Modular fluorescent benzobis(imidazolium) salts: Syntheses, photophysical analyses, and applications

Article abstract of DOI:10.1021/ja7102247

A series of benzobis(imidazolium) (BBI) salts has been prepared and studied as a new class of versatile fluorescent materials. Using a high yielding, modular synthetic strategy, BBI salts with a range of functionality poised for investigating fundamental

Full text of DOI:10.1021/ja7102247

Published on Web 02/14/2008
Modular Fluorescent Benzobis(imidazolium) Salts:
Syntheses, Photophysical Analyses, and Applications
Andrew J. Boydston, Peter D. Vu, Olga L. Dykhno, Vicki Chang, Alvin R. Wyatt, II,
Adam S. Stockett, Eric T. Ritschdorff, Jason B. Shear, and
Christopher W. Bielawski*
Department of Chemistry and Biochemistry, The UniVersity of Texas at Austin,
Austin, Texas 78712
Received November 11, 2007; E-mail: bielawski@cm.utexas.edu
Abstract: A series of benzobis(imidazolium) (BBI) salts has been prepared and studied as a new class of
versatile fluorescent materials. Using a high yielding, modular synthetic strategy, BBI salts with a range of
functionality poised for investigating fundamental and applications-oriented characteristics, including emission
wavelength tunability, solvatochromism, red-edge excitation, chemical stability, multiphoton excitation, and
protein conjugation, were prepared in overall yields of 40-97%. Through structural variation, the BBIs
exhibited λem ranging between 329 and 561 nm while displaying Φfs up to 0.91. In addition, the emission
characteristics of these salts were found to exhibit strong solvent dependencies with Stokes shifts ranging
from 4570 to 13 793 cm-1, depending on the nature of the BBI core. Although red-edge effects for BBI
salts with Br and BF4 counterions were found to be similar, unique characteristics were displayed by an
analogue with MeSO4 anions. The stability of an amphiphilic BBI was quantified in aqueous solutions of
varying pH, and >85% of the emission intensity was retained after 2 h at pH 3-9. Through multiphoton
excitation experiments in aqueous solutions, a BBI salt was found to exhibit three-photon fluorescence
action cross sections similar to serotonin. The application of BBI salts as fluorescent protein tags was
demonstrated by conjugating bovine serum albumin to a maleimide-functionalized derivative.
novel materials for display applications,7,8 multiphoton excitation
(MPE),9 and nanoscopic fluorescent ionic liquids.10 Overall,
Introduction
The discovery and development of novel organic emissive
materials is essential for advancement in a multitude of areas
of chemical research.1 As an emerging subclass, fluorescent
organic salts are receiving considerable attention due to their
unique attributes. They not only exhibit tunable electronic and
physical characteristics, including solution-based and solid-state
fluorescence, but also offer other advantages ascribed specifi-
cally to their charged nature. For example, the incorporation of
ionic moieties in emissive salts has been credited with imparting
high thermal stabilities, phase tunabilities, water-solubility,
chemoselective sensing via electrostatic interactions, and strength-
ened solid-state intermolecular interactions. These features have
already inspired research in a number of areas including
fundamental photophysical investigations,2,3 sensory materials,4-6
these materials encompass a tremendous breadth of structural
diversity, each targeting a specific application or area of study.
To complement these systems, we sought a single, modular
platform from which task-specific fluorophores could be
obtained rapidly and efficiently. Benzobis(imidazolium) (BBI)
salts are one class of such compounds that feature a number of
attributes poised for achieving this goal (see Figure 1). The BBI
architecture features two imidazolium ring systems annulated
to a common arene linker. An important consequence of this
arrangement is that the charged moieties are embodied within
the system’s chromophore, which distinguishes these materials
from common types of fluorescent organic salts. In addition,
BBIs feature four N-substituents, two C1-substituents, and two
(1) For excellent reviews, see the following: (a) Thomas, S. W., III.; Joly, G.
D.; Swager, T. M. Chem. ReV. 2007, 107, 1339. (b) Finney, N. S Curr.
Opin. Chem. Biol. 2006, 10, 238. (c) Glasbeek, M. Zhang, H. Chem. ReV.
2004, 104, 1929. (d) Lakowicz, J. R. Principles of Fluorescence Spectros-
copy, 2nd ed.; Plenum Publishing: New York, 1999.
(2) (a) Mandal, P. K.; Paul, A.; Samanta, A. J. Photochem. Photobiol., A 2006,
182,113. (b) Samanta, A. J. Phys. Chem. B 2006, 110, 13704. (c) Das, S.;
Fro¨lich, R.; Pramanik, A. Org. Lett. 2006, 8, 4263. (d) Paul, A.; Mandal,
P. K.; Samanta, A. J. Phys. Chem. B 2005, 109, 9148. (e) Paul, A.; Mandal,
P. K.; Samanta, A. Chem. Phys. Lett. 2005, 402, 375. (f) Mandal, P. K.;
Sarkar, M.; Samanta, A. J. Phys. Chem. A 2004, 108, 9048.
(6) (a) Harrison, B. S.; Ramey, M. B.; Reynolds, J. R.; Schanze, K. S. J. Am.
Chem. Soc. 2000, 122, 8561. (b) Balanda, P. B.; Ramey, M. B.; Reynolds,
J. R. Macromolecules 1999, 32, 3970.
(7) Xu, H.; Meng, R.; Xu, C.; Zhang, J.; He, G.; Cui, Y. Appl. Phys. Lett.
2003, 83, 1020.
(8) Chondroudis, K.; Mitzi, D. B. Appl. Phys. Lett. 2000, 76, 58.
(9) (a) Krishna, T. R.; Parent, M.; Wets, M. H. V.; Moreaux, L.; Gmouh, S.;
Charpak, S.; Caminade, A.-M.; Majoral, J.-P.; Blanchard-Desce, M. Angew.
Chem., Int. Ed. 2006, 45, 4645. (b) Woo, H. Y.; Liu, B.; Kohler, B.;
Korystov, D.; Mikhailovsky, A.; Bazan, G. C. J. Am. Chem. Soc. 2005,
127, 14721. (c) Woo, H. Y.; Korystov, D.; Mikhailovsky, A.; Nguyen,
T.-Q.; Bazan, G. C. J. Am. Chem. Soc. 2005, 127, 13794. (d) Abbotto, A.;
Beverina, L.; Bozio, R.; Facchetti, A.; Ferrante, C.; Pagani, G. A.; Pedron,
D.; Signorini, R. Org. Lett. 2002, 4, 1495.
(3) For excellent reviews, see the following: (a) Hu, Z.; Margulis, C. J. Acc.
Chem. Res. 2007, 40, 1097. (b) Demchenko, A. P. Luminescence 2002,
17, 19.
(4) Wang, S.; Chang, Y.-T. J. Am. Chem. Soc. 2006, 128, 10380.
(5) Chen, L.; McBranch, D. W.; Wang, H.-L.; Helgeson, R.; Wudl, F.; Whitten,
D. G. Proc. Nat. Acad. Sci. U.S.A. 1999, 96, 12287.
(10) For an example of an low-Tg, ionic PAMAM dendrimer exhibiting blue
photoluminescence, see the following: Huang, J.-F.; Luo, H.; Liang, C.;
Sun, I-W.; Baker, G. A.; Dai, S. J. Am. Chem. Soc. 2005, 127, 12784.
9
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J. AM. CHEM. SOC. 2008, 130, 3143-3156
3143

A R T I C L E S
Boydston et al.
Scheme 2. Synthesis of BBI Salts via Anion Metathesis
Figure 1. Key features of fluorescent benzobis(imidazolium) salts.
To further investigate the photophysics and applications of
fluorescent BBI salts, a systematic series of these materials was
prepared and studied. Syntheses and analyses were aimed at
understanding how electronic charge delocalizes throughout the
BBI chromophores, methods for controlling their emission
properties, and incorporation of task-specific functionalities.
Herein, we report a series of designer BBI fluorophores and
studies involving their absorption and emission characteristics,
solvatochromism, red-edge excitation, and chemical stability,
as well as applications in MPE and protein conjugation.
Scheme 1. Synthesis and Photoluminescence Properties of
Tetrabutyl BBI Salt 2‚Br and Tetraphenyl BBI Salt 4
Results and Discussion
Syntheses of Benzobis(imidazolium) Salts. To develop
comprehensive structure-photophysical property relationships
based upon BBI salts, focus was placed on controlling four areas
of structural modulation: (1) counterions; (2) regioisomers; (3)
N-aryl electronics; and (4) C1-aryl electronics. Photophysical
studies and applications of BBI salts are described in subsequent
sections. General syntheses utilized one of four strategies,
summarized in Schemes 2-5 below. Overall, syntheses of BBIs
2, 6, 9, 15, 16, and 26-32 required a maximum of four steps,
were chromatography-free, and afforded products in yields
ranging from 40 to 97% from commercially available starting
materials.
(a) Counterions. A unique feature of organic salts is that, in
principle, both their cation and anion constituents may be
independently tuned. While the anion has been shown to play
a key role in controlling physical properties,14,15 photophysical
anion dependencies have not been fully explored.16 This is likely
due to the fact that, for emissive organic salts, focus is placed
primarily on cationic chromophores. However, ion-pairing
interactions may greatly influence the photophysical character-
istics of an organic salt under specific conditions. For example,
red-edge effects (REEs)2,3 from charged compounds have been
attributed to different orientations of associated species.2,3 Thus,
one might expect that the counterion would influence a
fluorescent salt’s response to red-edge excitation.
counterions which can be independently varied. As such, the
physical, electronic, and chemical properties exhibited by these
materials may be finely tuned. Combined, these features may
synergistically open new opportunities for fluorescent organic
salts that not only display versatile photoluminescence charac-
teristics but also may be used in a broad array of applications.
Our preliminary interest in annulated bis(imidazolium) salts
was as precursors to facially opposed bis(N-heterocyclic car-
bene)s.11 As such, syntheses were designed to provide predomi-
nantly symmetric and exclusively C1-unsubstituted systems (i.e.,
R ) H, Figure 1). Initial efforts focused on two primary variants
exemplified in Scheme 1: one featuring four N-alkyl groups
(e.g., 2‚Br) and the other containing four N-aryl groups (e.g.,
4). In general, syntheses of the former were accomplished via
4-fold alkylation of benzobis(imidazole) (1) with an appropriate
alkyl halide.11d The latter were prepared from 1,2,4,5-tetrabro-
mobenzene (3) via Pd-catalyzed 4-fold aryl amination12,13 using
1,3-bis(2,6-diisopropylphenyl)imidazolium chloride (IPr‚HCl)
as a preligand. Subsequent formylative cyclization of the
tetraaminobenzene intermediates (not shown) resulted in BBIs
such as 4 in modest to excellent overall yields (39-88%).11c
During our initial studies, we observed that systems such as
2‚Br and 4 were highly photoluminescent with λem of 330 and
474 nm, respectively. In addition, high photoluminescence
efficiencies (Φf) were observed from each BBI, with Φf ) 0.64
and 0.41 for 2‚Br and 4, respectively. These results inspired
the design and synthesis of new subclasses of fluorescent, phase-
tunable (i.e., ionic liquid and ionic liquid crystal) BBI salts.14
To facilitate a comparative study involving counterion effects
on photophysical properties of BBIs, compounds with simple,
highly symmetric dicationic cores and either Br, BF4, or MeSO4
counterions were investigated. Capitalizing on the nucleophilic
nature of halides, 2‚Br was treated with either Et3O‚BF4 or Me2-
SO4 to produce anion metathesis17 products 2‚BF4 and 2‚MeSO4,
respectively, as shown in Scheme 2. Subsequent removal of
(11) (a) Khramov, D. M.; Boydston, A. J.; Bielawski, C. W. Angew. Chem.,
Int. Ed. 2006, 45, 6186. (b) Boydston, A. J.; Rice, J. D.; Sanderson, M.
D.; Dykhno, O. L.; Bielawski, C. W. Organometallics 2006, 25, 6087. (c)
Khramov, D. M.; Boydston, A. J.; Bielawski, C. W. Org. Lett. 2006, 8,
1831. (d) Boydston, A. J.; Williams, K. A.; Bielawski, C. W. J. Am. Chem.
Soc. 2005, 127, 12496.
(12) For additional reports regarding aryl amination, see the following: (a) Prim,
D.; Campagne, J.-M.; Joseph, D.; Andrioletti, B. Tetrahedron 2002, 58,
2041. (b) Wolfe, J. P.; Wagaw, S.; Marcox, J.-F.; Buchwald, S. L. Acc.
Chem. Res. 1998, 31, 805. (c) Hartwig, J. F. Angew. Chem., Int. Ed. 1998,
37, 2046.
(13) For another example of 4-fold aryl amination of tetrahaloarenes, see the
following: Wenderski, T.; Light, K. M.; Ogrin, D.; Bott, S. G.; Harlan, C.
J. Tetrahedron Lett. 2004, 45, 6851.
(14) Boydston, A. J.; Pecinovsky, C. S.; Chao, S. T.; Bielawski, C. W. J. Am.
Chem. Soc. 2007, 129, 14550.
(15) (a) Kouwer, P. H. J.; Swager, T. M. J. Am. Chem. Soc. 2007, 129, 14042.
(b) Lo´pez-Martin, I.; Burello, E.; Davey, P. N.; Seddon, K. R.; Rothenberg,
G. Chem. Phys. Chem. 2007, 8, 690. (c) Yoshio, M.; Ichikawa, T.; Shimura,
H.; Kagata, T.; Hamasaki, A.; Mukai, T.; Ohno, H.; Kato, T. Bull. Chem.
Soc. Jpn. 2007, 80, 1836. (d) Binnemans, K. Chem. ReV. 2005, 105, 4148.
(16) Fluorescent anion sensors require separate consideration, see ref 1a and
the following: (a) Gunnlaugsson, T.; Glynn, M.; Tocci, G. M.; Kruger, P.
E.; Pfeffer, F. M. Coord. Chem. ReV. 2006, 250, 3094. (b) Badugu, R.;
Lakowicz, J. R.; Geddes, C. D. Curr. Anal. Chem. 2005, 1, 157. (c)
Martinez-Ma´n˜ez, R.; Sanceno´n, F. Chem. ReV. 2003, 103, 4419. (d) Sessler,
J. L.; Tvermoes, N. A.; Davis, J.; Anzenbacher, P., Jr.; Jurs´ıkova´, K.; Sato,
W.; Siedel, D.; Lynch, V.; Black. C. B.; Try, A.; Andrioletti, B.; Hemmi,
G.; Mody, T. D.; Magda, D. J.; Kra´l, V. Pure Appl. Chem. 1999, 71, 2009.
(17) Vu, P. D.; Boydston, A. J.; Bielawski, C. W. Green Chem. 2007, 9, 1158.
9
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Modular Fluorescent Benzobis(imidazolium) Salts
A R T I C L E S
Scheme 4. Synthesis of BBI Salts with Electronically Modified
Scheme 3. Synthesis of Regioisomeric Diphenyl
Dimethylbenzobis(imidazolium) Salts
N-Aryl Substituents
nature of the N-substituent (cf., 2‚Br and 4 in Scheme 1).14 To
further explore the effects of differential N-aryl electronic
factors, BBIs bearing electron-donating and withdrawing N-aryl
groups were synthesized as shown in Scheme 4. This was
accomplished from 2,4-dinitro-1,5-dichlorobenzene (10), which
underwent SNAr reactions with benzocaine and p-anisidine to
provide dinitroarenes 11 and 12, respectively. Reduction fol-
lowed by in situ formylative cyclization afforded the corre-
sponding benzobis(imidazole)s 13 and 14. Finally, alkylation
with MeI afforded the BBI salts 15 and 16 in 94 and 74% overall
yield, respectively, from 10. As will be discussed below,
incorporation of electron-rich N-arenes bathochromically shifted
the λem of these compounds by as much as 61 nm (compare:
16, λem ) 469 nm, versus 15, λem ) 408 nm, in MeOH). In
contrast, electron-poor N-arenes resulted in significant hypso-
residual, volatile byproducts afforded each of the desired BBIs
in excellent isolated yields (g95%). As will be discussed below,
the counterions had little effect on the λem, provided excitation
was performed at the λabs; however, analysis of these compounds
ultimately revealed counterion-dependent REEs in dilute solu-
tions.
(b) Regioisomers. Incorporation of distinct functional groups
around the BBI core was expected to provide an additional
degree of control over the λem of the resulting BBIs. Prior to
engaging in the synthesis of compounds bearing substituents
with different electronic properties, however, we investigated
the effects of site-specific N-arylation. Hence, BBI regioisomers
bearing two N-phenyl and two N-methyl groups in various
substitution patterns were synthesized as shown in Scheme 3.
Benzobis(imidazole) 1 underwent Cu-catalyzed cross coupling
with PhI to provide a 1:1 mixture of 1,7-diphenyl- (5syn) and
1,5-diphenylbenzobis(imidazole)s (5anti) in 95% combined
yield.18,19 Separation of the two isomers was accomplished via
recrystallization from hot DMSO.20 Subsequent alkylation with
Me2SO4 afforded BBIs 6syn and 6anti, respectively.21 As shown
in Scheme 3, the other possible regioisomer (9) was obtained
from 5,6-dichloro-1-methylbenzimidazole (7).22 Aryl amination
with PhNH2, followed by formylative cyclization of the resulting
diamine with HC(OEt)3, afforded benzimidazolium chloride 8
in 86% yield. Treatment of 8 with 2.0 equiv of Me2SO4 not
only resulted in N-methylation but also effectively removed the
Cl anion (as gaseous MeCl), affording the desired bis(methyl
sulfate) salt (9) in 85% overall yield from 7. As will be discussed
below, BBI 6syn displayed a longer λem (447 nm) than 6anti (442
nm) and 9 (403 nm). Hence, further structural modulation
focused on 1,7-diaryl BBIs.
chromic shifts in the λem
.
(d) Differential N- and C1-Substitution Effects. The ability
to independently functionalize the N- and C1-positions of the
BBI structure provides an additional handle for precisely tuning
BBI photophysical properties. To facilitate solubility in common
organic solvents (see below), BBIs bearing N-(4-BuPh) groups
were targeted. Furthermore, these BBIs provided a platform for
independently comparing different C1-substituents while main-
taining constant N-groups (i.e., BBIs 26-29). In addition to
solubility enhancement, the N-aryl substituents were also used
to modulate the electronic characteristics of the BBI chro-
mophores. Specifically, N-(4-MeOPh) groups were incorporated
into C1-substituted BBIs 30 and 31 to ultimately provide longer
wavelength λem than other analogues (see below for a full
discussion).
Dinitroarenes 12 (Scheme 4) and 17 (Scheme 5) were each
synthesized from SNAr reactions of 1,5-dichloro-2,4-dinitroben-
zene (10) with the corresponding aryl amines. Reduction of the
nitro groups using Pd/C and hydrazine under acidic conditions
afforded the corresponding tetraaminobenzenes 18 and 19, which
were each isolated as their hydrochloride salts in >99% yield.23
Reaction of these tetraamines with various aroyl chlorides
afforded diamide intermediates (structures not shown), which
underwent dehydrocyclization in AcOH to give benzobis-
(imidazole)s 20-25 in yields ranging from 78 to 99% over the
two steps.24 The aroyl chlorides chosen for study were com-
mercially available and encompassed both electron-withdrawing
(e.g., R ) 4-CNPh) and -donating functional groups (e.g., R )
(c) N-Aryl Electronic Effects. As stated previously, the
photophysics of BBI salts were found to be dependent on the
(18) In this context, we use the subscripts “syn” and “anti” to indicate the relative
positions of the N-phenyl groups about the BBI core.
(19) For an example of this type of coupling using benzimidazole as a substrate,
see the following: Harkal, S.; Rataboul, F.; Zapf, A.; Fuhrmann, C.;
Riermeier, T.; Monsees, A.; Beller, M. AdV. Synth. Catal. 2004, 346, 1742.
(20) Chromatographic separation on silica gel (1:1 EtOAc/Et2O eluent) was also
effective. An alternate synthesis of 5syn has been previously reported.14
(21) An alternate synthesis of BBI 6syn has been previously reported.14
(22) For a synthesis of 7, see the following: Feitelson, B. N.; Mamalis, P.;
Moualim, R. J.; Petrow, V.; Stephenson, O.; Sturgeon, B. J. Chem. Soc.
1952, 2389.
(23) These compounds were found to decompose gradually upon storage under
ambient conditions. Under dry N2, they showed no signs of decomposition
for >2 months.
(24) For related synthetic approaches to C1-aryl benzimidazoles, see the
following: (a) Xin, Z. et al. J. Med. Chem. 2006, 49, 4459. (b) Gong, B.;
Hong, F.; Kohm, C.; Bonham, L.; Klein, P. Bioorg. Med. Chem. Lett. 2004,
14, 1455. (c) McKee, R.; Bost, R. J. Am. Chem. Soc. 1947, 69, 471.
9
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A R T I C L E S
Boydston et al.
Scheme 5. Synthesis of BBI Salts Bearing N- and C1-Aryl
Substituents
Figure 2. BBI salts used to evaluate the electronic impacts of regioisomeric
placement of N-aryl and N-alkyl substituents.
4-MeOPh, 2-thienyl, and 4-biphenyl). Subsequent alkylation of
bis(imidazole)s 20-25 with MeI provided a series of highly
functionalized BBI salts (26-31) in overall yields of 67-94%
based on 10. As discussed below, the different electron-donating
and -withdrawing capabilities of the C1-substituents lead to λem
tunability over a broad range of wavelengths (393-561 nm).
Ultimately, through judicious combinations of N- and C1-aryl
groups, we were able to prepare BBI salts with λem that were
bathochromically shifted by up to 227 nm relative to BBI 2‚
Br; specific details are discussed in the following sections.
Photophysical Analyses. With a systematic series of BBI
salts in hand, attention turned toward studying the electronic
and physical characteristics of these materials; a summary of
key results is shown in Table 1. Collectively, these BBI salts
displayed good Φfs and molar absorptivities as well as a broad
range of λem. The following sections summarize our investiga-
tions of probing how structural, substituent, and solvent varia-
tions influence the photophysical properties of BBI fluorophores.
The utilities of BBI salts in a variety of applications are also
discussed.
As stated previously, the emission properties of BBI salts
are strongly influenced by the nature of its N-substituents. In
particular, comparing 2‚Br versus 4 revealed a relatively large
∆λem (124 nm) by incorporating N-phenyl (4) versus N-alkyl
(2‚Br) groups. Hence, we aimed to better understand the impact
of systematically placing N-aryl groups at different positions
on the BBI core. Specifically, regioisomeric BBIs 6syn, 6anti, and
9 (see Figure 2) were analyzed by UV-vis and fluorescence
spectroscopies in methanolic solutions. While each of the
regioisomers displayed similar λabs (345-347 nm), the longest
wavelength λem were obtained when N-phenyl substituents were
placed on opposite imidazolium rings. In particular, BBIs 6syn
and 6anti showed λem at 447 and 442 nm, respectively, whereas
BBI 9, which bears two N-phenyl substituents on the same
imidazolium ring, exhibited a λem at 403 nm. Considering BBI
6syn displayed the longest λem and was straightforward to
synthesize, subsequent efforts focused on studying derivatives
of this compound to further tune its emissive properties (see
below).25
Figure 3. BBI salts used to evaluate N-aryl electronic effects on λem. Values
shown are λem (excitation at the λabs) in MeOH under ambient conditions.
uents, we compared BBIs 6syn, 15, and 16 (see Figure 3).
Whereas BBI 6syn showed a λem at 447 nm, incorporation of
electron-withdrawing groups on the N-aryl substituents (15)
resulted in a hypsochromically shifted λem at 408 nm. In contrast,
BBI 16, which bears electron-donating groups on the N-aryl
substituents, showed a λem at 469 nm. Thus, from this series, it
was apparent that increased electron density at the N-substituent
effectively increased the λem of BBIs.
To further study electronic communication within the BBI
chromophore, and to probe a new avenue for further tuning the
λem exhibited by these materials, we prepared an N-to-N′
“donor-acceptor” BBI (32) featuring N-(4-MeOPh) and
N′-(4-EtO2CPh) groups (see Figure 3 for the structure of this
compound). The synthesis (not shown) of 32 began with 1,5-
dichloro-2,4-dinitrobenzene (10), which was treated in a step-
wise fashion with benzocaine (to afford intermediate 33)
followed by p-anisidine to provide the respective dinitroarene
34 in 93% yield over the two steps. Following reductive
cyclization, the resulting benzobis(imidazole) 35 was alkylated
with MeI to provide BBI 32 in 78% overall yield from 10.
Analysis of the emission spectra of this compound revealed a
λ
em at 472 nm, representing a slight bathochromic shift relative
to 16. Collectively, comparison of various BBI salts used to
evaluate N-aryl electronic effects (shown in Figure 3) not only
revealed an ability to tune the emission characteristics through
N-aryl variation but also established a means to increase λem
primarily via incorporation of electron-rich N-arenes.
,
Next, we turned our attention toward evaluating the feasibility
of tuning fluorescence properties through C1-substitution.
Analysis of BBIs 26-31 (see Figure 4) revealed a strong
dependence of the λem on the nature of the C1-substituent. Since
C1-substituted BBI fluorophores were essentially unexplored,
we initially investigated the effects of incorporating phenyl
groups at these positions. Thus, we compared BBI 6syn, which
is unsubstituted at C1, with C1-Ph substituted BBI 26. The λem
of the latter appeared at a considerably longer wavelength (507
Effects of Incorporating Functional N-Aryl Groups. To
compare the effects of electronically tuning the N-aryl substit-
(25) For comparison, 1-methyl-3-phenylbenzimidazolium iodide exhibited λabs
at 270 nm and λem at 392 nm under identical conditions.
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Modular Fluorescent Benzobis(imidazolium) Salts
A R T I C L E S
Table 1. Photophysical Properties of Benzobis(imidazolium) Saltsa
BBI
R1
R2
R3
R4
R5
X
λ
abs (nm)a
log(ꢀ a
)
λ
em (nm)a
Φfb
yield (%)c
2‚Br
H
H
H
H
H
H
H
H
H
H
Ph
4-CNPh
4-MeOPh
2-Th
4-PhPh
4-CNPh
Bu
Bu
Bu
Ph
Me
Ph
Bu
Bu
Bu
Ph
Bu
Bu
Bu
Ph
Ph
Me
Ph
4-(CO2Et)Ph
4-MeOPh
4-MeOPh
4-BuPh
4-BuPh
4-BuPh
4-BuPh
4-MeOPh
4-MeOPh
Bu
Bu
Bu
Ph
Ph
Ph
Me
4-(CO2Et)Ph
4-MeOPh
4-(CO2Et)Ph
4-BuPh
4-BuPh
4-BuPh
4-BuPh
4-MeOPh
4-MeOPh
Br
BF4
MeSO4
Cl
MeSO4
MeSO4
MeSO4
I
I
I
I
I
I
I
I
I
288
290
289
352
345
345
347
360
343
362
311
388
332
349
331
389
4.29
4.25
4.26
3.94
3.31
4.05
3.92
4.13
3.93
3.60
4.54
4.94
4.51
4.00
4.61
4.91
330
329
331
474
447
442
403
408
469
472
507
551
393
400
461
561
0.64
0.55
0.63
0.41
0.91
0.38
0.31
0.02
0.05
0.01
0.14
0.43
0.55
0.18
0.05
0.06
97
92
96
64
86
40
85d
94
74
78
90
87
86
67
79
94
2‚BF4
2‚MeSO4
4
6syn
6anti
9
15
16
32
26
27
28
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
29
30
31
a Data taken in MeOH under ambient conditions. b Quantum efficiencies (Φf) were determined relative to E-stilbene, anthracene, or 9,10-
bis(phenylethynyl)anthracene. c Isolated overall yield from commercial material. d Isolated overall yield from 5,6-dichloro-1-methylbenzimidazole (7).
Figure 5. Diagram of the proposed major electronic communication
pathways throughout the BBI chromophore: D ) electron donor; A )
electron-acceptor.
the λem of these BBIs to appear at wavelengths that were lower
than even C1-unsubstituted BBI 6syn
.
Electron-withdrawing C1-substituents, on the other hand,
increased λem relative to BBI 26. For example, installation of
4-CNPh groups at C1 (27) resulted in a λem at 551 nm, which
is bathochromically shifted by 44 nm relative to 26. Overall, it
appeared that electronic influences from the N- and C1-
substituents were complementary in that increased electron
density at the N-positions and decreased electron density at C1
each resulted in longer λem. Considering these two factors, we
envisioned it should be possible to take advantage of a donor-
acceptor functionalized BBI featuring electron-donating N-aryl
groups in combination with electron-withdrawing C1-aryl groups
to further increase λem. Such a system was expected to display
electronic communication as depicted in Figure 5.
To investigate, we synthesized BBI 31 which features
4-MeOPh groups at the 1- and 7-positions and 4-CNPh groups
at C1. The λem of BBI 31 appeared at 561 nm, which is the
longest λem of all the BBIs described herein. Although the
emission spectra of 31 and 27 were very similar (see Figure 4),
close examination revealed that the λem for 31 was slightly
bathochromically shifted relative to 27 (λem ) 551 nm). Notably,
both BBI 27 and 31 undergo efficient excitation in the visible
region, as the absorption cutoff of each appeared at ap-
proximately 450 nm.
Figure 4. Photoluminescence spectra of C1-aryl BBI salts in MeOH,
excited at the absorption maximum, under ambient conditions. The
concentration is ca. 5 µM; signal intensities have been normalized.
nm) than the former (447 nm). Having revealed a strong
dependence of the λem on the C1-substituent, we considered
electronic variation at this position as an additional parameter
for tuning this property.
Installation of electron-donating C1-aryl groups such as
4-MeOPh (28), 2-thienyl (29), or 4-biphenyl (30) resulted in
hypsochromic shifts in the λem relative to BBI 26. The
magnitudes of these shifts were consistent with the relative
electron-donating abilities of the respective C1-arenes (i.e.,
4-MeOPh > 2-thienyl > 4-biphenyl). In other words, increased
electron density at C1 was consistently accompanied by a
decrease in λem. Interestingly, the effect of increased electron
density at C1 in BBIs 28 and 29 was significant enough to cause
Solvent Effects on Absorption and Emission Properties.
Many classes of molecules exhibit solvent-dependent photo-
9
J. AM. CHEM. SOC. VOL. 130, NO. 10, 2008 3147

A R T I C L E S
Boydston et al.
Scheme 6. Proposed Excited-State Reaction Giving Rise to Large
Stokes Shifts and Positive Solvatofluorochromism
Figure 6. BBI salts used to evaluate photophysical solvent effects as a
function of structure.
Table 2. Solvent Effects on the Photophysical Properties [λabs/λem
(nm)] of Various BBI Saltsa
BBI
MeCN
(MeO)3PO
DMF
DMSO
27
27‚BF4
26
28
21
312/437
312/446
310/420
331/392
350/436
313/439
313/436
310/412
331/390
336/438
315/551
315/550
312/412
332/394
358/439
315/557
316/561
314/438
333/395
354/442
Table 3. Stokes Shifts (cm-1) of BBIs 26-28 and Bis(imidazole)
21 in Different Solvents
BBI
MeCN
(MeO)3PO
DMF
DMSO
27
26
28
21
9168
8449
4701
5636
9170
7986
4570
6931
13597
7779
4740
5154
13793
9016
4714
5624
a BBIs 26-28 were used as their diiodide salts. Data were taken under
ambient conditions. Concentration were ca. 5 µM. Solvent donor numbers
and dielectric constants respectively are as follows: MeCN ) 14.1, 38.0;
(MeO)3PO ) 23.0, 20.6; DMF ) 26.6, 38.3; DMSO ) 29.8, 45.0.34
a Stokes shifts, in various solvents, given in cm-1 by 107(1/λem - 1/λabs).
Solvents are listed in order of increasing donor number (DN).
physical properties.26-28 Commonly, the λabs and λem respond
to changes in solvent dielectric in a manner consistent with
changes in the polarity of the chromophore as it transitions from
the ground state to an excited state. Other factors such as solvent
reorganization energies, solvent donor and acceptor abilities,
and chemical reactions taking place in an excited state can each
alter the Stokes shift, and thus the λem, as the solvent is
varied.29,30 Regardless of the origins, it is important to note that
solvent dependency can offer an excellent handle for tuning
the photophysical characteristics of a solute.31
To probe the effects of different solvents on the photophysical
properties of BBI salts, the absorption and emission spectra of
BBI 27 (see Figure 6) were analyzed in MeCN, (MeO)3PO,
DMF, and DMSO; limited solubilities precluded analysis in less
polar solvents (i.e., PhMe, THF, CH2Cl2, dioxane, etc.).32 For
comparison, more electron-rich BBIs 26 and 28, as well as
benzobis(imidazole) 21, were also investigated (see below for
further discussion). As shown in Table 2, the λabs of 27 remained
fairly constant upon increasing solvent polarity. The λem of this
compound, however, was found to be strongly dependent on
the nature of the solvent, displaying positive solvatofluoro-
chromism with λem ranging from 437 to 557 nm.33
reactions.35 For comparison, the Stokes shifts exhibited by BBIs
26 (7779-9016 cm-1) and 28 (4570-4740 cm-1) were smaller
than 27. The relative magnitudes of the Stokes shifts observed
for these BBIs were consistent with the trend in λem and may
be rationalized in terms of the relative electron densities on the
BBI cores.
As described in Scheme 6, one plausible reorganization
pathway may involve nucleophilic attack at one of the electro-
philic C1-positions. Such an event would result in a strongly
dipolar system capable of extended delocalization, features
commonly manifested in large positive solvatofluorochromism.26,27
The most likely nucleophilic species (Nuc) are either an iodide
counterion or a solvent molecule. To exclude the former from
consideration, we prepared 27‚BF4 from 27 (which contains
iodide counteranions) using the anion metathesis method
previously described.17 As shown in Table 2, nearly identical
spectroscopic signals were observed in each of the aforemen-
tioned solvents suggesting that excited-state structural changes
were independent of the anion.
We next considered the solvent as a possible nucleophile in
the mechanism described in Scheme 6. As stated above, an
interesting relationship was realized after comparing the sol-
vatofluorochromic shifts with solvent DNs, which is a semi-
quantitative comparison of solvent nucleophilicities,34 and
dielectric constants (κ).34 The λem for BBI 27 correlated better
with increasing solvent DN than with κ. For example, MeCN
and DMF have nearly identical κ (38.0 and 38.3, respectively),
but different DN (14.1 and 26.6, respectively). The λem of 27
appeared at 437 nm in MeCN but was shifted to 551 nm in
DMF. This strong dependence on solvent DN, in combination
with counterion independence (cf., 27 and 27‚BF4; see above),
further supported the proposed excited-state reaction described
in Scheme 6.
The Stokes shifts exhibited by BBI 27 ranged from 9168 to
13 793 cm-1, depending on the donor number (DN) of the
solvent used.34 These values are in the range of unusually large
Stokes shifts (5000-15 000 cm-1), commonly ascribed to
structural reorganization of the fluorophore or excited-state
(26) For excellent reviews, see the following: (a) Reichardt, C. Green Chem.
2005, 7, 339. (b) Minkin, V. I. Chem. ReV. 2004, 104, 2751. (c) Reichardt,
C. Chem. ReV. 1994, 94, 2319. (d) Buncel, E.; Rajagopal, S. Acc. Chem.
ReV. 1990, 23, 226.
(27) For a discussion of solvatochromism in quadrupolar fluorophores, see the
following: Terenziani, F.; Painelli, A.; Katan, C.; Charlot, M.; Blanchard-
Desce, M. J. Am. Chem. Soc. 2006, 128, 15742.
(28) Reichardt, C. SolVents and SolVent Effects in Organic Chemistry, 3rd ed.;
Wiley-VCH: Weinheim, Germany, 2003.
(29) Mertz, E. L.; Tikhomirov, V. A.; Krishtalik, L. I. J. Phys. Chem. A 1997,
101, 3433.
To further explore the source of the observed solvatofluoro-
chromism, we compared the solvent dependencies of the λem
and Stokes shifts exhibited by BBIs 26 and 28, as well as neutral
benzobis(imidazole) 21, with the more electron-deficient dicyano
(30) Marcus, R. A. J. Chem. Phys. 1963, 38, 1858.
(31) Joshi, H. S.; Jamshidi, R.; Tor, Y. Angew. Chem., Int. Ed. 1999, 38, 2722.
(32) Similarly, BBI 43 was excluded from comparative analyses due to poor
solubility. See Experimental Section for details.
(33) To clarify, the terms “solvatochromism” and “solvatofluorochromism” are
used to describe solvent-induced changes in λabs and λem, respectively.
(34) Gutmann, V. Coordination Chemistry in Non-Aqueous Solutions; Springer-
Verlag: New York, 1968.
(35) Doroshenko, A. O. Theor. Exp. Chem. 2002, 38, 135.
9
3148 J. AM. CHEM. SOC. VOL. 130, NO. 10, 2008

Modular Fluorescent Benzobis(imidazolium) Salts
A R T I C L E S
Figure 8. Electronic absorption (solid line), excitation (dashed line,
emission intensity monitored at 330 nm), and emission (dotted line,
excitation at 290 nm) spectra of 2‚MeSO4 in MeOH under ambient
conditions.
Figure 7. Solutions of BBIs 27 (left) and 31 (right) irradiated with a 365
nm (5 W) lamp. Solvents in each series from left to right: MeCN; EtOH;
pyridine.
BBI 27 (see Tables 2 and 3). In contrast with 27, BBI 26 showed
only relatively small changes in λem (412-438 nm) and Stokes
shifts (7779-9016 cm-1) upon varying the solvent (Table 3).
Similar effects were observed with BBI 28 and benzobis-
(imidazole) 21, which also exhibited relatively narrow ranges
of λem and Stokes shifts. However, more generally, no discern-
ible trends were observed between the λem or Stokes shifts for
21, 26, and 28 and solvent DN. In other words, a positive
correlation between λem (and Stokes shifts) and solvent DN was
observed only for a relatively electron deficient BBI (i.e., 27).
The other compounds studied were apparently not sufficiently
electrophilic to undergo nucleophilic attack from solvent
molecules (Scheme 6). Hence, the dicationic nature of BBI 27,
in combination with the electron-withdrawing C1-substituents,
appear to be primarily responsible for the large solvatofluoro-
chromic shifts. In particular, the electrophilic C1-position may
be involved in an excited-state reaction that is facilitated by
electron-withdrawing C1-substituents but impaired by electron-
donating C1-arenes.
Figure 9. Emission spectra of 2‚Br (1 µM) in MeOH under ambient
conditions excited at 330 (solid line), 340 (dashed line), and 350 nm (dotted
line).
involve different ground-state orientations of associated spe-
cies.2,37 Considering the BBI structure comprises two imidazo-
lium moieties, we envisioned REEs may be observed from these
materials. In addition, differential ion-pairing interactions of
BBIs 2‚Br, 2‚BF4, and 2‚MeSO4 may manifest in counterion-
dependent REEs.
Since REEs of BBI-type fluorophores were unexplored, we
began our investigations with a simple BBI structure (2) that
would also be amenable to probing counterion effects. To
determine if a REE was observable from BBI solutions, and
explore any resulting counterion dependencies, MeOH solutions
(ca. 1 µM) of BBIs 2‚Br, 2‚BF4, and 2‚MeSO4 were each excited
at various wavelengths along the red-edge of their excitation
spectra. As shown in Table 1, the absorption and emission
spectra (excitation at the λabs) for 2 in MeOH were independent
of the counterion; a representative set of spectra for 2‚MeSO4
is shown in Figure 8.
Since electron-withdrawing C1-substituents appeared to be
mandatory for observing solvatofluorochromism, we envisioned
tuning solvent effects by modulating the N-arenes while
maintaining constant C1-(4-CNPh) groups. Indeed, as can be
seen in Figure 7, marked differences in the photoluminescent
properties of BBIs 27 and 31 were observed upon irradiation
of these compounds in different solvents. Overall, these results
underscore the potential for solvent effects to influence the
emission characteristics of BBIs.
Both 2‚Br and 2‚BF4 gave nearly identical spectra upon red-
edge excitation; the resulting spectra for 2‚Br are depicted in
Figure 9. Excitation at 330 nm resulted in a shoulder protruding
Red-Edge Effects. A red-edge effect (REE) refers to shifting
of the λem to longer wavelengths in response to an increase in
the excitation wavelength, along the red-edge of the absorption
band.3,36 Multiple polar organic fluorophores have been found
to display REEs, phenomena that have found applications in a
number of areas.2a,3 Particularly interesting to our studies were
recent reports of REEs from imidazolium-based ionic liquids.2
The specific cause of the REE in imidazolium species is not
entirely understood, but it has been speculated that its origins
at ca. 410 nm, which became the only apparent peak at λex
g
340 nm. Notably, a mixture of 2‚Br and 2‚BF4 (1:1 molar ratio)
also gave the same response. In contrast, red-edge excitation
of 2‚MeSO4 gave bimodal emission spectra that continued to
shift bathochromically when excited at 330, 340, and 350 nm
(see Figure 10). The difference in fine structure and overall
response to red-edge excitation between 2‚MeSO4 and 2‚Br/
BF4 strongly suggested that the counterion plays a decisive role
in dictating the photophysical properties of 2 under these
(36) (a) Galley, W. C.; Purkey, R. M. Proc. Natl. Acad. Sci. U.S.A. 1970, 67,
1116. (b) Rubinov, A. N.; Tomin, V. I. Opt. Spektrosk. 1970, 29, 1082. (c)
Weber, G.; Shinitzky, M. Proc. Natl. Acad. Sci. U.S.A. 1970, 65, 823. (d)
Weber, G. Biochem. J. 1960, 75, 335.
(37) In solution, a REE is attributed to excitation of ground-state molecules
that are most strongly interacting with solvent molecules; see the follow-
ing: Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum
Publishing: New York, 1983.
9
J. AM. CHEM. SOC. VOL. 130, NO. 10, 2008 3149

A R T I C L E S
Boydston et al.
Figure 10. Emission spectra of 2‚MeSO4 (1 µM) in MeOH under ambient
conditions excited at 330 (solid line), 340 (dashed line), and 350 nm (dotted
line).
conditions. These results demonstrate, for the first time, REEs
in extended, highly photoluminescent imidazolium-based chro-
mophores and support the contention that REEs may be
influenced by cation-anion interactions.
Figure 11. Three-photon fluorescence action cross section spectrum of
BBI 4.
Scheme 7. Possible Reaction Pathways of BBI Salts under Basic
Aqueous Conditions
Multiphoton Excitation. Multiphoton-excited fluorescence
of organic chromophores has provided important capabilities
for three-dimensionally resolved microscopy,38,39 simultaneous
measurements of multiple fluorophores,39 and low-background
analytical assays.40 Although two- and three-photon fluorescence
action cross-section values are typically small, fluorescence can
be promoted with relatively low excitation powers by using high
peak-power pulsed sources such as a femtosecond titanium:
sapphire (Ti:S) laser. Recently, particular attention has been
given to water-soluble organic salt fluorophores for applications
in biological membranes, cells, and pH sensing.9 Since mul-
tiphoton excitation (MPE) in BBI chromophores has not been
investigated, we probed the ability of this new class of
fluorophore to undergo MPE, particularly in aqueous solutions.
Thus, multiphoton fluorescence action cross sections of BBI 4
were determined semiquantitatively for excitation between 740
and 860 nm.
pH Stability. The potential of BBI fluorophores will ulti-
mately depend on their stabilities in basic and acidic media.
We have previously demonstrated that BBI salts can be
deprotonated44 under strongly basic conditions to generate bis-
(N-heterocyclic carbene)s.45 When the N-substituents were
small, such as 1°-alkyl groups (e.g., 2‚Br), carbene dimerization
ensued which resulted in the formation of poly(enetetraamine)s
as depicted in Scheme 7. Enetetraamines are well-known to be
extremely oxygen sensitive and rapidly convert to ureas upon
exposure to air, a fate that would ultimately preclude the use
of BBI fluorophores at high pH.46 Decomposition of imidazo-
lium species under acidic conditions, on the other hand, has
not been investigated; hence, a lower pH limit of stability may
also exist for this class of molecules. Notably, an alternate
decomposition pathway to deprotonation may involve hydrolysis
of the BBI to generate a diamide, which upon further hydrolysis
and oxidation would result in a diamino-benzoquinonediimine.
The uncertainty of the pH tolerance of BBI fluorophores
prompted us to explore the pH stability of a representative BBI
(2‚Br) in aqueous solutions; key data are summarized in
Table 4.
To begin investigating MPE of BBI salts, BBI 4 was prepared
in unbuffered water at 265 µM. Power studies were then
performed at 740, 760, 820, and 860 nm to determine the laser
power dependence of multiphoton-excited fluorescence. Loga-
rithmic plots of the fluorescence emission versus incident laser
power yielded linear least-square fits whose slopes represent
the number of photons required for molecular excitation. At all
wavelengths examined herein, BBI 4 was found to be excited
primarily via a three-photon mechanism. Specifically, log-log
slopes at 740, 780, 820, and 860 nm were found to be 3.00,
3.00, 3.01, and 2.98, respectively.
Three-photon fluorescence action cross sections were deter-
mined over this wavelength range using estimates of the spectral
functions of the optics and detector, laser pulse width, and beam-
waist size.38 As shown in Figure 11, the cross section of BBI 4
was greatest at the short wavelength limit of these studies. These
results show absolute three-photon cross sections similar to those
determined for the native biological fluorophore, serotonin,38
a
compound that has been used in a range of microscopy38,41 and
microanalysis applications.42,43
(41) Williams, R. M.; Shear, J. B.; Zipfel, W. R.; Maiti, S.; Webb, W. W.
Biophys. J. 1999, 76, 1835.
(42) Gostkowski, M. L.; Wei, J.; Shear, J. B. Anal. Biochem. 1998, 260, 244.
(43) Wise, D. D.; Shear, J. B. J. Chromatogr., A 2006, 1111, 153.
(44) The pKa of imidazolium salts has been estimated to be 18-22; see the
following: Amyes, T. L.; Diver, S. T.; Richard, J. P.; Rivas, F. M.; Toth,
K. J. Am. Chem. Soc. 2004, 126, 4366.
(45) Kamplain, J. W.; Bielawski, C. W. Chem. Commun. 2006, 1727.
(46) (a) Shi, Z.; Thummel, R. P. J. Org. Chem. 1995, 60, 5935. (b) Winberg,
H. E.; Downing, J. R.; Coffman, D. D. J. Am. Chem. Soc. 1965, 87, 2054.
(38) Maiti, S.; Shear, J. B.; Williams, R. M.; Zipfel, W. R.; Webb, W. W. Science
1997, 275, 530.
(39) Zipfel, W. R.; Williams, R. M.; Webb, W. W. Nat. Biotechnol. 2003, 21,
1369.
(40) Okerberg, E.; Shear, J. B. Anal. Chem. 2001, 73, 1610.
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3150 J. AM. CHEM. SOC. VOL. 130, NO. 10, 2008

Modular Fluorescent Benzobis(imidazolium) Salts
A R T I C L E S
Table 4. Relative Emission Intensities Observed from BBI 2‚Br at
Scheme 8. Synthesis of Task-Specific BBI Salt 36
Various Conditions and Durationsa
Ix/I7 at time n
30 min
In/Io at pH X
120 min
pH
0 min
120 min
30 min
1
3
5
7
9
0.09
0.87
0.87
1.00
0.86
0.01
0.09
0.92
0.89
1.00
0.91
0.01
0.10
1.00
1.04
1.00
0.98
0.01
0.94
0.98
0.95
0.93
0.98
0.98
0.93
0.98
1.02
0.85
0.97
1.03
11
a Emission data collected under ambient conditions. Excitation at 290
nm. Ix ) total integrated emission intensity at pH ) X and time indicated.
I7 ) total integrated emission intensity at pH ) 7 and time indicated. Io )
total integrated emission intensity at time 0 for the pH indicated.
Initial efforts focused on studying BBI 2‚Br in neutral H2O.
Under these conditions, this compound showed absorption and
emission characteristics nearly identical with those observed in
MeOH. After 30 min in H2O (pH 7), emission intensity of 2‚
Br was at 93% of its initial value (see Table 4). After 2 h, 85%
of the emission intensity persisted.
Scheme 9. Conjugation of BBI Salt 36 with MPA
To investigate the emission properties of 2‚Br under basic
conditions, a fresh sample was dissolved in aqueous media
buffered to pH 9. The emission intensity remained essentially
unchanged from that at pH 7 and did not diminish significantly
even after 2 h. At pH 11, the photoluminescence disappeared
almost instantly and was not returned upon acidification to
pH 7. Thus, the upper limit of tolerable pH levels appeared to
be approximately 9. Furthermore, the irreversibility of the
fluorescence loss suggested that the BBI chromophore was in
fact destroyed via hydrolysis or oxidation, as described in
Scheme 7.
To elucidate the degradation pathway of BBI 2‚Br under basic
aqueous conditions, we performed NMR-scale experiments in
D2O/NaOH (pH ) 11). Analysis of the products after 2 h at
room temperature revealed complete loss of the C1-proton
signals (1H: δ ) 9.57 in D2O). In addition, signals attributable
to arene protons were found at δ ) 8.22, 7.47, 7.42, and 6.76
ppm. These four singlets, which were each upfield of the
respective BBI arene signal, likely resulted from isomeric
hydrolysis products. Indeed, mass-spectral analysis confirmed
products consistent with the bis(formamide) shown in
Scheme 7.
Next, we explored the stability of 2‚Br under acidic aqueous
conditions. At pH levels of 3 and 5, the emission intensities
remained essentially unchanged after 2 h and were similar to
the emission intensity observed at pH 7. Upon further acidifica-
tion (pH 1), however, photoluminescence was lost immediately
and was not regained upon neutralization to pH 7. Analysis of
the degradation products of 2‚Br in acidic D2O using NMR
spectroscopy revealed upfield shifts of the arene and alkyl
signals. In addition, there appeared to be loss of butyl groups
relative to arene signals. Mass-spectral analysis confirmed the
presence of dibutyl benzobis(imidazole) suggesting that the BBI
is subject to dealkylation under strongly acidic conditions.
Overall, despite reactive imidazolium moieties, BBI 2‚Br
showed excellent stability in aqueous solutions ranging in pH
from 3 to 9.
using a BBI fluorophore that featured a pendant substituent
predisposed to reacting with specific protein residues. Consider-
ing maleimides are commonly used to conjugate with cysteine
residues,47 initial efforts were focused on incorporating this
functional group into a BBI. Thus, we targeted 36 (see Scheme
8) which features (1) two N-aryl groups to provide emission in
the visible region, (2) small N-methyl substituents to facilitate
solubility in aqueous media, and (3) a pendant maleimide to
undergo reactions with thiols.
The synthesis of 36 is shown in Scheme 8 and was based
upon the SNAr/reductive cyclization/alkylation methodology
previously described.14 Starting from dinitroarene 37, SNAr
reaction with p-phenylenediamine provided dinitroarene 38 in
98% yield. Reductive cyclization of 37 at 110 °C gave benzobis-
(imidazole) 39 in 92% yield. To install a maleimide functional-
ity, 39 was first treated with anhydride 40. Condensation of 40
with amine 39 resulted in clean formation of imide 41, a
protected maleimide. Subsequent alkylation of the benzobis-
(imidazole) 41, and concomitant deprotection via a retro Diels-
Alder reaction at elevated temperature, provided BBI 36 in 67%
overall yield from 37.
The λabs and λem of 36 in MeOH appeared at 346 and 432
nm, respectively. Although the Φf was low (0.01), this may be
related to the maleimide functionality which has been found to
reduce quantum efficiencies in other fluorophores.47a To deter-
mine if 36 would be a feasible Michael acceptor for protein
conjugation, and to explore the photophysical impacts of altering
the maleimide moiety, we performed a control experiment using
3-mercaptopropionic acid (MPA) as a model for a nucleophilic
thiol residue. Thus, BBI 36 was dissolved in H2O and treated
with an equimolar amount of MPA as shown in Scheme 9. After
Protein Conjugation. Encouraged by the amphiphilic nature
and broad pH stability of the BBI salts, we next addressed the
challenge of synthesizing a task-specific BBI designed for
protein conjugation. To accomplish this goal, we envisioned
(47) (a) Guy, J.; Caron, K.; Dufresne, S.; Michnick, S. W.; Skene, W. G.; Keilor,
J. W. J. Am. Chem. Soc. 2007, 129, 11969. (b) Corrie, J. E. T. J. Chem.
Soc., Perkin Trans. 1994, 1, 2975.
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A R T I C L E S
Boydston et al.
functionalized fluorophore was successfully conjugated with
3-mercaptopropionic acid as a control experiment and was
ultimately attached to bovine serum albumin in aqueous media.
The absorption and emission spectra obtained after protein
conjugation, in comparison with those of the control experiment,
confirmed attachment of the fluorophore to the protein.
Collectively, this new class of fluorophores has many
desirable attributes that have been disclosed herein. In addition,
the ease with which the fluorogenic cores may be functionalized
and finely tuned should increase the efficiency with which they
may be deployed in a range of applications. These materials
are expected to open new opportunities and enable advance-
ments in a range of areas, including fundamental photophysical
studies, biological imaging, sensory materials, and electronic
display applications.
Figure 12. Absorption (solid lines) and emission (dotted lines) of 36‚MPA
(red) and 36‚BSA (black) in MeOH under ambient conditions.
4 h at room temperature, the solution was concentrated to
provide 36‚MPA in 95% yield. In comparison with free 36,
attachment of the thiol to the maleimide significantly increased
the Φf of the BBI to 0.34.
Experimental Section
1
General Considerations. H and 13C NMR spectra were recorded
The efficacy of 36 in protein conjugation was studied using
bovine serum albumin (BSA). BSA was dissolved in H2O and
treated with a slight excess of 36. After 4 h at room temperature,
the conjugate 36‚BSA was subjected to dialysis against distilled
H2O for 48 h. After removal of excess H2O under vacuum, the
conjugate product was isolated in 90% yield and analyzed by
NMR, UV-vis, and fluorescence spectroscopy. Confirmation
of successful conjugation was evidenced by an absence of
maleimide proton signals in the 1H NMR spectrum (diagnostic
signal at δ ) 7.27 in DMSO-d6). In addition, 36‚BSA displayed
λabs and λem at 287 (additional peak at 345 nm) and 445 nm,
which were consistent with those observed from 36 and 36‚
MPA (see Figure 12).
using a Varian Unity Plus 300 or 400 spectrometer. Chemical shifts
(δ) are expressed in ppm downfield from tetramethylsilane using the
1
residual protio solvent as an internal standard (CDCl3, H, 7.26 ppm,
and 13C, 77.0 ppm; DMSO-d6, 1H, 2.49 ppm, and 13C, 39.5 ppm).
Coupling constants are expressed in hertz (Hz). HRMS (ESI, CI) were
obtained with a VG analytical ZAB2-E instrument. UV-vis spectra
were recorded using a Perkin-Elmer Instruments Lambda 35 spectrom-
eter. Emission spectra were recorded using a QuantaMaster Photon
Technology International fluorometer. Unless otherwise noted, all
reactions were performed under ambient atmosphere. All starting
materials and solvents were of reagent quality and used as received
from commercial suppliers.
1,3,5,7-Tetrabutylbenzobis(imidazolium) Bis(tetrafluoroborate)
(2‚BF4). After suspension of BBI 2‚Br (992 mg, 1.82 mmol) in dry
CH2Cl2, Et3O‚BF4 (692 mg, 3.64 mmol) was added in a single portion.
Conclusions
1
The reaction progress was monitored by H NMR spectroscopy, and
We have synthesized and analyzed a series of benzobis-
(imidazolium) salts which represent a new class of highly
versatile, robust fluorophores. A modular synthetic design
facilitated access to fluorophores whose structures were tailored
to probe various photophysical and applications-based charac-
teristics, including emission wavelength tunability, solvatochro-
mic effects, red-edge excitation, multiphoton excitation, pH
stability, and protein conjugation. Additionally, the syntheses
provided each of the fluorophores in few synthetic manipulations
and without the need for chromatographic purification.
The fluorophores displayed structure-dependent λem ranging
from 329 to 561 nm in MeOH. Depending on the nature of the
fluorogenic core, solvent-dependent λem were also observed in
conjunction with large Stokes shifts. Although the nature of the
counterion had no detectable impacts in these studies, investiga-
tion of red-edge effects revealed a strong counterion dependency.
Specifically, while Br and BF4 counterions gave similar results,
a strikingly different REE was observed when MeSO4 counte-
rions were incorporated. Additionally, preliminary experiments
were conducted to investigate the BBI’s ability to display
multiphoton excited fluorescence. Aqueous solutions of tet-
raphenyl BBI 4 exhibited three-photon excitation and fluores-
cence action cross sections similar to those observed from
serotonin.
upon completion (ca. 10 min), the solution was concentrated to provide
969 mg (95% yield) of the desired product as a white powder. 1H NMR
(400 MHz, DMSO-d6): δ 10.10 (s, 2H), 8.97 (s, 2H), 4.59 (t, J ) 7.2
Hz, 8H), 2.00-1.93 (m, 8H), 1.42-1.33 (m, 8H), 0.94 (t, J ) 7.2 Hz,
12H). 13C NMR (75 MHz, DMSO-d6): δ 145.5, 130.2, 98.8, 47.1, 30.2,
19.1, 13.4. HRMS (m/z): calcd for BF4, 87.0029; found, 87.0026.
1,3,5,7-Tetrabutylbenzobis(imidazolium) Bis(methyl sulfate) (2‚
MeSO4). After suspension of BBI 2‚Br (488 mg, 0.90 mmol) in dry
CH2Cl2, Me2SO4 (0.17 mL, 1.80 mmol) was added in a single portion.
1
The reaction progress was monitored by H NMR spectroscopy, and
upon completion (ca. 4 h), the solution was concentrated to provide
542 mg (99% yield) of the desired product as a beige powder. 1H NMR
(400 MHz, CDCl3): δ 9.94 (s, 2H), 8.84 (s, 2H), 4.72, (t, J ) 7.0 Hz,
8H), 3.76 (s, 6H), 1.96-1.89 (m, 8H), 1.42-1.32 (m, 8H), 0.93 (t, J
) 7.2 Hz, 12H). 13C NMR (100 MHz, CDCl3 + DMSO-d6): δ 145.3,
130.3, 99.7, 54.5, 47.8, 31.1, 19.5, 13.4.
1,5-Diphenylbenzobis(imidazole) (5anti). Benzobis(imidazole) 1 (500
mg, 3.16 mmol) was dissolved in dry DMF (15 mL) in a screw-cap
vial. Under nitrogen atmosphere, PhI (1.42 mL, 12.6 mmol), CuI (60
mg, 0.32 mmol), 1,10-phenanthroline (114 mg, 0.63 mmol), and K2-
CO3 (1.75 g, 12.6 mmol) were added. The vial was then sealed with a
Teflon-lined cap, and the mixture was stirred at 130 °C for 17 h.
Afterward the mixture was poured into H2O (30 mL). Collection of
the precipitated solids via vacuum filtration afforded 932 mg (95%
yield) as a 1:1 mixture of two regioisomers 5syn and 5anti. Recrystalli-
zation of the mixture from hot DMSO provided 392 mg (40% yield
based on 1) of 5anti. Spectral data for 5syn were consistent with those
Encouraged by broad emission tunability, ease of function-
alization, and good pH stability, we synthesized a task-specific
fluorophore predisposed for protein conjugation. A maleimide-
previously reported.14 Data for 5anti
:
1H NMR (300 MHz, CDCl3): δ
8.20 (s, 2H), 8.00 (s, 2H), 7.63-7.62 (m, 8H), 7.52-7.45 (m, 2H).
9
3152 J. AM. CHEM. SOC. VOL. 130, NO. 10, 2008

Modular Fluorescent Benzobis(imidazolium) Salts
A R T I C L E S
13C NMR (75 MHz, CDCl3) δ 143.7, 142.2, 136.6, 131.5, 130.1, 128.0,
123.9, 100.4; HRMS m/z calcd for C20H15N4 [M + H+] 311.1297, found
311.1299.
filtration, rinsed with H2O, and dried under vacuum to provide 1.42 g
(96% yield) of the desired product as a red-orange powder. H NMR
1
(300 MHz, CDCl3): δ 9.57 (br s, 2H), 9.31 (s, 1H), 7.05 (d, J ) 8.9
Hz, 4H), 6.84 (d, J ) 8.9 Hz, 4H), 6.18 (s, 1H), 3.80 (s, 3H). 13C
NMR (75 MHz, CDCl3): δ 158.2, 1147.7, 129.8, 129.4, 126.7, 125.0,
114.8, 94.9, 55.5. HRMS (m/z): calcd for C20H19N4O6 [M + H+],
411.1305; found, 411.1301.
1,5-Dimethyl-3,7-diphenylbenzobis(imidazolium) Bis(methyl sul-
fate) (6anti). After diphenylbenzobis(imidazole) 5anti (50 mg, 0.15 mmol)
was dissolved in MeCN (1.0 mL) in a screw-cap vial, Me2SO4 (29 µL,
0.31 mmol) was added in a single portion. The vial was then sealed
with a Teflon-lined cap and placed in an oil bath at 80 °C for 4 h. The
reaction mixture was then cooled and concentrated to afford 84 mg
1,7-Bis(4-carboethoxyphenyl)benzobis(imidazole) (13). Dinitroare-
ne 11 (400 mg, 0.81 mmol) was dissolved in HC(OEt3) (10 mL) in a
screw-cap vial. To the solution was added HCO2H (88%, 1 mL), HCO2-
Na (1.65 g, 24.3 mmol), and Pd/C (172 mg, 5 wt %, 0.08 mmol Pd).
The vial was then sealed with a Teflon-lined cap, and the mixture was
heated in an oil bath at 110 °C for 48 h. Upon cooling of the mixture
to ambient temperature, it was filtered through Celite. The filtrate was
treated with 5% HCl, stirred for ca. 30 min, and then brought to pH 9
using saturated aqueous Na2CO3. Precipitated solids were subsequently
collected via vacuum filtration, rinsed with H2O, and dried under
vacuum to provide 352 mg (96% yield) of the desired product as a tan
powder. 1H NMR (400 MHz, CDCl3): δ 8.38 (s, 1H), 8.26 (d, J ) 8.4
Hz, 4H), 8.21 (s, 2H), 7.63 (s, 1H), 7.61 (d, J ) 8.4 Hz, 4H), 4.43
(quart, J ) 7.2 Hz, 4H), 1.43 (t, J ) 7.2 Hz, 6H). 13C NMR (75 MHz,
CDCl3): δ 165.5, 143.1, 141.8, 140.1, 131.8, 131.7, 129.9, 123.3, 111.6,
90.4, 61.4, 14.3. HRMS (m/z): calcd for C26H23N4O4 [M + H+],
455.1719; found, 455.1716.
1,7-Bis(4-methoxyphenyl)benzobis(imidazole) (14). Dinitroarene
12 (1.00 g, 2.44 mmol) was suspended in HCO2H (88%, 100 mL). To
the suspension was added HCO2Na (1.99 mg, 29.2 mmol) and Pd/C
(103 mg, 5 wt %, 0.05 mmol Pd). The mixture was heated in an oil
bath at 110 °C for 48 h. Upon completion, the cooled reaction mixture
was filtered through Celite and the filtrate volume was reduced to ca.
20 mL under vacuum. The solution was then added slowly into a
vigorously stirred saturated solution of aqueous Na2CO3 (150 mL).
Precipitated solids were collected via vacuum filtration, rinsed with
H2O, and dried under vacuum to provide 705 mg (78% yield) of the
desired product as a tan powder. 1H NMR (300 MHz, CDCl3): δ 8.31
(s, 1H), 8.08 (s, 2H), 7.41 (d, J ) 8.8 Hz, 4H), 7.35 (s, 1H), 7.06 (d,
J ) 8.8 Hz, 4H), 3.88 (s, 6H). 13C NMR (75 MHz, CDCl3): δ 159.2,
143.6, 141.2, 132.8, 129.3, 125.9, 115.2, 110.5, 89.8, 55.6. HRMS (m/
z): calcd for C22H19N4O2 [M + H+], 371.1508; found, 371.1511.
1,7-Bis(4-carboethoxyphenyl)-3,5-dimethylbenzobis(imidazoli-
um) Diiodide (15). Benzobis(imidazole) 13 (300 mg, 0.66 mmol) was
dissolved in MeCN (10 mL) and MeI (1.0 mL) in a screw-cap vial.
The vial was then sealed with a Teflon-lined cap, and the mixture was
heated at 80 °C for 8 h. Afterward, the mixture was concentrated under
vacuum to provide 485 mg (>99% yield) of the desired product as a
dark brown powder. 1H NMR (400 MHz, DMSO-d6): δ 10.48 (s, 2H),
9.12 (s, 1H), 8.27 (d, J ) 8.2 Hz, 4H), 8.24 (s, 1H), 8.03 (d, J ) 8.2
Hz, 4H), 4.38 (quart, J ) 7.2 Hz, 4H), 4.30 (s, 6H), 1.35 (t, J ) 7.0
Hz, 6H). 13C NMR (75 MHz, DMSO-d6): δ 164.7, 147.4, 136.6, 131.6,
131.3, 131.2, 129.9, 125.7, 107.3, 99.9, 98.5, 61.4, 34.4, 14.2. HRMS
(m/z): calcd for C28H27N4O4 [M - H+], 483.2032; found, 483.2033.
1,7-Bis(4-methoxyphenyl)-3,5-dimethylbenzobis(imidazolium) Di-
iodide (16). Benzobis(imidazole) 14 (500 mg, 1.35 mmol) was dissolved
in MeCN (10 mL) and MeI (1.0 mL) in a screw-cap vial. The vial was
then sealed with a Teflon-lined cap, and the mixture was heated at 80
°C for 8 h. Upon completion, the mixture was concentrated under
vacuum to provide 877 mg (99% yield) of the desired product as a
dark brown powder. 1H NMR (400 MHz, DMSO-d6): δ 10.30 (s, 2H),
9.06 (s, 1H), 7.87 (s, 1H), 7.76 (d, J ) 8.8 Hz, 4H), 7.25 (d, J ) 8.8
Hz, 4H), 4.27 (s, 6H), 3.86 (s, 6H). 13C NMR (100 MHz, DMSO-d6):
δ 160.7, 147.1, 130.9, 130.6, 127.0, 125.4, 115.5, 99.4, 97.8, 55.8, 34.2.
HRMS (m/z): calcd for C24H23N4O2 [M - H+], 399.1821; found,
399.1817.
1
(>99% yield) of the desired product as a beige wax. H NMR (400
MHz, DMSO-d6): δ 10.35 (s, 2H), 8.64 (s, 2H), 7.92 (d, J ) 7.6 Hz,
4H), 7.85-7.78 (m, 6H), 4.24 (s, 6H), 3.34 (s, 6H). 13C NMR (75 MHz,
DMSO-d6): δ 147.4, 146.3, 132.9, 131.2, 130.9, 130.5, 130.1, 125.7,
99.1, 52.9, 34.0. HRMS (m/z): calcd for C22H19N4 [M - H+], 339.1610;
found, 339.1609.
Benzimidazolium Chloride 8. In a nitrogen-filled drybox, a screw-
cap vial was charged with 5,6-dichloro-1-methylbenzimidazole (7) (375
mg, 1.87 mmol) and PhMe (10 mL). To the solution was added Pd-
(OAc)2 (4 mg, 0.02 mmol), 1,3-bis(2,6-diisopropylphenyl)imidazolium
chloride (16 mg, 0.04 mmol), NaOtBu (368 mg, 3.83 mmol), and PhNH2
(869 mg, 9.33 mmol). The vial was then sealed with a Teflon-lined
cap and placed in an oil bath at 120 °C for 12 h. After cooling of the
reaction mixture to ambient temperature, it was poured into a solution
of HC(OEt)3 (50 mL) and concentrated HCl (1.0 mL). The resulting
mixture was then stirred in an oil bath at 150 °C for 12 h, allowed to
cool, and then poured into Et2O (100 mL). The precipitated solids were
collected via vacuum filtration, rinsed with Et2O, and redissolved in a
i
saturated solution of Na2CO3 in PrOH. After standing for 1 h, the
mixture was filtered through Celite and the filtrate was concentrated
to provide 523 mg (86% yield) of the desired product as a light brown
1
powder. H NMR (400 MHz, DMSO-d6): δ 10.58 (s, 1H), 8.57 (s,
1H), 8.13 (s, 1H), 8.09 (s, 1H), 8.05-8.02 (m, 4H), 7.83-7.72 (m,
6H). 13C NMR (75 MHz, DMSO-d6): δ 149.2, 143.2, 143.0, 134.9,
133.5, 133.4, 130.5, 130.4, 128.1, 127.7, 125.6, 125.4, 102.6, 94.6,
31.5. HRMS (m/z): calcd for C21H17N4 [M+], 325.1453; found,
325.1454.
1,3-Dimethyl-5,7-diphenylbenzobis(imidazolium) Bis(methyl sul-
fate) (9). After benzimidazolium chloride 8 (50 mg, 0.15 mmol) was
dissolved in MeCN (1.0 mL), Me2SO4 (39 mg, 0.31 mmol) was added
in a single portion. After stirring the resulting solution at 80 °C for 2
h, it was concentrated to afford 84 mg (>99% yield) of the desired
1
product as a tan solid. H NMR (400 MHz, CDCl3): δ 10.81 (s, 1H),
10.04, (s, 1H), 8.60 (s, 2H), 8.04 (d, J ) 7.2 Hz, 4H), 7.85-7.78 (m,
6H), 4.18 (s, 6H), 3.35 (s, 6H). 13C NMR (75 MHz, DMSO-d6): δ
147.4, 146.3, 132.9, 131.2, 130.9, 130.5, 130.1, 125.7, 99.1, 52.9, 34.0.
HRMS (m/z): calcd for C22H19N4 [M - H+], 339.1610; found,
339.1607.
1,5-Bis(4-carboethoxyphenyl)amino-3,4-dinitrobenzene (11). A
screw-cap vial was charged with 1,5-dichloro-3,4-dinitrobenzene (10)
(413 mg, 1.74 mmol), benzocaine (1.44 g, 8.71 mmol), iPrOH (15 mL),
and a stir bar. The vial was sealed with a Teflon-lined cap, placed in
an oil bath at 120 °C, and stirred for 48 h. After the mixture was poured
into 5% HCl (50 mL), the precipitated solids were collected via vacuum
filtration, rinsed with H2O, and dried under vacuum to provide 1.66 g
(98% yield) of the desired product as a yellow powder. 1H NMR (400
MHz, CDCl3): δ 9.92 (br s, 2H), 9.32 (s, 1H), 8.05 (d, J ) 8.4 Hz,
4H), 7.25 (d, J ) 8.4 Hz, 4H), 6.83 (s, 1H), 4.38 (quart, J ) 7.2 Hz,
4H), 1.41 (t, J ) 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 165.4,
145.6, 141.2, 131.3, 129.2, 128.2, 126.1, 123.0, 96.7, 61.2, 14.3. HRMS
(m/z): calcd for C24H23N4O8 [M + H+], 495.1516; found, 495.1517.
1,5-Bis(4-methoxyphenyl)amino-3,4-dinitrobenzene (12). After
1,5-dichloro-3,4-dinitrobenzene (10) (1.00 g, 4.22 mmol) was dissolved
in EtOH (75 mL), p-anisidine (2.08 g, 16.9 mmol) was added in a
single portion. The mixture was then placed in an oil bath at 80 °C
and stirred for 48 h. The mixture was then poured into H2O (200 mL)
which caused solids to precipitate. The solids were collected via vacuum
1,5-Bis(4-butylphenyl)amino-3,4-dinitrobenzene (17). Following
a similar procedure used to prepare 12, 1,5-dichloro-3,4-dinitrobenzene
(10) (1.00 g, 4.22 mmol) and 4-butylaniline (2.52 g, 16.88 mmol)
9
J. AM. CHEM. SOC. VOL. 130, NO. 10, 2008 3153

A R T I C L E S
Boydston et al.
afforded 1.83 g (94% yield) of the desired product as a burnt orange
powder. 1H NMR (400 MHz, CDCl3): δ 9.68 (br s, 2H), 9.30 (s, 1H),
7.14 (d, J ) 8.2 Hz, 4H), 7.05 (d, J ) 8.2 Hz, 4H), 6.48 (s, 1H), 2.58
(t, J ) 7.6 Hz, 4H), 1.61-1.53 (m, 4H), 1.40-1.31 (m, 4H), 0.95 (t,
J ) 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 147.0, 141.5, 134.6,
129.4, 129.3, 125.1, 124.6, 95.1, 35.1, 33.6, 22.2, 13.9. HRMS (m/z):
calcd for C26H31N4O4 [M + H+], 463.2345; found, 463.2343.
General Procedure for the Reduction of Dinitroarenes 18 and
19. After the dinitroarene (1.0 mmol, 1.0 equiv) was dissolved in EtOH
(20 mL) in a 100 mL flask, Pd/C (5 wt %, 0.10 mmol Pd, 0.10 equiv)
was added. The flask was then fitted with a H2O-jacketed condenser,
and H2NNH2‚H2O (80%, 16 mmol, 16 equiv) was added dropwise
followed by 1.2 N HCl (7 mL, 8.0 mmol, 8.0 equiv). The solution was
stirred in an oil bath at 80 °C for 4 h. The cooled reaction mixture was
then filtered through Celite and concentrated under vacuum to provide
the corresponding tetraamines as their dihydrochloride salts. These
products were used directly in subsequent steps without further
purification. Note: these materials were found to decompose upon
prolonged standing under ambient atmosphere and thus were stored in
a nitrogen-filled desiccator prior to use.
Tetraaminobenzene Dihydrochloride 18. Yield: 4.20 g (>99%).
1H NMR (300 MHz, CDCl3): δ 6.97 (d, J ) 6.6 Hz, 4H), 6.85 (s,
1H), 6.56 (d, J ) 6.6 Hz, 4H), 6.26 (s, 1H), 4.86 (br s, 2H), 3.76 (br
s, 4H), 2.49 (br t, 4H), 1.59-1.49 (m, 4H), 1.40-1.27 (m, 4H), 0.91
(t, J ) 7.2 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 144.8, 142.5,
132.9, 129.1, 126.5, 119.6, 114.0, 102.3, 34.7, 34.0, 22.3, 14.0. HRMS
(m/z): calcd for C26H35N4 [M + H+], 403.2862; found, 403.2858.
Tetraaminobenzene Dihydrochloride 19. Yield: 1.15 g (>99%).
1H NMR (400 MHz, CDCl3): δ 6.79 (s, 1H), 6.75 (d, J ) 8.8 Hz,
4H), 6.59 (d, J ) 8.8 Hz, 4H), 6.25 (s, 4H), 4.74 (br s, 2H), 3.74 (s,
6H). 13C NMR (100 MHz, CDCl3): δ 152.8, 141.9, 140.8, 125.1, 120.5,
115.5, 114.8, 102.6, 55.7. HRMS (m/z): calcd for C20H23N4O2 [M +
H+], 351.1821; found, 351.1818.
General Procedure for the Synthesis of C1-Aryl Benzobis-
(imidazole)s 20-25. Under an atmosphere of dry nitrogen, the
corresponding tetraaminobenzene (1.0 mmol, 1.0 equiv) was suspended
in THF (10 mL). The respective aroyl chloride (2.0 mmol, 2.0 equiv)
was added, followed by Na2CO3 (10.0 mmol, 10.0 equiv). After stirring
of the solution for 12 h at ambient temperature, it was slowly poured
into AcOH (30 mL). The resulting suspension was then heated open-
air in an oil bath at 110 °C for 24 h, during which time AcOH was
added in portions to maintain the solution volume at ca. 30 mL. Upon
completion, the solvent volume was reduced to ca. 10 mL under reduced
pressure, and the resulting mixture was slowly poured into a vigorously
stirred solution of 10% NaOH containing an equal weight of crushed
ice. The resulting solids were collected via vacuum filtration, rinsed
with 10% NaOH and then H2O, and dried under vacuum.
8.28 (s, 1H), 7.50 (d, J ) 8.1 Hz, 4H), 7.27 (d, J ) 7.3 Hz, 4H), 7.19
(d, J ) 7.3 Hz, 4H), 6.90 (s, 1H), 6.80 (d, J ) 8.1 Hz, 4H), 3.80 (s,
6H), 2.68 (t, J ) 7.7 Hz, 4H), 1.70-1.60 (m, 4H), 1.45-1.33 (m, 4H),
0.96 (t, J ) 7.2 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 160.3, 152.9,
143.3, 140.5, 135.6, 135.0, 130.8, 129.8, 127.3, 122.7, 113.6, 108.4,
89.9, 55.2, 35.3, 33.3, 22.3, 13.9. HRMS (m/z): calcd for C42H43N4O2
[M + H+], 635.3386; found, 635.3390.
1,7-Bis(4-butylphenyl)-2,6-bis(2-thienyl)benzobis(imidazole) (23).
1
Yield: 200 mg (78%). H NMR (300 MHz, CDCl3): δ 8.25 (s, 1H),
7.37-7.26 (m, 10H), 6.89 (dd appearing as t, J ) 4.5 Hz, 2H), 6.77
(d, J ) 3.6 Hz, 2H), 6.59 (s, 1H), 2.73 (t, J ) 7.7 Hz, 4H), 1.74-1.64
(m, 4H), 1.48-1.35 (m, 4H), 0.97 (t, J ) 7.2 Hz, 6H). 13C NMR (75
MHz, CDCl3): δ 148.2, 144.6, 140.5, 136.4, 134.0, 132.9, 130.0,
128.25, 128.16, 127.9, 127.4, 108.4, 89.6, 35.4, 33.3, 22.4, 13.9. HRMS
(m/z): calcd for C36H35N4S2 [M + H+], 587.2303; found, 587.2307.
2,6-Bis(4-biphenyl)-1,7-bis(4-methoxyphenyl)benzobis(imida-
1
zole) (24). Yield: 492 mg (88%). H NMR (400 MHz, CDCl3): δ
8.36 (s, 1H), 7.67 (d, J ) 8.4 Hz, 4H), 7.59 (d, J ) 7.2 Hz, 4H), 7.54
(d, J ) 8.4 Hz, 4H), 7.45-7.41 (m, 4H), 7.37-7.33 (m, 2H), 7.25 (d,
J ) 6.8 Hz, 4H), 7.01 (d, J ) 8.8 Hz, 4H), 6.85 (s, 1H), 3.88 (s, 6H).
13C NMR (75 MHz, CDCl3): δ 159.4, 152.9, 141.8, 140.6, 140.1, 136.3,
130.0, 129.7, 129.0, 128.82, 128.78, 127.7, 127.0, 126.8, 115.2, 108.9,
90.0, 55.5. HRMS (m/z): calcd for C46H35N4O2 [M + H+], 675.2760;
found, 675.2763.
2,6-Bis(4-cyanophenyl)-1,7-bis(4-methoxyphenyl)benzobis(imida-
zole) (25). Yield: 469 mg (99%). 1H NMR (400 MHz, CDCl3
+
DMSO-d6): δ 8.29 (s, 1H), 7.68 (d, J ) 8.2 Hz, 4H), 7.55 (d, J ) 8.2
Hz, 4H), 7.16 (d, J ) 8.4 Hz, 4H), 6.97 (d, J ) 8.4 Hz, 4H), 6.80 (s,
1H), 3.84 (s, 6H). 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 159.7,
151.1, 140.6, 136.8, 134.2, 131.9, 129.5, 128.9, 128.5, 118.2, 115.4,
112.6, 109.6, 90.3, 55.5. HRMS (m/z): calcd for C36H25N6O2 [M +
H+], 573.2039; found, 573.2038.
General Procedure for the Alkylation of Benzobis(imidazole)s
20-25 To Provide BBI Salts 26-31. The respective benzobis-
(imidazole) (1.0 mmol, 1.0 equiv) was dissolved or suspended in MeCN
(10 mL) and MeI (5.0 mmol, 5.0 equiv) in a screw-cap vial. After the
vial was sealed with a Teflon-lined cap, the reaction mixture was stirred
in an oil bath at 100 °C for 2-10 h. The reaction progress was
monitored by No-D 1H NMR spectroscopy48 of aliquots taken periodi-
cally. Upon completion of the reaction, the reaction mixtures were
cooled to ambient temperature and concentrated. Crude materials were
then rinsed with cold EtOAc and then dried under vacuum to provide
the desired products.
1,7-Bis(4-butylphenyl)-3,5-dimethyl-2,6-diphenylbenzobis(imida-
zolium) Diiodide (26). Yield: 149 mg (>99%). 1H NMR (300 MHz,
CDCl3): δ 8.44 (s, 1H), 7.88 (d, J ) 7.5 Hz, 4H), 7.65-7.50 (m, 10H),
7.31 (s, 1H), 7.23 (d, J ) 8.4 Hz, 4H), 4.27 (s, 6H), 1.60-1.50 (m,
4H), 1.35-1.23 (m, 4H), 0.89 (t, J ) 7.2 Hz, 6H). 13C NMR (75 MHz,
CDCl3): δ 153.7, 146.4, 133.1, 132.9, 132.0, 131.4, 130.2, 129.5, 129.3,
127.6, 120.6, 99.9, 98.0, 35.2, 34.9, 32.9, 22.1, 13.8. HRMS (m/z):
calcd for C42H43N4 [M - H+], 603.3488; found, 603.3492.
1,7-Bis(4-butylphenyl)-2,6-diphenylbenzobis(imidazole) (20).
1
Yield: 580 mg (96%). H NMR (300 MHz, CDCl3): δ 8.36 (s, 1H),
7.58 (dd, J ) 6.3, 1.8 Hz, 4H), 7.37-7.26 (m, 10H), 7.20 (d, J ) 8.7
Hz, 4H), 6.97 (s, 1H), 2.68 (t, J ) 2.68 Hz, 4H), 1.70-1.60 (m, 4H),
1.45-1.32 (m, 4H), 0.96 (t, J ) 7.3 Hz, 6H). 13C NMR (75 MHz,
CDCl3): δ 153.1, 143.4, 135.8, 134.8, 130.1, 129.8, 129.4, 129.3, 128.2,
127.3, 109.0, 90.2, 35.3, 33.3, 22.3, 13.9. HRMS (m/z): calcd for
C40H39N4 [M + H+], 575.3175; found, 575.3176.
1,7-Bis(4-butylphenyl)-2,6-bis(4-cyanophenyl)benzobis(imidazo-
1
lium) Diiodide (27). Yield: 255 mg (>99%). H NMR (400 MHz,
DMSO-d6): δ 9.33 (s, 1H), 8.12 (d, J ) 7.8 Hz, 4H), 8.05 (d, J ) 7.8
Hz, 4H), 7.49 (d, J ) 8.2 Hz, 4H), 7.38 (s, 1H), 7.36 (d, J ) 8.2 Hz,
4H), 4.19 (s, 3H), 2.59 (t, J ) 7.4 Hz, 4H), 1.52-1.48 (m, 4H), 1.25-
1.20 (m, 4H), 0.84 (t, J ) 7.2 Hz, 6H). 13C NMR (75 MHz, CDCl3 +
DMSO-d6): δ 151.7, 146.5, 132.6, 132.3, 132.1, 131.8, 129.9, 128.7,
127.0, 124.8, 116.7, 116.3, 99.8, 97.3, 34.7, 34.3, 32.4, 21.7, 13.3.
HRMS (m/z): calcd for C44H41N6 [M - H+], 653.3393; found,
653.3398.
1,7-Bis(4-butylphenyl)-2,6-bis(4-cyanophenyl)benzobis(imida-
1
zole) (21). Yield: 308 mg (93%). H NMR (300 MHz, CDCl3): δ
8.37 (s, 1H), 7.69 (d, J ) 8.7 Hz, 4H), 7.58 (d, J ) 8.7 Hz, 4H), 7.32
(d, J ) 8.4 Hz, 4H), 7.19 (d, J ) 8.4 Hz, 4H), 6.96 (s, 1H), 2.71 (t, J
) 7.7 Hz, 4H), 1.72-1.61 (m, 4H), 1.46-1.34 (m, 4H), 0.97 (t, J )
7.2 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 151.2, 144.3, 140.8, 136.5,
134.3, 134.0, 132.0, 130.2, 129.7, 127.1, 118.4, 112.8, 109.9, 90.5,
35.3, 33.3, 22.4, 13.9. HRMS (m/z): calcd for C42H37N6 [M + H+],
625.3080; found, 625.3078.
1,7-Bis(4-butylphenyl)-2,6-bis(4-cyanophenyl)benzobis(imidazo-
lium) Bis(tetrafluoroborate) (27‚BF4). This compound was prepared
1,7-Bis(4-butylphenyl)-2,6-bis(4-methoxyphenyl)benzobis(imida-
zole) (22). Yield: 287 mg (91%). H NMR (300 MHz, CDCl3): δ
(48) Hoye, T. R.; Eklov, B. M.; Ryba, T. D.; Voloshin, M.; Yao, L. J. Org.
Lett. 2004, 6, 953.
1
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3154 J. AM. CHEM. SOC. VOL. 130, NO. 10, 2008

Modular Fluorescent Benzobis(imidazolium) Salts
A R T I C L E S
analogously to 2‚BF4 from BBI 27 (59 mg, 0.06 mmol) to provide 49
mg (98% yield) of the desired product. H NMR (300 MHz, DMSO-
9.90 (br s, 1H), 9.06 (s, 1H), 8.18 (d, J ) 8.8 Hz, 2H), 7.38 (d, J ) 8.8
Hz, 2H), 7.32 (s, 1H), 4.41 (quart, J ) 7.2 Hz, 2H), 1.42 (t, J ) 7.2
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 165.4, 144.2, 140.4, 136.7,
135.8, 131.7, 130.3, 129.4, 126.5, 124.0, 118.1, 61.4, 14.3. HRMS (m/
z): calcd for C15H13N3O6Cl [M + H+], 366.0493; found, 366.0489.
1-(4-Carboethoxyphenyl)amino-2,4-dinitro-5-(4-methoxyphenyl)-
aminobenzene (34). After aryl chloride 33 (380 mg, 1.04 mmol) was
dissolved in EtOH (10 mL), p-anisidine (256 mg, 2.08 mmol) was added
in a single portion. The mixture was then stirred at 80 °C for 24 h.
After being cooled to ambient temperature, the mixture was poured
into 5% HCl (50 mL). Precipitated solids were collected via vacuum
filtration, rinsed with H2O, and dried under vacuum to provide 462
mg (98% yield) of the desired product as an orange powder. 1H NMR
(300 MHz, CDCl3): δ 9.85 (br s, 1H), 9.65 (br s, 1H), 9.33 (s, 1H),
7.98 (d, J ) 8.7 Hz, 2H), 7.17 (d, J ) 8.7 Hz, 2H), 7.12 (d, J ) 8.7
Hz, 2H), 6.92 (d, J ) 9.0 Hz, 2H), 6.53 (s, 1H), 4.38 (quart, J ) 7.2
Hz, 2H), 3.81 (s, 3H), 1.41 (t, J ) 7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ 165.6, 158.7, 148.1, 145.1, 141.7, 131.1, 129.5, 129.3, 127.6,
127.0, 125.64, 125.55, 122.6, 115.1, 96.1, 61.1, 55.5, 14.3. HRMS (m/
z): calcd for C22H21N4O7 [M + H+], 453.1410; found, 453.1406.
1-(4-Carboethoxyphenyl)-7-(4-methoxyphenyl)benzobis(imida-
zole) (35). This compound was prepared analogously to 13 from
dinitroarene 34 (354 mg, 0.78 mmol) to provide 272 mg (84% yield)
of the desired product as an off-white solid. 1H NMR (300 MHz,
CDCl3): δ 8.33 (s, 1H), 8.24 (d, J ) 8.7 Hz, 2H), 8.19 (s, 1H), 8.10
(s, 1H), 7.61 (d, J ) 9.0 Hz, 2H), 7.49 (s, 1H), 7.41 (d, J ) 9.0 Hz,
2H), 7.08 (d, J ) 9.0 Hz, 2H), 4.42 (quart, J ) 7.2 Hz, 2H), 3.89 (s,
3H), 1.42 (t, J ) 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 165.5,
159.4, 143.9, 142.7, 141.6, 141.5, 140.3, 133.1, 131.6, 131.5, 129.6,
129.1, 125.9, 123.2, 115.3, 111.1, 90.1, 61.4, 55.6, 14.3. HRMS (m/z):
calcd for C24H21N4O3 [M + H+], 413.1614; found, 413.1611.
Benzobis(imidazolium) Diiodide (36). Benzobis(imidazole) 41 (2.00
g, 4.23 mmol), was dissolved in dry MeCN (20 mL) and MeI (5.99 g,
42.3 mmol) in a screw-cap vial. The vial was then sealed with a Teflon-
lined cap and placed in an oil bath at 95 °C for 12 h. The solution was
then cooled, opened to air, and then stirred in an oil bath at 95 °C until
the solvent volume reached ca. 1 mL. The residue was then dried under
vacuum to provide 2.88 g (99% yield) of the desired product. 1H NMR
(400 MHz, DMSO-d6): δ 10.45 (s, 1H), 10.43 (s, 1H), 9.15 (s, 1H),
8.13 (s, 1H), 8.04 (d, J ) 8 Hz), 7.93 (d, J ) 8 Hz, 2H), 7.73 (m, 5H),
7.27 (s, 2H), 4.32 (s, 2H), 4.31 (s, 2H). 13C NMR (100 MHz, DMSO-
d6): 169.6, 147.2, 147.1, 134.9, 133.4, 133.0, 131.8, 131.1, 131.1, 130.7,
130.5, 130.0, 129.9, 128.3, 125.8, 125.3, 99.7, 98.3. HRMS (m/z): calcd
for C26H21IN5O2 [M + I], 562.0740; found, 562.0716.
1
d6): δ 9.30 (s, 1H), 8.15 (d, J ) 8.4 Hz, 4H), 7.99 (d, J ) 8.4 Hz,
4H), 7.46 (d, J ) 8.1 Hz, 4H), 7.43 (s, 1H), 7.38 (d, J ) 8.7 Hz, 4H),
4.20 (s, 6H), 2.62 (t, J ) 7.8 Hz, 4H), 1.57-1.47 (m, 4H), 1.31-1.19
(m, 4H), 0.86 (t, J ) 7.2 Hz, 6H). 13C NMR (75 MHz, DMSO-d6): δ
152.4, 145.8, 132.8, 132.3, 131.9, 131.3, 130.2, 129.5, 127.4, 125.4,
117.6, 115.5, 99.4, 96.8, 34.3, 33.8, 32.5, 21.6, 13.7.
1,7-Bis(4-butylphenyl)-2,6-bis(4-methoxyphenyl)-3,5-dimethyl-
benzobis(imidazolium) Diiodide (28). Yield: 425 mg (95%). 1H NMR
(400 MHz, DMSO-d6): δ 9.22 (s, 1H), 7.73 (d, J ) 8.6 Hz, 4H), 7.48
(d, J ) 8.2 Hz, 4H), 7.37 (d, J ) 8.2 Hz, 4H), 7.15 (s, 1H), 7.13 (d,
J ) 8.6 Hz, 4H), 4.19 (s, 6H), 3.82 (s, 6H), 2.61 (t, J ) 7.6 Hz, 4H),
1.55-1.48 (m, 4H), 1.26-1.21 (m, 4H), 0.85 (t, J ) 7.2 Hz, 6H). 13
C
NMR (400 MHz, DMSO-d6): δ 162.5, 153.8, 145.4, 133.4, 131.7,
131.1, 130.14, 130.09, 127.5, 114.5, 112.5, 55.7, 34.3, 34.0, 32.5, 21.6,
13.7. HRMS (m/z): calcd for C44H47N4O2 [M - H+], 663.3699; found,
663.3701.
1,7-Bis(4-butylphenyl)-2,6-bis(2-thienyl)-3,5-dimethylbenzobis-
1
(imidazolium) Diiodide (29). Yield: 276 mg (93%). H NMR (400
MHz, CDCl3): δ 8.48 (s, 1H), 7.80 (d, J ) 3.6 Hz, 2H), 7.78 (d, J )
4.8 Hz, 2H), 7.39 (d, J ) 8.2 Hz, 4H), 7.31 (d, J ) 8.2 Hz, 4H), 7.25
(d, J ) 4.8 Hz, 2H), 7.11 (s, 1H), 4.32 (s, 6H), 2.67 (t, J ) 7.8 Hz,
4H), 1.64-1.56 (m, 4H), 1.38-1.29 (m, 4H), 0.92 (t, J ) 7.4 Hz, 6H).
13C NMR (100 MHz, CDCl3): δ 148.7, 147.1, 137.6, 135.5, 132.8,
132.2, 130.6, 129.5, 128.7, 127.8, 119.0, 98.8, 97.0, 35.3, 34.4, 33.0,
22.2, 13.8. HRMS (m/z): calcd for C38H39N4S2 [M - H+], 615.2616;
found, 615.2612.
2,6-Bis(4-biphenyl)-1,7-bis(4-methoxyphenyl)-3,5-dimethylbenzo-
bis(imidazolium) Diiodide (30). Yield: 186 mg (94%). 1H NMR (400
MHz, DMSO-d6): δ 9.28 (s, 1H), 7.95 (d, J ) 8.4 Hz, 4H), 7.90 (d,
J ) 8.4 Hz, 4H), 7.58-7.43 (m, 10H), 7.37 (s, 1H), 7.10 (d, J ) 9.2
Hz, 4H), 4.24 (s, 6H), 3.76 (s, 6H). 13C NMR (75 MHz, DMSO-d6):
δ 160.6, 153.8, 144.1, 138.0, 132.3, 132.0, 131.2, 129.2, 128.8, 127.0,
126.9, 124.7, 119.8, 115.5, 99.1, 96.4, 55.6, 34.1. HRMS (m/z): calcd
for C48H39N4O2 [M - H+], 703.3073; found, 703.3077.
2,6-Bis(4-cyanophenyl)-1,7-bis(4-methoxyphenyl)-3,5-dimethyl-
benzobis(imidazolium) Diiodide (31). Yield: 102 mg (99%). 1H NMR
(400 MHz, DMSO-d6): δ 9.30 (s, 1H), 8.14 (d, J ) 8.6 Hz, 4H), 8.04
(d, J ) 8.6 Hz, 4H), 7.51 (d, J ) 9.0 Hz, 4H), 7.49 (s, 1H), 7.07 (d,
J ) 9.0 Hz, 4H), 4.18 (s, 6H), 3.76 (s, 6H). 13C NMR (100 MHz,
DMSO-d6): δ 160.7, 152.5, 132.8, 132.4, 131.3, 129.1, 125.5, 124.2,
117.6, 115.5, 115.4, 96.9, 88.0, 55.6, 34.0. HRMS (m/z): calcd for
C38H29N6O2 [M - H+], 601.2352; found, 601.2355.
Conjugate 36‚MPA. BBI 36 (130 mg, 0.19 mmol) and 3-mercap-
topropionic acid (20 mg, 0.19 mmol) were dissolved in H2O (2.0 mL)
in a screw-cap vial. The mixture was stirred for 12 h at room
temperature and then concentrated under vacuum to provide 142 mg
1-(4-Carboethoxyphenyl)-7-(4-methoxyphenyl)-3,5-dimethylben-
zobis(imidazolium) Diiodide (32). This compound was prepared
analogously to BBI 15 from benzobis(imidazole) 35 (100 mg, 0.24
mmol) to provide 169 mg (>99% yield) of the desired product as a
pale yellow powder. 1H NMR (300 MHz, DMSO-d6): δ 10.48 (s, 1H),
10.33 (s, 1H), 9.1 (s, 1H), 8.27 (d, J ) 8.7 Hz, 2H), 8.07 (s, 1H), 8.04
(d, J ) 9.0 Hz, 2H), 7.80 (d, J ) 9.0 Hz, 2H), 7.25 (d, J ) 9.0 Hz,
2H), 4.38 (quart, J ) 6.9 Hz, 2H), 4.30 (s, 3H), 4.29 (s, 3H), 3.86 (s,
3H), 1.35 (t, J ) 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ
164.7, 160.6, 147.2, 136.7, 131.6, 131.3, 131.1, 131.0, 130.6, 129.8,
127.0, 125.8, 125.5, 115.5, 99.7, 98.2, 61.4, 55.8, 34.4, 34.3, 14.1.
HRMS (m/z): calcd for C26H25N4O3 [M - H+], 441.1927; found,
441.1930.
1
(95% yield) of the desired product. H NMR (400 MHz, DMSO-d6):
δ 10.47 (s, 1H), 10.43 (s, 1H), 9.16 (s, 1H), 8.14 (s, 1H) 8.07 (d, J )
8 Hz, 2H), 7.93 (d, J ) 8 Hz, 2H), 7.71 (m, 5H), 4.31 (s, 3H), 4.30 (s,
3H), 4.25-4.22 (m, 1H) 3.44-3.37 (m, 1H), 3.06-2.89 (m, 2H), 2.76-
2.70 (m, 1H), 2.65-2.60 (m, 2H). 13C NMR (100 MHz, DMSO-d6):
δ 175.8, 174.0, 172.8, 147.3, 147.1, 133.9, 133.0, 132.6, 131.1, 130.7,
130.5, 130.0, 129.8, 128.9, 125.9, 125.2, 99.8, 98.3, 36.2, 34.4, 34.3,
34.1, 26.3. HRMS (m/z): calcd for C29H27IN5O4S [M + I], 668.0828;
found, 668.0828.
Conjugate 36‚BSA. BBI 36 (61 mg, 0.08 mmol) and bovine serum
albumin (BSA) (200 mg, 0.003 mmol) were dissolved in distilled H2O
(3 mL). The resulting solution was stirred at 23 °C for 4 h. Upon
completion, the solution was transferred to a 6000 Da dialysis
membrane and dialyzed against distilled H2O for 48 h. Afterward, the
solution was concentrated under vacuum and analyzed.
1-(4-Carboethoxyphenyl)amino-5-chloro-2,4-dinitrobenzene (33).
A screw-cap vial was charged with 1,5-dichloro-3,4-dinitrobenzene (10)
(595 mg, 2.51 mmol), benzocaine (829 g, 5.02 mmol), EtOH (10 mL),
and a stir bar. The vial was then sealed with a Teflon-lined cap, and
the mixture was placed in an oil bath at 50 °C and stirred for 12 h.
Upon completion, the mixture was poured into 5% HCl (50 mL).
Precipitated solids were collected via vacuum filtration, rinsed with
H2O, and dried under vacuum to provide 875 mg (95% yield) of the
1-Chloro-2,4-dinitro-5-(phenylamino)benzene (37). 1,5-Dichloro-
2,4-dinitrobenzene (10) (10.0 g, 42.2 mmol) and PhNH2 (7.85 g, 84.4
1
i
desired product as a yellow powder. H NMR (400 MHz, CDCl3): δ
mmol) were dissolved in PrOH (250 mL), and the resulting mixture
9
J. AM. CHEM. SOC. VOL. 130, NO. 10, 2008 3155

A R T I C L E S
Boydston et al.
1
was stirred at room temperature for 48 h. The resulting slurry was then
poured into H2O (250 mL), and the solids were collected by vacuum
filtration, rinsed with H2O, and dried under vacuum to provide 11.6 g
provide 500 mg (96% yield) of the desired compound. H NMR (400
MHz, CDCl3): δ 8.39 (s, 1H), 7.70 (d, J ) 8.4 Hz, 4H), 7.59 (d, J )
8.4 Hz, 4H), 7.53-7.52 (m, 6H), 7.31-7.29 (m, 4H), 6.94 (s, 1H). 13
C
1
(94% yield) of the desired product. H NMR (400 MHz, DMSO-d6):
NMR (75 MHz, CDCl3): δ 151.2, 140.9, 136.6, 136.5, 134.2, 132.0,
130.4, 129.7, 129.2, 127.4, 118.3, 113.0, 110.1, 90.3. HRMS (m/z):
calcd for C34H21N6 [M + H+], 513.1828; found, 513.1822.
2,6-Bis(4-cyanophenyl)-1,7-dimethyl-3,5-diphenylbenzobis(imida-
zolium) Diiodide (43). This compound was prepared analogously to
BBIs 26-31 from benzobis(imidazole) 42 to provide 255 mg (>99%
δ 10.14, (s, 1H), 8.88 (s, 1H), 7.51 (m, 2H), 7.38 (m, 3H), 6.96 (s,
1H). 13C NMR (100 MHz, DMSO-d6): δ 145.3, 137.3, 134.6, 133.3,
130.0, 127.3, 126.6, 125.8, 117.9. HRMS (m/z): calcd for C12H9ClN3O4
[M + H+], 294.0282; found, 294.0283.
1-(4-Aminophenyl)amino-5-(phenyl)amino-2,4-dinitrobenzene (38).
In a flask fitted with a H2O-jacketed condenser, aryl chloride 37 (5.00
g, 17.1 mmol) and p-phenylenediamine (3.69 g, 34.1 mmol) were
1
yield) of the desired compound. H NMR (400 MHz, DMSO-d6): δ
9.35 (s, 1H), 8.13 (d, J ) 8.6 Hz, 4H), 8.06 (d, J ) 8.6 Hz, 4H),
7.61-7.55 (m, 10H), 7.48 (s, 1H), 4.20 (s, 6H). 13C NMR (100 MHz,
DMSO-d6): δ 152.3, 132.7, 132.5, 132.0, 131.9, 131.4, 130.5, 127.7,
125.4, 117.6, 115.5, 99.7, 96.9, 54.4, 34.1. HRMS (m/z): calcd for
C36H25N6 [M - H+], 541.2141; found, 541.2137. UV-vis and
fluorescence data in MeOH: λabs ) 384, log(ꢀ) ) 4.19, λem ) 547, Φf
) 0.33.
i
dissolved in PrOH (75 mL). The resulting mixture was stirred in an
oil bath at 80 °C for 24 h. Afterward, the mixture was poured into 100
mL of water, which caused solids to precipitate. After collection by
vacuum filtration, the solids were rinsed with H2O and then dried under
vacuum to provide 6.10 g (98% yield) of the desired product. 1H NMR
(400 MHz, DMSO-d6): δ 9.54 (s, 1H), 9.56 (s, 1H), 9.01 (s, 1H), 7.35-
7.16 (m, 2H), 7.25 (d, J ) 6.0 Hz, 2H), 7.16 (t, J ) 8.0 Hz, 2H) 6.89
(d, J ) 8.0 Hz, 2H), 6.21 (d, J ) 8.0 Hz, 2H), 6.33 (s, 1H), 5.17 (s,
2H). 13C NMR (100 MHz, DMSO-d6): δ 147.4, 147.3, 145.6, 137.8,
129.3, 128.5, 126.7, 125.7, 125.3, 124.7, 124.6, 124.3, 114.1, 95.4.
HRMS (m/z): calcd for C18H16N5O4 [M + H+], 366.1202; found,
366.1197.
Multiphoton Excitation. All experiments were performed using a
mode-locked Ti:S oscillator (Coherent Mira 900F) pumped by a 532
nm 10 W frequency-doubled neodymium-vanadate (Coherent Verdi)
laser. This source produces ca. 150 fs pulses at a repetition rate of 76
MHz. The near-IR output of the Ti:S was attenuated to desired powers
using a half-wave plate/polarizing beam splitting pair. After attenuation,
the beam was passed through a long-pass dichroic mirror (Chroma
Technology Corp., 575DCXR) and directed into the back aperture of
a 100× 1.3-NA oil-immersion objective (Fluar, Zeiss). Fluorescence
generated at the beam focus within a sample cuvette was collected by
the Fluar objective and reflected by the dichroic mirror through a series
of filters (5 cm saturated aqueous CuSO4 and two 3-mm thick BG-39
filters) before striking a bialkali photomultiplier tube (PMT; Hamamatsu
HC-125) detector. Signal was sent to a photon counter (Stanford
Research Systems SR-400), which collected data in 1 s intervals. Data
were corrected for background counts from the cuvette and solvent.
The collection efficiency of the instrument was estimated from the
fluorescence emission spectrum of BBI 4 and measured/reported
transmission spectra of the optics, filters, and PMT.
1-(4-Aminophenyl)-7-(phenyl)benzobis(imidazole) (39). This com-
pound was prepared analogously to benzobis(imidazole) 14 from
dinitroarene 38 (5.00 g, 15.4 mmol). Analysis of the crude product
1
mixture by H NMR spectroscopy and LC-MS revealed the presence
of a 1-(4-formamidophenyl) analogue of the desired product. To effect
hydrolysis of the formamide, the crude product was suspended in 10%
aqueous H2SO4 (100 mL) and heated at 100 °C for 3 h. The cooled
solution was then neutralized with K2CO3 powder, and the resulting
precipitate was collected via vacuum filtration, rinsed with H2O, and
dried under vacuum to provide 4.14 g (92% yield) of the desired
product. 1H NMR (400 MHz, DMSO-d6): δ 8.54 (s, 1H), 8.36 (s, 1H),
8.06 (s, 1H) 7.69 (d, J ) 8.0 Hz, 2H), 7.63-7.59 (m, 2H), 7.47-7.43
(m, 2H), 7.27 (d, J ) 8.0 Hz, 2H), 6.72 (d, J ) 8.0 Hz, 2H), 5.42 (s,
2H). 13C NMR (100 MHz, DMSO-d6): δ 148.7, 144.5, 143.9, 140.9,
140.8, 136.3, 132.2, 131.1, 130.1, 127.6, 125.3, 124.2, 123.6, 114.4,
109.0, 90.0. HRMS (m/z): calcd for C20H16N5 [M + H+], 326.1406;
found, 326.1407.
Benzobis(imidazole) 41. Benzobis(imidazole) 39 (3.00 g, 9.23
mmol) and 4,10-dioxatricyclo[5.2.1.0]dec-8-ene-3,5-dione (40) (1.53
g, 9.23 mmol) were dissolved in AcOH (150 mL), and the resulting
mixture was stirred at ambient temperature for 12 h. Upon completion,
the solution was neutralized with K2CO3 powder and the resulting
precipitate was collected via vacuum filtration, rinsed with H2O, and
dried under vacuum to provide 3.49 g (80% yield) of the desired
product. 1H NMR (400 MHz, DMSO-d6): δ 8.64 (s, 1H), 8.59 (s, 1H),
8.13 (s, 1H), 7.87 (d, J ) 8 Hz, 2H), 7.74 (d, J ) 8 Hz, 2H), 7.70 (s,
1H), 7.65-7.61 (m, 2H), 7.49-7.44 (m, 3H), 6.62 (s, 2H), 5.27 (s,
2H), 3.12 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 175.7, 172.2,
144.4, 141.23, 141.18, 136.7, 136.2, 136.0, 131.3, 131.0, 130.2, 128.5,
127.7, 124.1, 123.7, 109.5, 90.6, 80.8, 64.8, 47.6. HRMS (m/z): calcd
for C28H20N5O3 [M + H+], 474.1566; found, 474.1560.
Acknowledgment. We are grateful to the United States Army
Research Office (Grants W911NF-05-1-0430 and W911NF-06-
1-0147), the National Science Foundation (Grant CHE-
0645563), the Petroleum Research Fund as administered by the
American Chemical Society (Grant 44077-G1), and the Robert
A. Welch Foundation (Grant F-1621), for their generous
financial support. J.B.S. gratefully acknowledges support for
these studies from the Robert A. Welch Foundation (Grant
F-1331). C.W.B. is a Beckman Young Investigator, a DuPont
Young Professor, and a Cottrell Scholar of Research Corpora-
tion. J.B.S. is a Fellow of the Institute for Cellular and Molecular
Biology.
Supporting Information Available: A full list of authors for
ref 24a as well as 1H and 13C NMR spectra for new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
2,6-Bis(4-cyanophenyl)-1,7-diphenylbenzobis(imidazole) (42). This
compound was prepared analogously to benzobis(imidazole)s 20-25
from 1,5-diamino-2,4-bis(phenylamino)benzene dihydrochloride to
JA7102247
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3156 J. AM. CHEM. SOC. VOL. 130, NO. 10, 2008