Combinatorial synthesis of benzimidazolium dyes and its diversity directed application toward GTP-selective fluorescent chemosensors

Article abstract of DOI:10.1021/ja063733d

Highly selective fluorescence turn-on GTP sensor, GTP Green, was discovered by a diversity directed sensor approach, combined by solid-phase combinatorial synthesis of a benzimidazolium library and high-throughput screening. Copyright

Full text of DOI:10.1021/ja063733d

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Published on Web 07/22/2006
Combinatorial Synthesis of Benzimidazolium Dyes and Its Diversity Directed
Application toward GTP-Selective Fluorescent Chemosensors
Shenliang Wang and Young-Tae Chang*
Department of Chemistry, New York UniVersity, New York, New York 10003
Received May 28, 2006; E-mail: yt.chang@nyu.edu
Scheme 1. Synthesis of Benzimidazolium Dyesa
Fluorescent chemosensors are dye molecules whose fluorescence
excitation/emission changes in response to the surrounding medium
or through specific molecular recognition events.1 Due to their
simplicity and high sensitivity, fluorescent sensors have been widely
utilized as popular tools for chemical, biological, and medical
applications.1,2 The most general strategy for fluorescent sensor
design is to combine fluorescence dye molecules with designed
receptors for specific analytes, in hopes that the recognition event
between receptor and analyte will lead to a fluorescence property
change of the dye moiety. Although many fluorescent sensors have
been successfully developed through this approach, each individual
development requires a major effort in both the design and synthesis
of the sensors. Also, the sensor’s scope of application is limited to
the selected specific analytes that the sensor was rationally designed
for, so-called Analyte Directed Sensors.3,4 Combinatorial dye library
synthesis offers one of the most promising alternatives as DiVersity
Directed Sensors, once an efficient synthetic route can be developed
for a diverse set of dyes. Combinatorial chemistry is now widely
a Reagents and conditions: (a) triethyl orthoacetate, H+, toluene, reflux;
(b) KOH, MeI, acetone; (c) Tf2O, poly(4-vinylpyridine), DCM; (d) 4, DCM;
(e) 48% HBr, 65 °C; (f) 2-chlorotrityl alcohol resin sequentially treated
with thionyl chloride in DCM and ethylenediamine in DCM; (g) 8, HATU,
DIPEA, 30% DMF/DCM; (h) R-CHO (96 aromatic aldehydes, Supporting
Information Chart S1), pyrrolidine, NMP; (i) 5% TFA/DCM.
being used in the chemical biology and medicinal/pharmaceutical
fields for the discovery of biologically active molecules or drug
candidates, yet the application of this method to fluorescent dyes
is only in its infancy.5 Herein, we report a synthesis of a com-
binatorial benzimidazolium dye library and the discovery of the
first turn-on fluorescent GTP sensor reported thus far.
Nucleotide anion detection has long intrigued researchers and
witnessed continuous growth.6,7 Assuming the cationic hemicyanine
dye is a potential receptor of nucleotides due to electrostatic
interactions, we chose the benzimidazolium motif as the library
scaffold of the fluorescent sensors. Condensation of benzimidazo-
lium ring with 96 aromatic aldehydes provides extended conjugation
and structural diversity. To achieve longer wavelengths of the final
fluorophore, which may be more useful for possible biological
application, we introduced two Cl groups to the benzimidazolium
ring (green-red range of emission) rather than using an unsubsti-
tuted benzimidazolium ring (UV-blue range of emission). It is
noteworthy that the diversity elements (from aldehydes) constitute
part of the conjugation system of the dye products and will also
serve as recognition motifs for analyte binding. Without linking
two separate motifs, as in common analyte directed sensors, our
diversity directed sensors can be smaller in size and may respond
more directly to their conformational change upon analyte binding.
To facilitate the synthetic procedure, securing high purity
compounds without further purification, we developed a novel solid-
phase synthesis pathway for the benzimidazolium library. The
optimized synthetic procedure is described in Scheme 1. The
benzimidazolium scaffold with linker was prepared in solution phase
and loaded onto ethylenediamine derivatized 2-chlorotrityl poly-
styrene solid support. Various lengths of the linker were tested and
optimized for best loading of the benzimidazolium compound onto
the resin. Aromatic aldehyde building blocks were then coupled to
the benzimidazolium ring on solid support, and final products were
Figure 1. Structure of G32 and G49 and their quinolinium analogues.
collected by acidic cleavage. Due to the structural diversity, various
excitation/emission wavelengths were observed (see Supporting
Information).
For a primary screening, the synthesized dye compounds were
tested at 20 µM for 100 µM of AMP, ADP, ATP, UTP, CTP, and
GTP in 10 mM HEPES buffer (pH ) 7.4) with 1% DMSO in 384-
well microplates using a fluorescence plate reader. Two structurally
related compounds (G32 and G49, Figure 1) showed dramatically
increased fluorescence upon addition of GTP, while not responding
to other nucleotides. Although GTP plays an important role in
biological processes,8 very little work has been done on the
development of fluorescent sensors for it.7 Thus far, the best
reported GTP sensor, which was designed rationally, showed around
90% quenching response at around mM concentration of GTP,7b
and most of the known GTP sensors compete with ATP to some
extent. To our knowledge, no turn-on fluorescent sensors for GTP
have been reported yet.
9
10380
J. AM. CHEM. SOC. 2006, 128, 10380-10381
10.1021/ja063733d CCC: $33.50 © 2006 American Chemical Society

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
C O M M U N I C A T I O N S
In conclusion, we discovered the first turn-on GTP fluorescent
sensor thus far from a semi-designed diversity directed sensor
approach. On the basis of this unprecedented high selectivity of
G49 to GTP and its visual green fluorescence increase, we propose
to dub this compound “GTP Green.” The details of molecular
recognition between GTP Green and GTP, structure-activity
relationships, and biological application studies are in progress.
Acknowledgment. This work was supported by the National
Institutes of Health (P20GM072029). Components of this work were
conducted in a Shared Instrumentation Facility constructed with
support from Research Facilities Improvement Grant C06 RR-16572
from the NCRR/NIH.
Supporting Information Available: Complete experimental details;
spectral data for G32, G49, and precursors thereof; fluorescence
emission spectra of G32 upon addition of 16 analytes; fluorescent
titration of G32 and G49 upon GTP. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 2. Fluorescence emission spectra (excitation ) 480 nm, cutoff )
515 nm) of G49 (1 µM) with 100 µM of GTP (green), ATP (red), all other
14 analytes and blank control (all in black) in 10 mM HEPES buffer
(pH ) 7.4) with 1% DMSO; 96-well picture was taken using 5 µM of G49
for better visualization, otherwise in the same condition, under 365 nm UV
lamp light.
References
(1) Valeur, B. Molecular Fluorescence: Principles and Applications; Wiley-
VCH: New York, 2001.
(2) (a) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.;
Kluwer Academic/Plenum: New York, 1999. (b) Czarnik, A. W.
Fluorescent Chemosensors for Ion and Molecule Recognition; American
Chemical Society: Washington, DC, 1993.
(3) (a) Srinivasan, N.; Kilburn, J. D. Curr. Opin. Chem. Biol. 2004, 8, 305.
(b) Rurack, K.; Resch-Genger, U. Chem. Soc. ReV. 2002, 31, 116. (c)
Valeur, B.; Leray, I. Coord. Chem. ReV. 2000, 205, 3.
(4) (a) Martinez-Manez, R.; Sancenon, F. Chem. ReV. 2003, 103, 4419. (b)
de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J.
M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97,
1515.
(5) (a) Li, Q.; Lee, J. S.; Ha, C.; Park, C. B.; Yang, G.; Gan, W. B.; Chang,
Y. T. Angew. Chem., Int. Ed. 2004, 43, 6331. (b) Gao, J.; Watanabe, S.;
Kool, E. T. J. Am. Chem. Soc. 2004, 126, 12748. (c) Rosania, G. R.; Lee,
J. W.; Ding, L.; Yoon, H. S.; Chang, Y. T. J. Am. Chem. Soc. 2003, 125,
1130. (d) Zhu, Q.; Yoon, H. S.; Parikh, P. B.; Chang, Y. T.; Yao, S. Q.
Tetrahedron Lett. 2002, 43, 5083.
(6) Recent examples of nucleotide fluorescent detection: (a) Li, C.; Numata,
M.; Takeuchi, M.; Shinkai, S. Angew. Chem., Int. Ed. 2005, 44, 6371.
(b) Descalzo, A. B.; Marcos, M. D.; Martinez-Manez, R.; Soto, J.; Beltran,
D.; Amoros, P. J. Mater. Chem. 2005, 15, 2721. (c) Mizukami, S.; Nagano,
T.; Urano, Y.; Odani, A.; Kikuchi, K. J. Am. Chem. Soc. 2002, 124, 3920.
(d) Ojida, A.; Park, S.; Mito-oka, Y.; Hamachi, I. Tetrahedron Lett. 2002,
43, 6193. (e) Sancenon, F.; Benito, A.; Lloris, J. M.; Martinez-Manez,
R.; Pardo, T.; Soto, J. HelV. Chim. Acta 2002, 85, 1505. (f) Thanh, N.;
Rathbone, D.; Billington, D.; Hartell, N. Anal. Lett. 2002, 35, 2499. (g)
Turkewitsch, P.; Wandelt, B.; Darling, G.; Powell, W. J. Photochem.
Photobiol., A 1998, 117, 199.
(7) (a) Kim, S. K.; Moon, B. S.; Park, J. H.; Seo, Y.; Koh, H. S.; Yoon, Y.
J.; Lee, K. D.; Yoon, J. Tetrahedron Lett. 2005, 46, 6617. (b) Kwon, J.
Y.; Singh, N. J.; Kim, H. N.; Kim, S. K.; Kim, K. S.; Yoon, J. Y. J. Am.
Chem. Soc. 2004, 126, 8892. (c) McCleskey, S. C.; Griffin, M. J.;
Schneider, S. E.; McDevitt, J. T.; Anslyn, E. V. J. Am. Chem. Soc. 2003,
125, 1114. (d) Amemiya, S.; Buhlmann, P.; Umezawa, Y. Chem. Commun.
1997, 1027.
To fully check the selectivity of the two hit compounds, all the
nucleosides (adenosine, uridine, cytidine, guanosine) and nucleotides
(XNP, where X ) A, U, C, G, and N ) mono, di, tri) were tested
systematically in a 96-well plate. High selectivity of both G49 and
G32 only to GTP was clearly exhibited without any obvious cross
response to any of other nucleotides or nucleosides (Figure 2 for
G49 and Figure S9 in Supporting Information for G32). As we
observed, G32 suffered from significant photobleaching under
strong irradiation light, so we decided to focus on G49 for further
analysis. Upon addition of GTP (100 µM) to G49 (1 µM), a red
shift for both λex (from 450 to 480 nm) and λem (from 520 to 540
nm) was observed (the full titration curve is available in Supporting
Information). When excited at 480 nm, an approximately 80-fold
fluorescence increase at an emission wavelength of 540 nm was
observed only for GTP, while only two (ATP) or fewer fold changes
were observed for all other analytes. In the same condition, dGTP
showed a little weaker (70-fold increase) but almost similar response
to that of GTP. This indicates that the 2′-hydroxyl group of GTP
is crucial for the molecular interaction with G49. The quantum
yields (Φ) of G49 before and after addition of GTP were 0.003
and 0.074, respectively.9 A visual distinction was also possible when
5 µM of G49 was used (picture in Figure 2).
The quinolinium analogues of G32 and G49 were also prepared
as control compounds, and neither of them showed any fluorescence
change in the presence of GTP. Combined with the fact that none
of the other 94 library members that originated from different
aldehyde groups showed strong fluorescent response to GTP, we
postulate that both the imidazolium and the 2-phenylindole moiety
are important for selective GTP recognition.
(8) (a) Burma, D. P. J. Sci. Ind. Res. 1988, 47, 65. (b) Pogson, C. I. Am. J.
Clin. Nutr. 1974, 27, 380.
(9) Fluorescence quantum yields were determined by reference to fluorescein
in 0.1 N NaOH (Φ ) 0.95) with excitation at 450 nm following these
references: (a) Fery-Forgues, S.; Lavabre, D. J. Chem. Educ. 1999, 76,
1260. (b) Brannon, J. H.; Magde, D. J. Phys. Chem. 1978, 82, 705.
JA063733D
9
J. AM. CHEM. SOC. VOL. 128, NO. 32, 2006 10381