C L E S
Scheme 1. A Simple and Efficient Synthesis of Xanthene Dyes
Scheme 2. Synthesis of Precursors of Type [a]- and [b]Benzoxanthenes
When BBr3 is used to demethylate 4, compound 10 is
obtained in 61% yield. When 7 is demethylated, 12 is obtained
quantitatively. Treatment of 12 using 20 more equiv of BBr3
affords 13 in 56% yield. When 8 is demethylated, 14 is obtained
in a yield of 15% (Scheme 3). Structure assignments of 10 and
13 are confirmed by single-crystal X-ray structural analysis
(Figure 3).
UV-Visible Absorption Properties of SNAFR-1 as a
Function of pH in 99.75% Buffer Solution. Absorption spectra
of SNAFR-1 as a function of varying pH are shown (Figure
4A). SNAFR-1 is dissolved in 50 mM phosphate buffer with
0.25% DMSO. As the pH increases, the absorption band
centered at 550 nm increases. This band is assigned to the
anionic form (A, Scheme 4). At the same time, the absorption
at ca. 460 nm decreases. The 460 nm band is assigned to the
neutral form (N). Four isosbestic points at 484, 394, 327, and
304 nm are observed. The 484 nm isosbestic point is near the
488 nm Ar ion laser line. These latter results show that
ratiometric experiments using SNAFR-1 as a probe may be
performed with common, commercially available filter sets (vide
infra).
SNAFR-1 thus also exhibits dual emission bands that are pH-
sensitive. Upon increasing the solution pH, the intensity of the
red emission band (620 nm), corresponding to the anionic form
(A) increases, while that of the green emission band (540 nm),
assigned to the neutral (N) form decreases (Figure 4E-H). The
two emission bands are well-separated and show clear iso-
emissive points at ca. 600 nm when excited at 325, 488, and
514 nm (Figure 4E-G). When excited at 543 nm, only the tail
of the emission from the neutral form plus the emission of the
anion is observed, and a clear isoemissive point is present at
560 nm (Figure 4H). Excitation at longer wavelengths would
further reduce the emission from the neutral form and eventually
lead to red emission from the anion only with no isoemissive
point. Most organic fluorophores are limited by their narrow
excitation which hinders their use in multiplexing with other
fluorophores. The absorption and excitation spectra of SNAFR-1
demonstrate that it can be excited from 260 nm up to 600 nm
in buffer including various common laser lines, for example,
HeCd at 325 nm, Ar ion at 488 and 514 nm, and HeNe at 543
nm (Figure 4A-D). This is further evidence that SNAFR-1
would be compatible with commercially available filter sets used
in various spectroscopic instruments and fluorescence micro-
scopes (vide infra). The pKa values for SNAFR-1 based on
absorption and emission data are summarized in Table 1a. The
Fluorescence Properties of SNAFR-1 as a Function of pH
in 99.75% Buffer Solution. Benzoxanthenes are known to
afford dual emission bands whose intensities are pH dependent.
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Yang et al.
Figure 2. Ortep drawings and numbering schemes of compounds 4-9.
Figure 3. Ortep drawings and numbering schemes of compounds 10 (with two acetone molecules) and 13.
Scheme 3. Synthesis of Type [a]- and [b]Benzoxanthenes
pKa values calculated based on emission spectra using different
excitation wavelengths vary from 8.31 to 8.38 with an average
pKa-em of 8.34 ( 0.02. The pKa values calculated from
absorption spectra based on different isosbestic points vary from
8.47 to 8.68, with an average pKa-abs of 8.53 ( 0.06. The
mismatch of the pKa-abs values and pKa-em values suggests the
presence of excited-state proton transfer (ESPT). Upon excita-
tion, the acidity of SNAFR-1 increases, as measured in
phosphate buffer containing 0.25% DMSO. The pK/a values for
SNAFR-1 estimated using the Forster equation are summarized
in Table 1b. When different methods were applied to calculate
the optical frequency of the light needed to excite the molecule
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An Organic White Light-Emitting Fluorophore
A R T I C L E S
Figure 4. Spectral properties of 30 µM SNAFR-1 in 50 mM phosphate buffer at various pH values with 0.25% DMSO. The trend of spectral changes is
indicated by arrows as the pH increases from 7 to 10. A ) anionic form; N ) neutral form. (A) Absorption spectra is shown; the / indicates the position
of isosbestic points. (B-D) Excitation spectra is shown with emission monitored at 400, 540, and 620 nm, respectively. (E-H) Emission spectra excited at
325, 488, 514, and 543 nm, respectively, corresponding to common laser lines is shown. The legends for panels A-H are shown in panel H. All fluorescence
spectra are normalized to the maximum of each data set.
Scheme 4. Acid-Base Equilibria of SNAFR-1a
) 390 nm), green (λem ) 540 nm), and red (λem ) 620 nm)
spectral regions. The violet-blue emission is of relatively low
intensity compared to the emission from the neutral and anionic
forms. However, the intensity of the blue emission is increased
in organic solvents (vide infra).
Spectral Properties of SNAFR-1 in Organic Solvents.
SNAFR-1 is fluorescent in organic solvents such as MeOH and
DMSO. It exhibits dual emission with two emission bands
located at 390 and 560 nm in DMSO and blue shifted to 385
and 550 nm in MeOH. Excitation-emission matrixes (EEMs)
of SNAFR-1 in MeOH and DMSO are shown in Figure 5.
Spectral properties of SNAFR-1 in DMSO and MeOH are
summarized in Table 2. Violet-blue emission is observed to
increase in these organic solvents compared to buffer. The green
emission is attributed to the neutral form. As expected, the
emission corresponding to the anionic form is diminished in
a N ) neutral; A ) anionic.
both solvents.
from its ground state to its lowest excited state, the pK/a value
was found to vary from 3.3 to 6.3. It is interesting to note that
When phosphate buffer32 (0.25% final volume) is added to
the DMSO solution of SNAFR-1, deep red emission attributed
when the 0-0 excitation method is used, ν˜anion - ν˜neutral of 1813
to the anionic form appears with a corresponding decrease in
cm-1 is obtained, which is in close agreement with 1700 cm-1
calculated for C.SNAFL-1.4a The pK/a is calculated to be 4.54
green emission attributed to the neutral form. Most interestingly,
three emissions of nearly equal intensity in the violet-blue,
or 4.73 at room temperature, when pKa-em and pKa-abs are used,
green, and red spectral region appear when excited in the UV
respectively. These results demonstrate that the acidity of
SNAFR-1 increases in the excited state.
SNAFR-1 Is a Single Component Red-Green-Blue
(RGB) Fluorophore. Importantly, a third emission band is
region, when ca. neutral buffer solutions are added (Figure 6).
As a result, the emission is nearly white. SNAFR-1 can be
excited over a 400 nm spectral window anywhere from ca. 260
present at 390 nm when SNAFR-1 is excited in the UV region
(Figure 4B,E). Thus, SNAFR-1 emits in the violet-blue (λem
(32) Adding 0.25% distilled water instead of 0.25% phosphate buffer does not
lead to any significant change of spectral shape.
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A R T I C L E S
Yang et al.
Table 1a. The pKa Values for SNAFR-1 Based on Absorption and
Emission Dataa
method
λex
λisosbestic
λ1/
λ2
Rmax
Rmin
pKa
em
em
em
em
em
em
em
em
em
abs
abs
abs
abs
abs
abs
abs
abs
abs
abs
abs
325
325
325
488
488
488
514
514
514
627/597
575/597
541/597
630/600
575/600
542/600
623/595
575/595
539/595
543/484
511/484
469/484
380/394
344/394
380/327
344/327
312/327
312/304
296/304
265/304
1.33
1.42
2.31
1.16
1.47
2.47
1.44
1.33
2.16
3.76
2.04
0.48
1.41
2.73
0.52
0.99
1.17
0.91
1.38
2.79
0.54
0.35
0.06
0.51
0.32
0.05
0.57
0.36
0.02
0.88
1.23
0.37
1.14
1.90
0.40
0.67
0.99
0.75
1.13
2.07
8.38
8.32
8.32
8.35
8.36
8.33
8.34
8.32
8.31
8.52
8.54
8.47
8.68
8.58
8.51
8.51
8.54
8.48
8.54
8.48
484
484
484
394
394
327
327
327
304
304
304
Figure 5. Excitation-emission matrixes (EEM) of 30 µM SNAFR-1 in
DMSO (left) and MeOH (right).
Table 2. A Summary of Spectral Properties of SNAFR-1 in DMSO
and MeOH
DMSO
MeOH
λabsa (nm)
ꢀb (M-1 cm-1
λemc (nm)
λemd (nm)
Φe
493, 528
7500, 6000
390, 560
560
489, 523
11800, 13200
385, 550
550
)
0.33
0.41
Ia
Ib
R - Rmin
Rmax - R
pH ) pKa + c log
+ log
[
]
a λabs are peak locations in the absorbance spectra. b ꢀ is the molar
extinction coefficient corresponding to each λabs.
c λem are the peak locations
in the emission spectra when excited at 325 nm. d λem are peak locations in
the emission spectra when excited at 488 and 514 nm. e Quantum yields of
compound SNAFR-1 relative to rhodamine 6G in EtOH (Φ ) 1). The
excitation wavelength was 514 nm for both SNAFR-1 and rhodamine 6G.
a The pKa of SNAFR-1 in 50 mM phosphate buffer containing 0.25%
DMSO was determined using the above equation.4a The pKa is taken to be
the intercept of the plot of pH versus the first log term in the equation,
where c is the slope; R is the ratio from the spectral data at λ1 and λ2; Rmax
and Rmin are the limiting values of this ratio; and Ia/Ib is the ratio of the
spectral intensity in acid to that in base at the wavelength chosen for the
denominator of R. This last term may be neglected by choosing an isosbestic
or isoemissive point as the denominator of R.
Table 1b. The pK/a Values for SNAFR-1 Estimated Using the
Forster Equationa
neutral form
anionic form
/
/
methods
ν˜neutral
λneutral
ν˜anion
λanion
ν˜
anion − νneutral
˜
pKa
pKa
-em
-abs
absorption
excitation
emission
19 495 512.94 18 451 541.99
19 608 510.00 18 519 540.00
18 484 541.00 16 103 621.00
1045
1089
2381
1541
1813
6.15 6.34
6.05 6.25
3.34 3.54
5.11 5.30
4.54 4.73
0-0 absorption 18 789 532.23 17 248 579.77
0-0 excitation 19 084 524.00 17 271 579.00
NAhc(ν˜anion - ν˜neutral
2.303RT
)
pK/a ) pKa -
a The pK/a of SNAFR-1 in 50 mM phosphate buffer containing 0.25%
DMSO was determined using the above equation11e with ν˜anion - ν˜neutral
determined from various methods. pK/ is the pKa at the first electronically
a
excited state (S1). NA is the Avogadro’s number. The h is the Plank’s
constant. The c is the light speed in the unit of cm/s. The ν˜anion and ν˜neutral
(cm-1) are the optical frequencies of the 0-0 transition in the neutral and
anionic form, respectively. The R is the ideal gas constant. The T (K) is
the temperature where absorption and fluorescence spectra were collected.
The λ (nm) is the wavelength. The pK/a-em is the pKa of S1 state calculated
using pKa-em ) 8.34. The pKa/-abs is the pKa of S1 state calculated using
pKa-abs ) 8.53. The 0-0 absorption/excitation is the intersection of the
emission with absorption/excitation spectra of neutral or anionic form,
respectively.
to 660 nm with corresponding emissions ranging from violet,
white, yellow, green to deep red.
Figure 6. (A) Excitation emission matrix for 30 µM SNAFR-1 in DMSO
with 0.25% phosphate buffer (50 mM, pH 7). (B) When excited at 310
nm, SNAFR-1 shows violet-blue, green, and red emission of approximately
equal intensities.
Spectral Properties of SNAFR-1 in DMSO. The absorption
and fluorescence spectra as a function of phosphate buffer pH
added to DMSO are shown in Figure 7. When the pH of the
added buffer is low, the absorption from both the neutral and
anionic forms is distinct. Above pH ) 6, absorption from the
anion dominates. Four clear isosbestic points are observed at
311, 345, 427, and 538 nm (Figure 7A). Emission spectra
excited at or below the 538 nm isosbestic point show that the
green emission at 560 nm from the neutral form decreases as
the pH of the phosphate buffer increases. The opposite trend is
observed for the emission at 670 nm, assigned to the anionic
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14086 J. AM. CHEM. SOC. VOL. 128, NO. 43, 2006

An Organic White Light-Emitting Fluorophore
A R T I C L E S
Figure 7. Spectral properties of 30 µM SNAFR-1 in DMSO with 0.25% phosphate buffer (50 mM) at various pH values. The trend of spectral changes is
indicated by arrows as the pH increases from 4 to 8. A ) anionic form; N ) neutral form. (A) Absorption spectra is shown; the / in panel A indicates the
positions of isosbestic points in absorbance spectra. (B-D) Excitation spectra with emission monitored at 400, 560, and 670 nm, respectively, is shown.
(E-I) Data are emission spectra excited at 325, 488, 514, 543, and 633 nm, respectively, corresponding to common laser lines. All fluorescence spectra are
normalized to the maximum of each data set.
form of SNAFR-1 (Figure 7E-I). Figure 7I shows that
excitation at 633 nm, much longer than the 538 nm isosbestic
point, excites only the anion yielding only the emission at 670
nm with no isoemissive point. A clear isoemissive point is likely
not apparent in Figure 7F because excitation at 488 nm results
in only minimal emission from the anion. Under these condi-
tions, emission from the neutral form dominates at all pH values
investigated. The best ratiometric behavior would occur when
excitation is at 538 nm (an isosbestic point), similar to the 543
nm output of a green HeNe laser (Figure 7H).
Compared to the red and green emissions, the violet-blue
emission does not show a strong dependence on the added buffer
pH (Figure 7E). Fluorescence excitation spectra, monitored at
400 nm, also verify that the violet-blue emission is independent
of pH (Figure 7B).
Lower pH results in greater excitation intensity at 490 nm
when monitored at the 560 nm peak of green emission band
(Figure 7C). This is explained by the increased formation of
the neutral form at low pH. At the same time, the opposite trend
is observed at 610 nm for the excitation spectra of the anion
form monitored at the peak of the deep red band (Figure 7D).
The similar pH-responsive behavior of SNAFR-1 in 0.25%
DMSO in buffer solutions (Figure 4) and 0.25% buffer in
DMSO solutions (Figure 7) is attributed to the preferential
solvation of SNAFR-1 by water in water-poor DMSO solutions.
Chromaticity33 of SNAFR-1 Emission Is Sensitive to
Excitation Wavelength and pH. By changing either the
excitation wavelength or the pH of the added buffer in DMSO,
a variety of emission colors can be obtained. Excitation from
270 to 340 nm leads to three emission bands of varying
intensities. Thus, the emission can appear either violet or near
white. When excited from 340 to 410 nm, the color changes
from near white to yellow owing to the disappearance of violet-
blue emission and the increase of green emission. When excited
from 420 to 540 nm, SNAFR-1 exhibits intensified green
emission and comparatively weak red emission. As the excita-
tion moves to longer wavelengths than 550 nm, that is, up to
650 nm, it exhibits red emission (Figure 8). As one can see
from Figure 7, green and red emission is dependent on pH
though blue emission is not. As a result, emission colors change
significantly when the pH of the added buffer solution changes.
The chromaticity of the emission as a function of both pH and
excitation from 270 to 340 nm is summarized in Table 3. A
complete table is located in Supporting Information. The
chromaticity diagram obtained in DMSO using added buffer
solution adjusted to pH 7 is shown in Figure 8A as an example.
9
J. AM. CHEM. SOC. VOL. 128, NO. 43, 2006 14087

A R T I C L E S
Yang et al.
Figure 9. (A) Structures of compound 14 and 15; (B) emission spectra of
14 and 15 excited at 325 nm, excitation spectrum of 14 with emission
monitored at 470 nm; absorption spectrum of 15; all compounds in 0.1 M
NaOH; (C) emission spectra of 14 and 15 excited at 325 nm; excitation
spectrum of 14 with emission monitored at 375 nm; absorption spectrum
of 15. The concentrations of 14 in both solution are 10 µM. All the other
solutions are 30 µM. All spectra are normalized to allow comparison.
fluorescein (14) and 15 as model naphthyl-containing fluoro-
phores. The blue emission of compound 14 is similar to that of
compound 15 (Figure 9). Importantly, the excitation spectrum
of 14 with emission monitored at 470 nm (blue band) overlays
with the absorption spectrum of 15 (in 0.1 M NaOH, Figure
9B). A similar trend is observed in MeOH (Figure 9C). This
implies that 14 and 15 have a similar mechanism of blue
emission in both solvents.
Figure 8. (A) Chromaticity coordinates for emission spectra collected with
excitation wavelength between 270 and 650 nm plotted in a 1931 CIE
(Commission Internationale de L’Eclairage) chromaticity diagram for a
solution of 30 µM SNAFR-1 in DMSO with 0.25% phosphate buffer (50
mM, pH 7). (B) Photographs of 30 µM SNAFR-1 in DMSO with 0.25%
phosphate buffer (50 mM, pH 7) when excited at 300, 320, 460, and 560
nm, respectively (from left to right), to afford emissions of near white,
yellow, green, and red color. (C) The emission spectra corresponding to
the photographs in panel B are shown.
We thus hypothesize that the blue emission of SNAFR-1 as
well as known 14 arises from the isolated naphthyl moiety. In
14, the lactone form would isolate the naphthyl fluorophore. In
organic solvents, it is well-known that the lactone form of
xanthenes predominates. Thus, as expected, we observe a more
intense blue emission than green emission in MeOH (Figure
9C). In 0.1 M NaOH solution, the equilibrium favors the for-
mation of carboxylate anion instead of the lactone, leading to a
more intense red emission (Figure 9B). However, the presence
of some lactone (or covalently attached hydroxide, vide infra)
apparently still induces some blue, naphthyl-derived emission.
Though SNAFR-1 possesses no lactone to isolate the
naphthalene fluorophore, its blue emission in various solvents
resembles those of 14 and 15 (Figures 10C and D). This suggests
an alternative mechanism to isolate the naphthalene unit,
accounting for the violet-blue emission of SNAFR-1. Our
experimental evidence shows that intermolecular nucleophilic
addition readily occurs in the naphtho[a]- and [b]xanthene series.
First, compound 10, the isolated adduct possessing a methyl
ether at the bridging benzylic carbon, exhibits primarily violet-
blue emission (Figure 10A). Second, compound 12, the methyl
ether analogue of one tautomer of SNAFR-1, also undergoes
nucleophilic addition to its central carbon, furnishing 16
(Scheme 5).
Table 3. Chromaticity of 30 µM SNAFR-1 in DMSO with 0.25%
Buffer as a Function of pH of Added Phosphate Buffer Solutions
pH 4
pH 5
pH 6
pH 7
pH 8
λex
x
y
x
y
x
y
x
y
x
y
270 nm 0.37 0.34 0.36 0.32 0.33 0.27 0.36 0.30 0.30 0.18
280 nm 0.40 0.39 0.39 0.38 0.37 0.33 0.36 0.30 0.33 0.23
290 nm 0.40 0.39 0.39 0.38 0.37 0.33 0.39 0.34 0.33 0.22
300 nm 0.42 0.42 0.41 0.41 0.40 0.36 0.41 0.40 0.36 0.25
310 nm 0.44 0.46 0.44 0.45 0.42 0.42 0.43 0.44 0.38 0.31
320 nm 0.45 0.48 0.45 0.47 0.43 0.45 0.43 0.46 0.39 0.36
330 nm 0.46 0.49 0.45 0.48 0.44 0.47 0.40 0.38 0.39 0.38
340 nm 0.43 0.44 0.42 0.43 0.40 0.40 0.44 0.43 0.35 0.28
The data corresponding to other buffer pH values are also
included in the Supporting Information.
Origin of the Violet-Blue Emission. The origin of the green
and red emission is discussed above. To investigate the origin
of the violet-blue emission, we employ known benzo[c]-
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14088 J. AM. CHEM. SOC. VOL. 128, NO. 43, 2006

An Organic White Light-Emitting Fluorophore
A R T I C L E S
tautomerizable SNAFR-1. An overlay of the violet-blue
emissions arising from 10, 12, 15, and SNAFR-1 in various
solvents is shown in Figure 10. The similar spectral features,
along with NMR and single-crystal X-ray structure evidence
(e.g., the structure of 10 in Figure 3 possesses a predominant
isolated naphthyl), help confirm the hypothesis that the isolated
naphthyl is responsible for the violet-blue emission of this
series of compounds. The presence of a trace amount of H2O
apparently serves as the nucleophile in DMSO which leads to
the isolated naphthyl unit. Alternative mechanisms (such as local
excitation34) that might also lead to blue emission are currently
under investigation.
Dual Emission White Light Arising from Compound 12.
Figure 10B demonstrates that 12 in MeOH exhibits dual
emission in the violet-blue and green spectral regions. In MeOH
only a minimal amount of 12 remains in solution with the
majority in the form of 16 (Scheme 5), leading to a dominant
violet-blue emission (from ca. 330 to 450 nm) with a green
emission (from ca. 520 to 620 nm) when excited below 340
nm. However, as has been shown for SNAFR-1, the relative
intensities of these emission bands are dependent on excitation
wavelength (Figure 12A). Excitation beyond 340 nm dramati-
cally decreases the relative intensity of the blue emission band
as excitation of the naphthyl unit becomes less efficient. When
excited at 350 nm, the ratio of the two emission bands is nearly
equal. The mixing of these emission bands again results in near
white emission. The chromaticity diagram of 50 µM 12 in
MeOH is shown in Figure 12B. A photograph of the white
emission is shown in Figure 12C. Although two band white
light has a poorer color rendering index (CRI) owing to the
unbalanced red color,35 this result is of interest since currently
two band white light is the main origin of white emission in
white polymer light emitting diodes (WPLEDs).22a
Figure 10. (A) Fluorescence emission of 30 µM 10 in DMSO and MeOH;
(B) fluorescence emission of 25 µM 12 in DMSO and MeOH; (C) overlay
of the violet-blue emission of SNAFR-1 with other compounds possessing
the isolated naphthalene moiety in DMSO; (D) overlay of the violet-blue
emission of SNAFR-1 with other compounds possessing the isolated
naphthalene moiety in MeOH. All fluorescence spectra are collected with
an excitation wavelength at 325 nm. Solutions of SNAFR-1 and 15 are 30
µM. All fluorescence spectra are normalized to the maximum of the violet-
blue peak.
Scheme 5. Equilibrium between Compounds 12 and 16a
a Methyl groups and corresponding peaks in Figure 11 are shown in the
same color.
Live Cell Imaging. Organic fluorophores have found exten-
sive applications in cell imaging. However, their drawbacks,
such as narrow excitation, single, wide emission with a long
tail, poor photostability, etc., limited their applications in some
sophisticated applications such as multiplexing and real-time
measurements. Few water soluble, long wavelength (λem > 600
nm), and photostable probes were reported. Though SNAFR-1
also shows a wide emission in neutral buffer solution, its unique
properties of multiple emissions, wide excitation window, low
cytotoxicity, and excellent photostability (vide infra) make it
an ideal candidate for cellular imaging.
Cellular imaging studies reveal that SNAFR-1 readily enters
HEp2 cells (Figure 13). SNAFR-1 localization is directed
toward the lipophilic compartments, as there is a strong signal
from the endoplasmic reticulum (ER) as well as some signal
from mitochondria. The nuclei of the cells also show some
SNAFR-1 fluorescence. This could be due to accumulation in
Figure 11. 1H NMR study of the equilibrium between 12 and 16 in DMSO-
d6. The concentration of compound 12 decreases as MeOH increases.
(33) (a) Chromaticity coordinates x, y, and z are derived by calculating the
fractional components of the tristimulus values thus: x ) X/(X + Y + Z),
y ) Y/(X + Y + Z), z ) Z/(X + Y + Z). Tristimulus values are the amounts
of three primaries that specify a color stimulus. The CIE 1931 tristimulus
values are denoted as X, Y, and Z. All possible sets of tristimulus values
can be represented in a two-dimensional plot of two of these chromaticity
coordinates and by convention x and y are always used. A plot of this type
is referred to as a chromaticity diagram; http://www.colourware.co.uk/. (b)
http://hyperphysics.phy-astr.gsu.edu/hbase/vision/cie.html#c2. (c) Wyszecki,
G. Colorimetry. In Handbook of Optics; Driscoll, W. G., Vaughan, W.,
Eds.; McGraw-Hill Book Company: New York, 1978; pp 1-15.
(34) Zilberg, S.; Haas, Y. J. Phys. Chem. A 2002, 106, 1-10.
A study of the equilibrium between 12 and 16 using 1H NMR
is shown in Figure 11. The resonances of the three methyl ether
moieties of 12 and 16 are well-resolved. As the MeOH
concentration increases, the relative proportion of compound
12 is observed to decrease. A MeOH solution of 12 displays
an intense blue emission as well as a green emission (Figure
10B). The red emission of 12 is diminished compared to
(35) Zakauskas, A.; Shur, M.; Gaska, R. Introduction to solid-state lighting;
Wiley-Interscience: New York, 2002, pp 130.
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A R T I C L E S
Yang et al.
Figure 13. (a) Phase contrast of cells stained with SNAFR-1; (b)
fluorescence through Texas Red filter set with excitation 540-580 nm and
emission 610 nm long pass; (c) fluorescence through DAPI filter set with
excitation 330-370 nm and emission 420 nm long pass; (d) fluorescence
through FITC filter set with excitation 460-500 nm and emission 510-
560 nm; (e) endoplasmic reticulum stained with ERTracer Blue White
(Molecular Probes); (f) mitochondria stained with MitoTracker Green
(Molecular Probes). Scale bar is 10 µm.
compound signal. At the concentrations tested, SNAFR-1
exhibits no toxic effects.
SNAFR-1 Exhibits Excellent Photostability. The photosta-
bility of SNAFR-1 is investigated in MeOH. It is much more
photostable compared to fluorescein in 0.1 M NaOH. In MeOH
SNAFR-1 can be excited efficiently at 488 nm, corresponding
to an Ar ion laser line. This wavelength matches well with
absorbance maximum (λabs ) 493 nm) for fluorescein in aqueous
base as well as that of SNAFR-1 (λabs ) 489 nm) in MeOH.
The photostability studies are conducted with solutions prepared
to have an absorbance of 0.03 at 488 nm so that both SNAFR-1
and fluorescein solutions absorb the same number of photons
when excited at 488 nm. The excitation band-pass is opened to
14 nm, the maximum allowed by the instrument. Fluorescence
signal is collected using both the s and t channels of the
instrument. S channel data are collected through the dual mono-
chromator set at the emission maximum of a given fluorophore.
T channel data are collected through a 550 nm long pass filter.
High voltage for the photomultiplier tube (PMT) is set at 950
and 500 V for the s and t channels, respectively. Data are
collected with a 0.1 s integration time at 0.1 s intervals for at
least 1800 s. The signal is maintained within an acceptable range
using neutral density filters (Omega optical, Brattleboro, VT).
The photobleaching decay of each dye is plotted in Figure 14.
SNAFR-1 shows excellent photostability during the time
monitored, while fluorescein loses 20% of its fluorescence in
only 30 min.
Figure 12. (A) The emission of 50 µM 12 in MeOH. When excited at
350 nm, 12 shows two emission bands of approximately equal intensity.
(B) Shown are the chromaticity coordinates for emission spectra collected
with excitation wavelength between 275 and 375 nm plotted in a 1931 CIE
chromaticity diagram for a solution of 50 µM 12 in MeOH. (C) Photograph
of white light of compound 12 in MeOH.
the nuclear membrane or to intercalation into DNA. No obvious
staining of the plasma membrane is observed. This is not
surprising as the plasma membrane has a large surface area and,
as such, a low surface brightness signal may not be detected.
Three different filter sets, DAPI, FITC, and Texas Red, are
applied corresponding to the blue, green, and red emission of
SNAFR-1. Some autofluorescence from the cell is observed
when DAPI is used. However, autofluorescence is minimal when
longer wavelength filter sets such as FITC and Texas Red are
used.
Conclusion
Overnight incubation of cells with SNAFR-1 shows an overall
increase in signal intensity compared to 30 min incubation and
large vesicular formation within cells that do not colocalize with
Wolfbeis and co-workers had predicted that novel benzan-
nulated xanthenes would prove useful synthetic targets, par-
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14090 J. AM. CHEM. SOC. VOL. 128, NO. 43, 2006

An Organic White Light-Emitting Fluorophore
A R T I C L E S
using graphite monochromated Mo KR radiation (λ ) 0.71073 Å) on
a Nonius KappaCCD diffractometer fitted with an Oxford Cryostream
cooler. Structures were solved by direct methods and refined by full-
matrix least-squares, using SHELXL97.37 For all compounds, H atoms
were visible in difference maps but were placed in idealized positions,
torsional parameters were refined for methyl groups, and OH hydrogen
positions were refined individually.
UV-Visible Absorption and Fluorescence Spectroscopy.
UV-vis spectra were collected on Cary 50 Bio UV-vis spectropho-
tometer at room temperature using 1 cm quartz cuvette. Fluorescence
spectra were collected on a Fluorolog-22Tau3 (Horiba Jobin Yvon,
Edison, NJ). Solutions were placed in a 1 cm path length quartz cell
(Starna Cells, Atascadero, CA). Emission spectra were collected
following excitation with a 450 W xenon arc lamp. A dual monochro-
mator was used to select light of 325, 488, 514, 543, and 633 nm (band-
pass ) 1 nm). Emission wavelengths were scanned with 1 nm step
sizes using a dual monochromator (band-pass ) 4 nm). Integration
time was set to 0.1 s per point, and 950 V was applied to a Hamamatsu
R928 PMT. Excitation spectra were monitored with an emission and
excitation band-pass of 4 and 1 nm, respectively. All spectra were
corrected through division by signal from a reference channel. Excitation
emission matrixes (EEMs) were collected over various spectral regions
using 2 nm step sizes for emission and 10 nm step sizes for excitation.
An estimate of the fluorescence quantum yield was obtained as
reported.38 Quantum yields are relative to rhodamine 6G in EtOH. An
assumption was made that rhodamine 6G has a quantum yield of 1
when dissolved in EtOH.
Live-Cell Measurements. HEp2 cells were obtained from ATCC
and maintained in a 50:50 mixture of DMEM/Advanced MEM
(Invitrogen) supplemented with 5% FBS (Invitrogen) and Primocin as
antibiotic (Invivogen). Cells were subcultured twice weekly to maintain
working stocks. HEp2 cells were seeded onto LabTek ll chamber
coverslips and allowed to grow for 48 h. SNAFR-1 was first prepared
as a 10 mM stock in DMSO and then diluted directly into medium to
10 µM. Cells were then fed medium containing SNAFR-1 for 30 min.
Slides were then washed three times with medium containing 50 mM
HEPES pH 7.2 and examined using a Zeiss Labovert 200 M microscope
fitted with a standard Texas Red filter set (Chroma Technology Corp.).
The images were acquired using a Zeiss AxioCam MRm digital camera.
Mitochondria were stained using a 250 nM MitoTracker Green FM,
and endoplasmic reticulum were stained using 1 µM ER-Tracker Green
(both from Molecular Probes) by incubating cells for 30 min with the
appropriate dye and washing as above. The organelle stains were
visualized using a standard FITC filter set (Chroma Technology Corp.)
Compound toxicity was tested by plating 7500 HEp2 cells per well on
a Costar 96-well plate. The cells were allowed to grow for 48 h and
then were fed medium containing 2-fold dilutions of SNAFR-1 ranging
from 10 to 1.25 µM. Saponin (0.1%, Sigma) was used as a negative
control. Cells were then incubated for 24 h. For normalization purposes,
untreated cells were considered 100% viable, and cells treated with
0.1% saponin were considered 0% viable. Viability was measured using
the CellTiter Blue Cell Viability assay (Promega) as per manufacturer’s
instructions. Fluorescence signal was detected using an excitation
wavelength of 520 nm and emission wavelength of 584 nm.
Figure 14. Photobleaching decay of SNAFR-1 in MeOH and fluorescein
in 0.1 M NaOH monitored at emission maximum of each compound (top)
and through a 550 nm long pass filter (bottom). The fluorescence intensity
is normalized at time 0 s for each compound.
ticularly as intracellular pH probes. A new methodology for
the synthesis of “missing” [a]- and [b]benzoxanthene regio-
isomers was described herein. We have shown that a type
[a]benzofluorone, SNAFR-1, exhibits unique properties, includ-
ing white light emission as well as excitation over a 400 nm
range. Additionally, this compound shows promise for use in
cellular imaging studies.
The dual visible emission maxima in the green and red
spectral regions of the benzannulated xanthenes reported herein
are dependent on both solution pH and excitation wavelength.
We have found that benzannulated xanthenes emit blue light
based on the equilibria involving nucleophilic addition at their
benzylic carbon. Thus, tuning the intensities of the red, green,
and blue emission wavelengths of SNAFR-1 to approximately
equal intensities, via optimizing both solution parameters and
excitation, renders it a red-green-blue (RGB) dye. This
embodies a unique example of a simple dye that emits white
light upon UV excitation. To realize materials applications,
studies of SNAFR-1 and congeners in the solid state are needed.
The synthesis and properties of these and related new fluoro-
phores will be reported in due course.
Experimental Section
General Methods. 1,6-Dimethoxynaphthalene was purchased from
Wilshire Technologies. 2,4-Dimethoxybenzenephenone was prepared
by a known procedure.36 All other reagents were purchased from Sigma-
Aldrich and used as received without further purification except THF
and CH2Cl2 which were dried using the Innovative Technologies solvent
purification system. EtOAc, hexane, CHCl3, acetone, and MeOH were
purchased from EMD biosciences and used as received. All reactions
were carried out under an Ar atmosphere unless otherwise indicated.
The TLC plates used were silica XHL TLC plates, glass backed, 250
µm, from Sorbent Technologies. The silica gel used was 60 Å standard
grade with a distribution of 40-63 µm, from Sorbent Technologies.
High-resolution MS (HRMS) and ESI spectra were obtained at the Mass
Spectrometry Facility of Georgia State University, Atlanta, GA. NMR
spectra were acquired in DMSO-d6 or CDCl3 on a Bruker DPX-250,
DPX-300, or Avance-400 spectrometer. All δ values are reported with
Acknowledgment. The authors thank Dr. William E. Crowe
and Dr. Rafael Cueto for insightful discussions and Alexander
D. Wong, Michael C. Touchy II, Louise A. Corle, and Francis
S. Sicard for their diligent lab work and also very gratefully
acknowledge the generous support of the National Institutes of
Health (Grant R01 EB002044). The purchase of the Nonius
Kappa CCD diffractometer was made possible by Grant No.
1
DMSO referenced at 2.49 ppm for H NMR and at 39.5 ppm for 13C
NMR, or with CDCl3 referenced at 7.26 ppm for 1H NMR and at 77.0
ppm for 13C NMR.
X-ray Crystallography. Intensity data for X-ray crystallography
was collected for compounds 4, 5, 6, 7, 8, 9, 10, and 13 at T ) 110 K
(37) Sheldrick, G. M. SHELXL97; University of Go¨ttingen: Go¨ttingen, Germany,
1997.
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A R T I C L E S
Yang et al.
LEQSF-(1999-2000)-ESH-TR-13, administered by the Louisiana
Board of Regents.
information of 10, SNAFR-1, 12, 14; apparent pKa values in
DMSO containing 0.25% phosphate buffer; chromaticity dia-
gram of 10, SNAFR-1, 12, and 14 at various conditions are
included. This material is available free of charge via the Internet
at http://pubs.acs.org.
Supporting Information Available: 1H, 13C NMR spectra,
mass spectra of compounds 4-10, 12, SNAFR-1, and 13;
crystallographic information files (CIFs) and ORTEP graphs
for compounds 4-10 and 13; cytotoxicity study; spectral
JA0632207
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14092 J. AM. CHEM. SOC. VOL. 128, NO. 43, 2006