Fluorescence Properties of Benz[f]indole, a Wavelength and Quenching Selective Tryptophan Analog

Article abstract of DOI:10.1021/jp9925536

Tryptophan analogues with unique spectral and photophysical properties offer intrinsic fluorescent probes for studying peptide-protein and protein-protein interactions. Two benzannulated indole derivatives, benz-[f]indole and 3-methylbenz[f]indole, were synthesized and their fluorescence was characterized. The absorption and fluorescence emission spectra are red shifted about 75 nm to the red of the spectra of indole and 3-methylindole. INDO/S-CIS computations indicate two nearly degenerate lowest excited singlet states, analogous to the ¹L_a and ¹L_b transitions of indoles but with almost collinear transition moments. The limiting excitation anisotropy spectra increase and the emission anisotropy spectra decrease with increasing wavelength, probably due to vibronic coupling of the ¹L_a state with higher energy B states. Solvent and temperature effects on the wavelength of maximum emission argue that emission occurs from ¹L_a in polar environments. The fluorescence quantum yields and lifetimes are high and essentially independent of temperature and solvent isotope. Quantum yields are 0.80 for both compounds. Lifetimes are 19 ns for benz[f]indole and 25 ns for 3-methylbenz[f]indole. The fluorescence is not quenched by most protein functional groups in Stern-Volmer experiments, though it is quenched weakly by strong electron acceptors, such as histidine and cysteine. These results indicate that two nonradiative processes of indoles, solvent quenching and excited-state proton transfer, are not important decay pathways of benzannulated indoles. A third nonradiative process, excited-state electron transfer, occurs in benzannulated indoles under more limited conditions than in indoles.

Full text of DOI:10.1021/jp9925536

J. Phys. Chem. B 2000, 104, 1837-1843
1837
Fluorescence Properties of Benz[f]indole, a Wavelength and Quenching Selective
Tryptophan Analog
Bo Liu and Mary D. Barkley*
Department of Chemistry, Case Western ReserVe UniVersity, 10900 Euclid AVenue, CleVeland, Ohio 44106
Guillermo A. Morales and Mark L. McLaughlin
Department of Chemistry, Louisiana State UniVersity, Baton Rouge, Louisiana 70803
Patrik R. Callis
Department of Chemistry & Biochemistry, Montana State UniVersity, Bozeman, Montana 59717
ReceiVed: July 23, 1999; In Final Form: December 20, 1999
Tryptophan analogues with unique spectral and photophysical properties offer intrinsic fluorescent probes
for studying peptide-protein and protein-protein interactions. Two benzannulated indole derivatives, benz-
[f]indole and 3-methylbenz[f]indole, were synthesized and their fluorescence was characterized. The absorption
and fluorescence emission spectra are red shifted about 75 nm to the red of the spectra of indole and
-methylindole. INDO/S-CIS computations indicate two nearly degenerate lowest excited singlet states,
3
1
1
analogous to the L
a b
and L transitions of indoles but with almost collinear transition moments. The limiting
excitation anisotropy spectra increase and the emission anisotropy spectra decrease with increasing wavelength,
1
probably due to vibronic coupling of the L
a
state with higher energy B states. Solvent and temperature
1
effects on the wavelength of maximum emission argue that emission occurs from L
a
in polar environments.
The fluorescence quantum yields and lifetimes are high and essentially independent of temperature and solvent
isotope. Quantum yields are 0.80 for both compounds. Lifetimes are 19 ns for benz[f]indole and 25 ns for
3-methylbenz[f]indole. The fluorescence is not quenched by most protein functional groups in Stern-Volmer
experiments, though it is quenched weakly by strong electron acceptors, such as histidine and cysteine. These
results indicate that two nonradiative processes of indoles, solvent quenching and excited-state proton transfer,
are not important decay pathways of benzannulated indoles. A third nonradiative process, excited-state electron
transfer, occurs in benzannulated indoles under more limited conditions than in indoles.
Introduction
ing eight amino acid side chains and the peptide bond quench
indole fluorescence: two by excited-state proton transfer and
six by excited-state electron transfer. Given the complex
Being an intrinsic fluorescent probe in peptides and proteins,
tryptophan has attracted extensive study. The fluorescence
wavelength maximum, quantum yield, and lifetime are highly
sensitive to the local environment of the indole chromophore.
The first absorption band of indole around 280 nm consists of
4
photophysics, structural interpretation of fluorescence data from
even a single tryptophan in proteins remains a daunting task.
Spectrally enhanced proteins produced by biosynthetic in-
corporation of tryptophan analogues are useful to study protein-
1
1
two overlapping π f π* electronic transitions, La and Lb. In
most proteins and polar solvents, fluorescence occurs from La.
5,6
1
1
protein and protein-nucleic acid interactions. Analogues with
minor modifications to the indole ring, such as 5-hydroxytryp-
tophan and 7-azatryptophan, can be selectively excited at 310
nm in the presence of natural tryptophans. Like tryptophan, the
absorption spectrum of 5-hydroxytryptophan has a maximum
at 280 nm, but the shoulder on the red edge extends to about
320 nm. The absorption spectrum of 7-azatryptophan is less
structured and is red-shifted about 10 nm compared to tryp-
tophan. In benzannulated indole derivatives, such as the
benzindoles and carbazoles, the long wavelength absorption
1
The large increase in permanent dipole moment in the La state
compared to the ground state accounts for the large Stokes’ shift
in polar solvent due to inhomogeneous broadening and solvent
relaxation. Consequently, in proteins the fluorescence wave-
length maximum reports the local electrostatic environment due
2
to an internal Stark effect. Environmental sensitivity of the
fluorescence quantum yield and lifetime comes from the multiple
nonradiative decay pathways that compete with emission for
deactivation of excited indole: intersystem crossing, solvent
quenching, and excited-state proton and electron transfer reac-
tions. The latter three quenching processes depend on type and
proximity of solvent molecules, proton donors, and electron
acceptors. Water quenching refers to an isotopically sensitive
temperature-dependent nonradiative process that occurs in all
7
maximum is shifted even further to the red. Moreover, the
smaller energy gap between the ground and first excited singlet
states may simplify the photophysics of benzannulated tryp-
tophan derivatives.
This paper reports the spectroscopic and photophysical
properties of benz[f]indole (BzIn) and 3-methylbenz[f]indole (3-
MeBzIn). Fluorescence emission, excitation, and limiting ani-
sotropy spectra are presented and interpreted in terms of two
3
indoles. In proteins, six amino acid functional groups compris-
*
Corresponding author e-mail: mdb4@po.cwru.edu
1
0.1021/jp9925536 CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/08/2000

1838 J. Phys. Chem. B, Vol. 104, No. 8, 2000
Liu et al.
nearly degenerate low-lying excited states, which are shown to
3-Methylbenz[f]indole. 3-Bromo-2-aminonaphthalene (0.221
g), 1-trimethylsilylpropyne (0.224 g), n-Bu4NCl (0.278 g), Na2-
CO3 (0.265 g), Pd(OAc)2 (22 mg), and PPh3 (26 mg) were
placed in a 100 mL three-necked round-bottom flask under
argon, and 30 mL of anhydrous acetonitrile was cannulated into
the flask with argon. The mixture was refluxed under argon at
100 °C for 4 h. Hydrogen chloride was bubbled through the
solution for 2 min and the solution was refluxed for 1 h and
then poured into 250 mL of 3 N NH4OH. The final solution
was extracted by ethyl acetate. The combined organic extract
was washed and evaporated. The brown residue was taken up
in CH Cl and separated by silica gel chromatography (1:1
1
1
be closely related to the La and Lb states of indole and
8
4
tryptophan by INDO/S-CIS computations. As before, we
identify the nonradiative processes which these chromophores
undergo from the dependence of fluorescence quantum yield
and lifetime on temperature, solvent isotope, and solute quench-
er. The unique fluorescence properties and potential utility of
benzannulated tryptophans as probes of peptide structure and
peptide-protein interactions are discussed.
Experimental Section
Chemicals. 3-Methylcarbazole was prepared according to a
2
2
9
literature procedure. The final product was recrystallized twice
2 2
hexanes/CH Cl ) (0.146 g, 80.8% yield). The final product was
from warm methanol. Indole (Sigma) was recrystallized twice
from aqueous ethanol. 3-Methylindole (Aldrich, 99+%) was
used as received. All indole derivatives were stored in an inert
atmosphere at 4 °C in the dark. Aqueous solutions of benzan-
nulated indoles contain 10% methanol (vol/vol) to increase
solubility. Acetone (Fisher), 3-methyl-2-butanone (Aldrich), and
sublimed twice at 80-90 °C under reduced pressure: mp 192-
1
193 °C. H NMR (CDCl , 300 MHz): δ 8.03 (s, 1H), 7.98-
3
7.94 (dd, 1H), 7.88-7.85 (dd, 1H), 7.71 (s, 2H), 7.39-7.23
1
3
(m, 2H), 7.11 (s, 1H), 2.42 (s, 3H). C NMR (CDCl , 75
3
MHz): δ 137.36, 130.67, 130.52, 128.46, 127.47, 125.63,
123.87, 122.65, 111.26, 106.30, 10.08. GC-MS: m/z 181, 152,
3
,3-dimethyl-2-butanone (Aldrich) were distilled twice. Amino
139, 127, 90, 76, 63, 51. Anal. (C H N) H, N; C: calcd, C
1
3
11
acids and N-acetyl derivatives were purchased from Sigma.
Malonamide (Aldrich), succinamide (Aldrich), N-acetyltyrosine,
histidine, N-acetylphenylalanine, N-acetylcysteine, and lysine
were recrystallized from water. Glutamic and aspartic acids were
recrystallized by carefully adjusting monosodium glutamate and
aspartate solutions to pH 3.0 with 6 N HCl. N-Acetylglutamine
and N-acetylasparagine were recrystallized from methanol/ether
mixture. Glycerol (glass distilled) was from EM Science. Other
chemicals from Sigma and Aldrich were highest grade available.
86.15; found, C 85.33.
Absorbance and Fluorescence. Absorption spectra were
measured at 25 °C on a Cary 3E UV-vis spectrophotometer.
Extinction coefficients of benzindole and 3-methylbenzindole
were determined from the absorbance of solutions containing
1.5 mg of benzannulated indole dissolved in 500 mL of 9:1
3
-1
-1
water/methanol: ꢀ3 ) 4.4 × 10 M cm for benzindole
44
3
-1
-1
and ꢀ353 ) 4.5 × 10 M cm for 3-methylbenzindole. Sample
absorbance was below 0.1 at the absorption maximum for
steady-state fluorescence measurements. Fluorescence spectra
were measured on an SLM 8000 photon counting spectrofluo-
rometer with a single emission monochromator (1- or 4-nm
band-pass). The excitation polarizer was set at 54.7°, and the
emission polarizer was set at 0° to eliminate anisotropic effects.
Background fluorescence from a solvent blank was subtracted.
Between about 300 and 400 nm, excitation spectra were
corrected by the internal quantum counter in the SLM operating
in the ratio mode as judged by comparing the absorption and
excitation spectra of anthracene in ethanol. Emission spectra
were corrected for wavelength-dependent instrument response
using correction factors determined with a standard lamp from
Optronics. Quantum yields Φ were determined relative to
quinine sulfate (Fisher) in 1 N sulfuric acid (GFS Chemicals,
double distilled) at 365 nm excitation wavelength, 25 °C. The
quantum yield of quinine sulfate was taken to be 0.546.10
Low temperature fluorescence spectra were measured using
an Oxford model DN1704 liquid nitrogen cryostat with a model
ITC-4 temperature controller. The sample cell was a quartz
NMR tube cut to about 4 cm length. Solutions were placed in
the cryostat at room temperature and the temperature was
lowered to 130 K. Solutions in ethanol (AAPER) were prepared
immediately prior to use and saturated with nitrogen. Limiting
o
2-Methyl-3-nitro-naphthalene-bis(hexachlorocyclopentadiene)-
adduct. 2-Methylnaphthalene-bis(hexachlorocyclopentadiene)-
adduct (68.77 g, technical grade, Aldrich) was dissolved in 1.1
L of dichloromethane. The solution was filtered and 990 g of
fuming nitric acid was added while stirring. After 30 min the
reaction mixture was poured into 4 L of ice-water. The organic
layer was washed and evaporated and the residue was recrystal-
lized from toluene (62.23 g, 84.9% yield): mp 249-250 °C.
1
H NMR (CDCl3, 250 MHz): δ 8.41 (s, 1H), 7.73 (s, 1H),
.03-3.96 (t, 1H), 3.57-3.54 (d, 1H), 2.65 (s, 3H). C NMR
1
3
4
(CDCl3, 62.5 MHz): δ 147.75, 133.86, 133.80, 133.01, 132.80,
1
4
29.52, 129.40, 127.95, 125.23, 101.14, 84.52, 84.46, 82.15,
6.89, 46.67, 41.58, 41.45, 20.43.
2-Methyl-3-nitronaphthalene. 2-Methyl-3-nitro-naphthalene-
bis(hexachlorocyclopentadiene)adduct (82.21 g) was pyrolyzed
at 350 °C under vacuum. The resulting slurry was cooled in an
ice-water bath, washed with cold hexanes, and crystallized from
1
hexanes (20.8 g, 99.3% yield): mp 119-121 °C. H NMR
(
CDCl3, 250 MHz): δ 8.50 (s, 1H), 7.91-7.88 (d, 1H), 7.82-
7
.78 (d, 1H), 7.72 (s, 1H), 7.65-7.51 (m, 2H), 2.71 (s, 3H).
C NMR (CDCl3, 62.5 MHz): δ 147.75, 135.01, 131.22,
30.77, 129.28, 128.98, 127.05, 125.13, 20.44.
1
3
1
Benz[f]indole. 2-Methyl-3-nitronaphthalene (0.37 g), N,N-
dimethylformamide dimethyl acetal (2.38 g), and pyrrolidine
1.42 g) were mixed in 10 mL of dry DMF and refluxed for 6
anisotropy spectra r (λ) were calculated from
(
h under argon. After removing the solvent the dark residue was
dissolved in methanol, mixed with Pd-C (10%), and hydro-
genated at 30 psi for 9 h at room temperature. The mixture was
filtered, evaporated, and separated by flash chromatography on
ro(λ) ) {IVV(λ) - G(λ)IVH(λ)}/{IVV(λ) + 2G(λ)IVH(λ)} (1)
where the subscripts V and H denote vertically (0°) and
horizontally (90°) polarized components of the intensity at
wavelength λ, and G(λ) ) IHV(λ)/IHH(λ) is an instrumental
correction factor.
a silica gel column (1:1 hexanes/CH2Cl2) to afford white flakes
1
(
0.04 g, 12% yield): mp 185-186 °C. H NMR (CD3OD, 400
MHz): δ 8.02 (s, 1H), 7.86-7.79 (m, 3H), 7.44 (t, 1H), 7.27-
Fluorescence decays were measured by time-correlated single
.19 (m, 3H), 6.56 (s, 1H). 13C NMR (CD3OD, 100 MHz): δ
38.49, 131.42, 131.30, 130.09, 129.78, 128.88, 128.16, 123.96,
22.87, 118.06, 106.99, 101.46. GC-MS: m/z 167, 139, 83,
0.
photon counting and deconvolved as described elsewhere. The
11
7
1
1
7
laser system was a Coherent Antares 76-S mode-locked Nd:
YAG laser and a model 701 dye laser. For experiments on
benzannulated indoles, the laser dye was DCM, excitation

Fluorescence Properties of Benz[f]indole
J. Phys. Chem. B, Vol. 104, No. 8, 2000 1839
wavelength was 332 nm; emission wavelength was 450 or 470
nm (8-nm band-pass), and the instrument response was about
350 ps full width half-maximum (fwhm). For experiments on
indoles, the laser dye was R6G, excitation wavelength was 290
nm, emission wavelength was 350 or 370 nm (8-nm band-pass),
and the instrument response was about 100 ps fwhm. All decays
gave good fits to monoexponential functions with reduced chi-
square values of 1.1-1.6.
Stern-Volmer quenching rate constants kq were determined
from fluorescence quantum yield Φ and lifetime τ data in the
absence (subscript 0) and presence of various concentrations
of quencher Q in 9:1 methanol/water at 25 °C.
Φ /Φ ) τ /τ ) 1 + k τ [Q]
(2)
0
0
q 0
Computations. Absorbing and emitting states of benzindole
and 3-methylbenzindole were modeled using the same programs,
1
2
parameters, and analysis previously applied to indole and
3
-methylindole. The INDO/S-CIS method was used with 196
singly excited configurations. The Mataga-Nishimoto electron
repulsion parameterization and the default interaction factors
appropriate to that scheme were used. Geometries were obtained
by MOPAC1
state.
3,14
using the AM1 Hamiltonian for the ground
Results and Discussion
Synthesis. Benzindole synthesis was first attempted more than
a century ago by Fischer synthesis with acetone 2-naphthylhy-
15,16
drazone and was later improved.
Among the three isomeric
benzindoles, benz[e]-, benz[f]- and benz[g]-indoles, the linear
benz[f]indole is the most difficult to synthesize, because the
Fischer indole synthesis methods are not effective in this case.
When incorporated in place of indole in tryptophan, the linear
benz[f]indole moiety offers the best mimic of the side-chain
conformation and planar ring system of tryptophan. Recent
Figure 1. Absorption and emission spectra of benz[f]indole (s) and
3
2 3
-methylbenz[f]indole (‚‚‚) in 9:1 H O/CH OH, 25 °C. Upper panel:
-5
-5
2.3 × 10 M BzIn, 2.2 × 10 M 3-MeBzIn. Lower panel: excitation
wavelength 348 nm.
17,18
syntheses of benz[f]indole are long synthetic routes.
Batcho
19
and Leimgruber reported a general, mild, two-step process for
indole synthesis from 2-methylnitrobenzenes by condensation
with formamide acetals followed by reduction. We have adapted
this method to synthesize benz[f]indole. The required starting
material, 2-methyl-3-nitronaphthalene, is prepared from the
commercially available 2-methylnaphthalene-bis(hexachloro-
cyclopentadiene)adduct via nitration and high vacuum pyrolysis
to unmask the naphthalene ring. The condensation reaction gave
poor, but acceptable yields of benz[f]indole as other routes gave
good yields of 3-substituted derivatives. For instance, the
palladium-catalyzed hydroarylation of 1-trimethylsilylpropyne
with 3-bromo-2-aminonaphthalene provides a good yield of
-methylbenz[f]indoles.1
7,20,21
3
Absorption and Fluorescence Spectra. Figure 1 shows the
absorption and fluorescence emission spectra of benzindole and
-methylbenzindole in 10% CH3OH. The absorption spectrum
of benzindole is structured with a maximum at 344 nm and
peaks at 330 and 358 nm. The spectrum of 3-methylbenzindole
is less structured and red-shifted about 10 nm. The emission
spectra of both compounds are broad and featureless with the
3
Figure 2. Temperature dependence of emission spectrum of benz[f]-
indole in glycerol. 150 K (a), 253 K (b), 273 K (c), 298 K (d). Excitation
wavelength 340 nm.
mirror images with almost no Stokes’ shift (Figure 3). The
absorption spectrum has a maximum at 354 nm, a sharp peak
at 338 nm, and a peak at 323 nm. The emission spectrum has
three well-resolved peaks: a maximum at 357 nm, a sharp peak
at 377, and a peak at 396 nm. The solvent and temperature
dependence of the emission spectrum of benzindole is charac-
teristic of indole and its derivatives. Indeed, INDO/S-CIS
computations reveal a strong parallel between indole and
benzindole and the corresponding 3-methyl derivatives.1 The
3
-methylbenzindole spectrum about 25 nm to the red of the
benzindole spectrum. Both compounds show large Stokes’
shifts: about 70 nm for benzindole and 86 nm for 3-methyl-
benzindole. As temperature decreases from 298 to 150 K, the
emission spectrum of benzindole in glycerol narrows from 57
to 45 nm fwhm and shifts to the blue (Figure 2). Vibrational
structure begins to emerge below 253 K. In cyclohexane, the
absorption and emission spectra of benzindole are structured
,12

1840 J. Phys. Chem. B, Vol. 104, No. 8, 2000
Liu et al.
Figure 3. Absorption (left curve) and emission (right curve) spectra
of benz[f]indole in cyclohexane, 25 °C. Excitation wavelength 330 nm.
two lowest excited singlet states are nearly degenerate, and their
1
1
transition densities are characteristic of the La and Lb transi-
1
1
tions of indoles. As with indole, La has slightly lower energy,
the energy gap between La and Lb narrows upon methylation
at the 3 position, and the transition to La is several times more
intense than to Lb. The permanent dipole of La is several
Debyes larger than those of Lb and the ground state. More
specifically, the ground and Lb state permanent dipoles are
1
1
1
1
1
1
1
about 2 D, pointing toward the nitrogen from the center of the
molecule, with the positive end pointing to the N. There is more
1
electron transfer for La, almost entirely along the long axis, in
a direction away from the pyrrole end. The sensitivity of the
fluorescence of the benzindoles to solvent and temperature is
consistent with emission from La.
Figure 4. Excitation (s) and limiting excitation anisotropy (‚‚‚) spectra
of benz[f]indole (upper) and 3-methylbenz[f]indole (lower) in ethanol,
130 K. Emission wavelength 390 nm.
1
Anisotropy Spectra. Figure 4 shows the excitation and
limiting excitation anisotropy spectra of benzindole and 3-me-
thylbenzindole in ethanol glass at 130 K observed at 390 nm
emission wavelength. For both compounds the vibrational
structure is better resolved in the glass than in ethanol solution
at room temperature. The limiting anisotropy ro(λ) drops from
a maximum value of 0.33 for benzindole and 0.4 for 3-meth-
ylbenzindole on the red edge of the excitation spectrum to
around zero at 300 nm. The apparent rise in ro(λ) values of
and tryptophan in propylene glycol at 245 K to resolve the indole
1
1
absorption bands into L and L components by assuming a
a
b
1
1
constant angle between the L and L transition moments. Their
a
b
analysis was consistent with an angle of about 90° between the
two moments, as also determined from polarized absorption
2
3
spectra of single crystals. An angle near 90° has been found
by a variety of theoretical procedures, including the INDO/S-
1
CIS method used in this study. In contrast, the INDO/S-CIS
3
-methylbenzindole above the theoretical limit of 0.4 is due to
computed transition dipole moment directions for benzindole
and 3-methylbenzindole are seen in Figure 6 to be nearly parallel
errors in measurements below 375 nm including light scattering.
Figure 5 shows the emission and limiting emission anisotropy
spectra excited at 360 nm for benzindole and at 370 nm for
1
and approximately short-axis polarized. For naphthalene, La
is short-axis polarized by symmetry. The integrated absorptivity
1
a
3
-methylbenzindole. The anisotropy decreases monotonically
is computed to be an order of magnitude stronger for L relative
1
b
with increasing emission wavelength in both cases. For ben-
to L , similar to the case of indole and naphthalene. Because
zindole, ro(λ) drops from 0.27 near the 0-0 band to ∼0.1 at
of the wavelength dependence of the emission anisotropy, it is
not justified to carry out the Valeur-Weber type of analysis
for the benzindoles. In addition, an attempt to do so by us gave
an angle of ∼45° between the moments, and worse, gave about
equal integrated absorptivities for the two transitions.
4
90 nm. For 3-methylbenzindole, the corresponding drop is from
0
.3 to ∼0.15. The anisotropy spectra of benzindole in glycerol
at 298 K measured in a 1 cm cell were also wavelength
dependent (not shown). The excitation anisotropy spectrum
observed at 410 nm emission wavelength dropped from about
We believe that naphthalene rather than indole is a better
model for the anisotropy behavior of the benzindoles. It is
0
.17 at 390 nm to 0.05 at 300 nm; the emission anisotropy
1
a
spectrum excited at 360 nm dropped from about 0.15 at 370
nm to 0.08 at 500 nm.
known that the L state of naphthalene shows strong vibronic
coupling with intense B states lying at somewhat higher
2
4
25
Although many aspects of the spectral behavior of benzan-
nulated indoles are similar to those of indoles, the anisotropy
is qualitatively different. For indole and 3-methylindole, the
excitation anisotropy spectra show a sharp dip at the position
energies, both experimentally and theoretically. The experi-
mental excitation anisotropy spectrum of naphthalene drops from
1
0.21 to zero across the La band, with some modulation. When
the naphthalene La band is excited, the emission anisotropy
1
1
of the Lb origin, followed by a considerable increase at shorter
spectrum drops from 0.21 to 0.13 with increasing wavlength,
with considerable modulation because of the better resolved
vibrational structure typical of nonpolar molecules. Similar large
wavelengths. The emission anisotropy spectra are virtually
22
independent of emission wavelength. Valeur and Weber were
therefore able to use the excitation anisotropy spectra of indole
1
monotonic anisotropy changes across the La band have been

Fluorescence Properties of Benz[f]indole
J. Phys. Chem. B, Vol. 104, No. 8, 2000 1841
coupling was confirmed by measuring the limiting emission
anisotropy spectrum of benzindole at 320 nm excitation
wavelength, where ro ) 0.1 when observed at 390 nm emission
wavelength. The emission anisotropy spectrum was wavelength
independent, as expected when equal amounts of two overlapped
perpendicular oscillators are excited.
Thus, our interpretation of the limiting excitation anisotropy
spectra in Figure 4 is that most of the drop at shorter
1
wavelengths comes from vibronic coupling of La with a higher
lying B state having essentially long-axis polarization, and that
this effect dominates depolarization caused by the weaker, and
1
almost collinearly polarized, Lb absorption. A possible explana-
tion for why such vibronic coupling effects are much less
noticeable for indole is that in the latter there is greater charge
1
transfer and localized C2-C3 ethylenic nature for the La
1
transition, whereas for benzannulated indole the La transition
is more dominated by the naphthalene part of the molecule.
Photophysics. Benzindole and 3-methylbenzindole have high
quantum yields and long fluorescence lifetimes in 10% metha-
nol, which are independent of solvent isotope for benzindole
and nearly so for 3-methylbenzindole (Table 1). The quantum
yield and lifetime of 3-methylcarbazole are likewise independent
7
-1
of solvent isotope. The radiative rate kr ) Φ/τ is 4.2 × 10 s
7
-1
for benzindole, 3.2 × 10 s for 3-methylbenzindole, and 2.4
7
-1
×
10 s for 3-methylcarbazole. The lifetimes of benzindole
and 3-methylbenzindole are very slightly temperature dependent,
decreasing with increasing temperature in the range 5-55 °C
from 19.6 to 18.7 ns for benzindole and from 25.7 to 23.8 ns
for 3-methylbenzindole. The lifetime of 3-methylcarbazole is
independent of temperature from 5 to 55 °C. This behavior
contrasts with simple indoles, whose quantum yield and lifetime
in aqueous solution are isotopically sensitive and temperature
dependent. Indoles have two isotopically sensitive temperature-
dependent nonradiative processes: solvent or water quenching
Figure 5. Emission (s) and limiting emission anisotropy (‚‚‚) spectra
of benz[f]indole (upper) and 3-methylbenz[f]indole (lower) in ethanol,
1
30 K. Excitation wavelength 360 nm for benz[f]indole and 370 nm
for 3-methylbenz[f]indole.
3
,29
and excited-state proton transfer.
Water quenching occurs
in aqueous solution at neutral pH in the absence of a strong
proton donor. The mechanism of water quenching is unknown,
perhaps involving an exciplex or incomplete proton transfer.
The fluorescence quantum yield of benzindole was measured
from pH(D) 1.0 to 12.5 in 10% CH3OH and in 10% CH3OD at
25 °C (Figure 7). At the extremes of pH, the quantum yields
show steep drops, which depend on solvent isotope. Isotopically
sensitive drops in quantum yield at low and high pH are also
exhibited by simple indoles and have been attributed to two
excited-state proton-transfer reactions: acid-catalyzed protona-
tion of the indole ring and base-catalyzed deprotonation of the
Figure 6. Transition moment directions calculated by INDO/S-CIS.
reported for quinoline, isoquinoline,24 tetraphene,26 and car-
24
2
7
bazole. The basis for such anisotropic behavior involving a
28
single electronic state is well understood. Zero point vibrational
motion of one (or a few) normal modes of the right symmetry
3
0
1
indole nitrogen. Assuming that analogous quenching mech-
anisms apply to benzindole, the excited-state pK*s in 10% CH3-
and form can mix strong, higher lying B state character into
1
the La state with an amplitude proportional to the nuclear
OH are about 1.1 for protonation and 11.2 for deprotonation.
displacement. This, in turn, causes the electronic transition
moment from the zero point vibrational wave function of the
excited state to have a nonzero Herzberg-Teller integral with
vibrational states of the ground electronic state that have one
quantum of excitation in the active modes. The transition
moment to the latter will have the polarization of the electronic
The corresponding pK*s for indole in aqueous buffer are 2.1
1
4
and 12.3.
Solute quenching experiments on benzindole, 3-methylindole,
and 3-methylcarbazole in 10% CH3OH using glycine, lysine,
and phenol as quenchers showed that excited-state proton
transfer does not occur in benzannulated indoles in aqueous
solution at neutral pH. No change in quantum yield or lifetime
was observed in the presence of 0.2 M quencher. In the presence
of good proton donors, such as ammonium and phenol, simple
1
28
state to which the mode couples the La state. Thus, in the
simplified case of only one vibronically active mode coupling
1
to a state polarized perpendicular to La, the emission spectrum
will have two progressions of the same shape: one beginning
1
31,32
4,29
at the 0-0 having La polarization; and the other displaced to
indoles can undergo intramolecular
or intermolecular
longer wavelengths by one quantum of the coupling mode,
photochemical H-D exchange at C2, C3, C4, and C7. The
quenching rate constant and the proton exchange rate measured
by mass spectrometry or NMR are the same, indicating that
proton transfer is the fluorescence quenching mechanism.4
Excited-state H-D exchange also occurs in simple indoles in
1
having polarization perpendicular to La. The ratio of the second
to the first of these therefore increases with decreasing excitation
wavelength or increasing emission wavelength, thereby resulting
in decreasing anisotropy. The presence of strong vibronic

1
842 J. Phys. Chem. B, Vol. 104, No. 8, 2000
TABLE 1: Quantum Yield and Lifetime Data at 25 °C
compound solvent
benz[f]indole
Liu et al.
A
max, nm
F
max, nm
Φa
τa, ns
9:1 H
:1 D
2
O/CH
O/CD
3
OH
OD
344
344
344
347
354
353
353
353
329
329
329
268
268
280
277
415
415
395
407
357
439
439
412
368
368
367
341
341
326
364
0.79 ( 0.03
0.80 ( 0.03
19.1 ( 0.1
19.3 ( 0.1
9
2
3
ethanol
glycerol
cyclohexane
3
3
-methylbenz[f]indole
9:1 H
:1 D
2
O/CH
O/CH
3
OH
OD
0.80 ( 0.03
0.84 ( 0.03
24.7 ( 0.1
25.9 ( 0.1
9
2
3
ethanol
-methylcarbazole
9:1 H
:1 D
2
O/CH
O/CH
3
OH
OD
0.23 ( 0.02
0.23 ( 0.02
9.5 ( 0.1
9.8 ( 0.1
9
2
3
ethanol
indole
9:1 H
:1 D
2
O/CH
O/CH
3
OH
OD
0.30 ( 0.01
0.37 ( 0.01
4.7 ( 0.1
6.0 ( 0.1
9
2
3
ethanol
3
-methylindole
9:1 H
:1 D
2
O/CH
O/CH
3
OH
OD
0.45 ( 0.02
0.60 ( 0.02
9.6 ( 0.1
12.7 ( 0.1
9
2
3
ethanol
Values with standard deviation are from 3 to 5 experiments.
TABLE 2: Stern-Volmer Quenching Rate Constants
282
346
a
, 10 M s-1
8
-1
k
q
quenchera
His, pH 4.7
N-acetyl-cys, pH 6.0
KI
benz[f]indole
3-methylbenz[f]indole
3-methylcarbazole
0.72
0.54
1.2
0.52
0.35
9.2
0.82
5.6
acrylamide, CH
2
dCHCONH
2
54
71
57
51
28
CH CHdCHCOOCH
3
3
NCCOOCH
acetone
3
7.1
4.5
3
3
-methyl-2-butanone
,3-dimethyl-2-butanone
4.7
2.0
6.6
YbCl
3
a
2 3
Solvent is 9:1 H O/CH OH.
examined. No ground-state complexation was detected in the
absorption spectra of benzannulated indoles in the presence of
0
.2 M quencher. Only strong electron acceptors were effective
quenchers. Weak quenchers of indole fluorescence, such as ethyl
acetate, methylcyanoacetate, N-acetylglycineamide, malonamide,
and GdCl3, do not quench the fluorescence of benzannulated
indoles. The lower limit of detection in the case of benzannulated
6
-1 -1
indoles is kq ∼ 10 M s . The side chains of two amino
acids, protonated histidine and cysteine, are weak quenchers
8
-1 -1
with quenching rate constants <10 M s . Ketones are
moderate quenchers with bulkier alkyl groups decreasing the
quenching rate constant. However, compounds with double or
triple bonds conjugated to an ester or amide group have
quenching rates approaching the diffusion limit.
Conclusions
Figure 7. pH profile of fluorescence quantum yield of benz[f]indole
in 9:1 H O/CH OH (- -b- -) and 9:1 D O/CH OD (- -O- -), 25 °C.
Excitation wavelength 365 nm, pD ) pH + 0.4. Lines drawn to connect
the points.
Benzannulation of the indole ring shifts the absorption
spectrum about 75 nm to the red, allowing more selective
excitation in the presence of tryptophan than tryptophan
analogues, such as 5-hydroxytryptophan and 7-azatryptophan.
It also raises the possibility of using tryptophan and benzindole
derivatives as energy transfer donor/acceptor pairs. We have
reported a synthesis of racemic t-Boc-benz[f]tryptophan.21 Like
tryptophan, the emission spectrum of benzindole is highly
sensitive to solvent polarity with large Stokes’ shifts in polar
solvents. Our INDO/S computations say that there is about a 4
2
3
2
3
trifluoroethanol, a solvent with higher acidity than water.33 The
quantum yield of benzindole drops in trifluoroethanol (Φ )
0
.32 in CF3CH2OH) and shows a solvent isotope effect (Φ )
0.58 in CF3CH2OD), possibly due to excited-state proton transfer
in this solvent.
Solute quenching experiments on benzannulated indoles in
1
10% CH3OH using electrophilic compounds as quenchers
D increase in the permanent dipole of La, with the pyrrole end
suggest that excited-state electron transfer is an important
fluorescence quenching mechanism (Table 2). Quenching by
ester, amide, and carbonyl groups and by lanthanides was
becoming more positive. This means that if benz[f]tryptophan
replaces a tryptophan in a protein and conserves the long axis
orientation, the local protein field is predicted to shift the

Fluorescence Properties of Benz[f]indole
J. Phys. Chem. B, Vol. 104, No. 8, 2000 1843
fluorescence in the same direction and approximate magnitude
as for tryptophan.
(5) Ross, J. B. A.; Senear, D. F.; Waxman, E.; Kombo, B. B.; Rusinova,
E.; Huang, Y. T.; Laws, W. R.; Hasselbacher, C. A. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 12023.
2
The photophysics of benzannulated indoles is simplified
somewhat compared to indoles, presumably a consequence of
the lower energy gap between ground and excited states. The
weak, if any, dependence of quantum yield and lifetime on
temperature and solvent isotope suggests that solvent quenching
is negligible in benzannulated indoles. If intersystem crossing
is the only nonradiative process, we can estimate the intersystem
(6) Ross, J. B. A.; Szabo, A. G.; Hogue, C. W. Methods Enzymol.
1997, 278, 151.
(
(
(
7) Ahmad, A.; Durocher, G. Photochem. Photobiol. 1981, 34, 573.
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(
-
1
crossing rate kisc from the nonradiative rate knr, where knr ) τ
(
7
-1
-
kr. This gives kisc values of 1.0 × 10 s for benzindole, 8.5
(13) Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. J.
Am. Chem. Soc. 1985, 107, 3902.
6
-1
7
-1
×
10 s for 3-methylbenzindole, and 8.1 × 10 s for
(
14) Stewart, J. J. P. QCPE Bull. 1990, 10, 86.
3
-methylcarbazole. Two other nonradiative processes, excited-
(15) Robinson, B. The Fischer Indole Synthesis; Wiley: London, 1982.
state proton and electron-transfer reactions, can also occur in
benzannulated indoles, but over a much narrower range of
conditions than in indoles. For example, tryptophan fluorescence
is quenched by six protein functional groups representing eight
amino acid side chains plus the peptide bond.4 However, only
two amino acid functional groups quench benzannulated indole
fluorescence in intermolecular quenching experiments. The
relative insensitivity of quantum yield and lifetime of benzan-
nulated indoles to solvent and protein functional groups make
them potentially useful as probes of peptide-protein interac-
tions. Their efficient quenching by acrylamide allows solute
accessibility studies of the spectrally enhanced species.
(16) Rydon, H. N.; Siddappa, S. J. Chem. Soc. 1951, 2462.
17) Sakamoto, T.; Kondo, Y.; Yamanaka, H. Heterocycles 1986, 24,
845.
(
1
(
18) Watanabe, T.; Miyagi, C.; Murakami, Y. J. Heterocycl. Chem. 1993,
3
0, 217.
,34
(19) Batcho, A. D.; Leimgruber, W. Org. Synth. 1985, 63, 214.
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3
1.
(
21) Yokum, T. S.; Tungaturthi, P. K.; McLaughlin, M. L. Tetrahedron
Lett. 1997, 38, 5111.
(22) Valeur, B.; Weber, G. Photochem. Photobiol. 1977, 25, 441.
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Chem. Phys. 1977, 23, 207.
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(
27) Sch u¨ tt, H.-U.; Zimmerman, H. Ber. Bunsen-Ges. Phys. Chem. 1963,
Acknowledgment. This work was supported by NIH grants
GM31824 and GM42101. We thank Mr. Paul Adams for
conducting low-temperature fluorescence measurements, Dr. T.
Scott Yokum for synthesizing 3-methylcarbazole, and Dr. Yu
Chen for helpful discussions.
6
7, 54.
(28) Fischer, G. Vibronic Coupling: The Interaction Between the
Electronic and Nuclear Motion; Academic Press: New York, 1984.
29) Yu, H.-T.; Colucci, W. J.; McLaughlin, M. L.; Barkley, M. D. J.
Am. Chem. Soc. 1992, 114, 8449.
(
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References and Notes
(
(
(
(
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(