9-Aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one (Seoul-Fluor) as a smart platform for colorful ratiometric fluorescent pH sensors

Article abstract of DOI:10.1039/c1cc12618k

In this communication, we report that 9-aryl-1,2-dihydropyrrolo[3,4-b] indolizin-3-one (Seoul-Fluor) can serve as a potential platform for colorful ratiometric fluorescent pH sensors by simple incorporation of pH responsive elements on Seoul-Fluor. Seoul-

Full text of DOI:10.1039/c1cc12618k

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COMMUNICATION
9-Aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one (Seoul-Fluor) as a smart
platform for colorful ratiometric fluorescent pH sensorsw
Eunha Kim,a Sanghee Leea and Seung Bum Park*ab
Received 4th May 2011, Accepted 23rd May 2011
DOI: 10.1039/c1cc12618k
In this communication, we report that 9-aryl-1,2-dihydropyrrolo-
[3,4-b]indolizin-3-one (Seoul-Fluor) can serve as a potential
platform for colorful ratiometric fluorescent pH sensors by simple
incorporation of pH responsive elements on Seoul-Fluor.
Seoul-Fluor-based fluorescent pH sensors allow the emission- and
pH-tuning ability upon protonation by varying their pKa values and
electronic characteristics of substituents by a rational design.
the R1 and R2 positions of Seoul-Fluor result in dramatic
emission changes from blue to red (Fig. 1).15,16 On the basis of
our previous studies, we envisioned that the unique features
of Seoul-Fluor could lead to new applications for the Seoul-
Fluor core skeleton, as a colorful ratiometric fluorescent
sensor platform. In other words, in biological systems, target
events associated with changes in the electronic states of
specific functional groups at the molecular level can induce
changes in the photophysical properties of Seoul-Fluor,
especially the emission wavelength.
The investigation of pivotal events in a much smaller world
than the one to which we are adapted has been of prime
interest to scientific communities, therefore various molecular
devices have been invented to convert invisible molecular events
into visible signals.1 Owing to their advantageous features,
fluorescent molecules have been widely used in various bio-
medical applications, including cellular imaging,2,3 biological
assays,4 medical diagnostics,5 and environmental monitoring.6
However, despite the high demands for these molecules, only a
limited number of fluorescent core skeletons are used.7,8
Moreover, most of the reported fluorescent sensors use a
turn-on/off sensing mechanism via photoinduced electron
transfer (PET) events of the traditional fluorophores.9,10
However, the signal of turn-on/off sensors, which show a
single increase or decrease in the emission intensity, can be
complicated by external influence. Owing to increasing attention
on multiplexing capability in image-based biological applications,
it is important to develop a series of colorful ratiometric
fluorescent sensors for the better understanding of biological
systems at the molecular level.11–14 Therefore, the development
of a new molecular framework for fluorophores with a tunable
emission wavelength and the subsequent use of the fluorophores
in the design of ratiometric sensors for the study of individual
events in a complex environment can help satisfy current
demands of the biomedical community.
For a proof-of-concept study, we chose pH changes as the
target molecular level event and designed novel ratiometric
fluorescent pH sensors through the simple introduction of pH
responsive elements at specific positions of the Seoul-Fluor core
skeleton. The protonation of these pH responsive elements leads
to changes in their electronic characteristics in accordance with
their pKa values and subsequent dramatic shifts in the emission
wavelength of Seoul-Fluor. We first introduced an amine moiety
as a pH responsive element at the R1 position of Seoul-Fluor,
because we envisioned that the protonation of the dialkylamino
group leads to the hypsochromic shift of the emission wavelength
Recently, we reported Seoul-Fluor (SF), a novel fluorescent
core skeleton with emission wavelength tunability covering the
full color range and simple changes in the electronic state at
a Department of Chemistry, Seoul National University, Seoul 151-747,
Korea. E-mail: sbpark@snu.ac.kr; Fax: +82 2 884 4025;
Tel: +82 2 880 9090
b Department of Biophysics and Chemical Biology,
Seoul National University, Seoul 151-747, Korea
w Electronic supplementary information (ESI) available: Detailed
synthetic procedures, photophysical property data of all compounds,
and spectroscopic data of all new compounds. See DOI: 10.1039/
c1cc12618k
Fig. 1 (a) Chemical structure of Seoul-Fluor and schematic illustration
of systematic changes in the emission wavelength with the protonation of
pH responsive moieties at the R1 and R2 positions of Seoul-Fluor.
(b) Structures of Seoul-Fluor derivatives and corresponding photography
images taken under irradiation at 365 nm.
c
7734 Chem. Commun., 2011, 47, 7734–7736
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through the reduction of electron donating characters (EDG) at
the R1 position (Fig. 1a). Seoul-Fluor with a dipropylamino
group at the R1 position and an acetyl group at the R2 position,
which we refer to as SF24, is a candidate for a ratiometric
fluorescent pH biosensor. As shown in Fig. 2a, SF24 showed a
clear hypsochromic shift of the emission wavelength from
619 nm to 532 nm with a pH change from 7.0 to 3.0 (Fig. S1,
ESIw). This confirmed that the protonation of the dipropylamino
group on SF24 perturbs the electronic states of Seoul-Fluor,
leading to the drastic changes in the emission properties. We also
examined the ratiometric response of SF24 in the pH range
3.0–6.0 and observed a gradual reduction in the emission
intensity at 619 nm as the acidity increased. Concurrently, a
gradual increase in the emission intensity at a wavelength of
532 nm was observed as the pH decreased. The sigmoid curves
obtained from the emission spectra of SF24 at variable pH
clearly confirmed the potential of Seoul-Fluor as a ratiometric
sensor platform (Fig. S2, ESIw). The inflection point of the two
sigmoid curves for SF24 is pH 3.7.
larger shift in the emission wavelength compared to that in the
case of carboxylate (SF45). However, no shift was observed in
the emission wavelength if Seoul-Fluor was not modified to
contain a pH responsive element, as in the case of the compound
SF46 (Fig. 2c and Fig. S1, ESIw). This result confirmed that the
Seoul-Fluor skeleton could serve as an appealing molecular
framework for ratiometric fluorescent sensors through the
introduction of electronic state perturbing elements such as pH
responsive moieties.
The first interesting feature of Seoul-Fluor is that its pH
response is directly correlated with the pKa of the pH responsive
element used for its modification. This allows the fine-tuning
of the pH sensitivity of Seoul-Fluor through the introduction
of substituents having different pKa values. To validate this
statement, we incorporated several amine-based, pH responsive
elements with different pKa values at the R1 position of Seoul-
Fluor by simple modifications to the alkyl groups of the
aniline moiety, from propyl (SF24) to ethyl (SF44) to methyl
(SF47), leading to fine changes in the pKa value of each
fluorescent compound.17 As expected, the emission wavelengths
of SF24, SF44, and SF47 are almost identical (Fig. S3, ESIw)
and all three sigmoid curves in the pH-response scatter plot of
the emission intensity exhibited the same pattern, but the
inflection points of each curve were shifted from pH 3.7 to
pH 4.0 and pH 4.8 (Fig. 3). This result confirmed that Seoul-
Fluor-based pH sensors can be designed to have a unique pH
response with identical photophysical properties through the
incorporation of desired pH responsive elements with appropriate
pKa values at the R1 or R2 position of Seoul-Fluor.
Introduction of a carboxylic acid moiety as a pH responsive
element at the R2 position of Seoul-Fluor (CF3 moiety at
the R1 position, hereafter referred to as SF45) resulted in a
bathochromic shift of the emission wavelength from 473 nm to
503 nm for a change in the pH from 7.0 to 3.0 (Fig. 2b and
Fig. S1, ESIw). The protonation of the carboxylic acid reduces
the electron-donating character, which perturbs the electronic
state of Seoul-Fluor. However, in this case, an electronic change
occurs at the R2 position, causing a bathochromic shift rather
than a hypsochromic shift. We observed a 30 nm bathochromic
shift of the emission wavelength upon protonation of the
carboxylic acid at the R2 position (SF45), while protonation of
the amine moiety at the R1 position (SF24) caused an 87 nm
hypsochromic shift of the emission wavelength. This observation
can be explained by the pronounced change in the electronic state
of dialkylamine (SF24) upon protonation and the subsequent
The second interesting feature of Seoul-Fluor is that the
electronic characters of substituents at the R1 and R2 positions
display opposite trends with regard to changes in their emission
wavelengths. While the enhancement of the EWG character at
the R2 position triggers a bathochromic shift of the emission
wavelength, the same change at the R1 position causes a
hypsochromic shift (Fig. 1a). Therefore, we can control the
direction of color change (hypsochromic or bathochromic
shift) corresponding to a change in the pH, simply by choosing
the location of pH responsive elements between the R1 and R2
positions of the Seoul-Fluor skeleton (Fig. 2). Considering
that the current ratiometric fluorescent pH sensors have quite
limited flexibility with regard to the range in which their
emission can vary18 and considering that it has now become
Fig. 2 Schematic representation of structural changes in Seoul-Fluor
derivatives with a change in the pH from 7.0 to 3.0. The pH responsive
elements are either introduced at the R1 (a: SF24) or R2 (b: SF45)
position or not introduced at all (c: SF46). Photography images of
Seoul-Fluor derivatives in buffer solutions with different pH values
were taken under irradiation at 365 nm.
Fig. 3 Scatter plot of the emission intensity about SF24 (red), SF44
(black), and SF47 (blue) for a change in the pH from 3.0 to 6.0 with
the excitation/emission wavelength at 417/532 nm.
c
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the desired emission wavelength and identical pH response by
carrying out simple substituent modifications on the Seoul-Fluor
skeleton.
In this study, we showed that Seoul-Fluor derivatives can
function as a novel molecular framework for developing
ratiometric fluorescent pH sensors when they are incorporated
with pH-responsive elements at specific positions of Seoul-Fluor.
Each pH sensor exhibited dramatic changes in the emission
wavelength with changes in the solution pH. Moreover, the
direction of emission shift (bathochromic or hypsochromic
shift) and the desired emission color can be controlled by
inducing simple changes in the electronic state or the position
of substituents. In addition, a unique pH response can be
achieved by fine-tuning the pKa values of the substituents on
Seoul-Fluor. Therefore, Seoul-Fluor can serve as a colorful
palette for ratiometric fluorescent sensors, not only for pH,
but also for various biological events that can change the
electronic state of substituents on Seoul-Fluor. We envision
that the tunable photophysical properties of Seoul-Fluor
with a rational design will lead to the diverse application of
Seoul-Fluor for the development of unique ratiometric
fluorescent sensor platforms, which can be quite useful in
high-content screening and multiplex monitoring applications.
This study was supported by the National Research
Foundation of Korea (NRF) and the WCU program of the
NRF, funded by the Korean Ministry of Education, Science,
and Technology (MEST). E. Kim and S. Lee are grateful for
the fellowships awarded by the BK21 Program and the Seoul
Science Fellowship.
Fig. 4 Normalized emission spectra and photography images of
fluorescent pH sensors in a pH 7.0 buffer solution. The images of
each compound in the buffer solution were taken under irradiation at
365 nm.
possible to control both the hypsochromic or bathochromic
shift of the emission wavelength, our results are of specific
interest and clearly show that the incorporation of electronic-
state-perturbing elements at specific locations in Seoul-Fluor
makes Seoul-Fluor a remarkable ratiometric and emission-
tunable fluorescent sensor platform.
Notes and references
1 S. M. Borisov and O. S. Wolfbeis, Chem. Rev., 2008, 108,
423–461.
2 X. Chen, K.-A. Lee, E.-M. Ha, K. M. Lee, Y. Y. Seo, H. K. Choi,
H. N. Kim, M. J. Kim, C.-S. Cho, S. Y. Lee, W.-J. Lee and
J. Yoon, Chem. Commun., 2011, 47, 4373–4375.
3 B. C. Dickinson, J. Peltier, D. Stone, D. V. Schaffer and
C. J. Chang, Nat. Chem. Biol., 2011, 7, 106–112.
4 K. K. Ghosh, E. Yap, H. Kim, J.-S. Lee and Y. T. Chang, Chem.
Commun., 2011, 47, 4001–4003.
5 R. Adhikary, P. Mukherjee, G. Krishnamoorthy, R. A. Kunkle,
T. A. Casey, M. A. Rasmussen and J. W. Petrich, Anal. Chem.,
2010, 82, 4097–4101.
6 D. T. Quang and J. S. Kim, Chem. Rev., 2010, 110, 6280–6301.
7 M. S. T. Gonc¸ alves, Chem. Rev., 2009, 109, 190–212.
8 E. Kim and S. B. Park, Chem.–Asian J., 2009, 4, 1646–1658.
9 A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M.
Huxley, C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem.
Rev., 1997, 97, 1515–1566.
10 Y. Urano, M. Kamiya, K. Kanda, T. Ueno, K. Hirose and
T. Nagano, J. Am. Chem. Soc., 2005, 127, 4888–4894.
11 B. Liu and H. Tian, Chem. Commun., 2005, 3156–3158.
12 M. Taki, J. L. Wolford and T. V. O’Halloran, J. Am. Chem. Soc.,
2004, 126, 712–713.
13 Z. Xu, Y. Xiao, X. Qian, J. Cui and D. Cui, Org. Lett., 2005, 7,
889–892.
14 C. J. Chang, J. Jaworski, E. M. Nolan, M. Sheng and
S. J. Lippard, Proc. Natl. Acad. Sci. U. S. A., 2004, 101,
1129–1134.
We then directed our attention to color-tuning of Seoul-
Fluor-based fluorescent pH sensors. As discussed, the Seoul-
Fluor system has two different sites (R1 and R2) that can be
modified to control the emission wavelength. Therefore, we
can consider one site as a pH responsive position and the other
site as a color tuning position. As shown in Fig. 4a, substituent
changes at the R2 position from hydrogen (SF48) to phenyl
(SF49) and acetyl (SF24) for a fixed pH-responsive dipropyl-
amino group at the R1 position triggered a bathochromic shift
of the emission maximum from 502 nm (SF48) to 550 nm
(SF49) and 619 nm (SF24) at pH 7.0. This significant batho-
chromic shift might be caused by strengthening the intra-
molecular charge transfer (ICT) process involving the enrichment
of electron-withdrawing characters at the R2 position of Seoul-
Fluor. All three compounds exhibited the hypsochromic shift
of the emission wavelength for a change in the pH from 7.0 to
3.0 (Fig. S4, ESIw). Likewise, with a fixed pH-responsive
carboxylic acid group at the R2 position, substituent changes
at the R1 position from CN (SF50) to Me (SF51) and to OMe
(SF52) triggered the bathochromic shift of the emission
maximum from 474 nm (SF50) to 496 nm (SF51) and 506 nm
(SF52) at pH 7.0 (Fig. 4b). All three compounds also exhibited
the bathochromic shift of the emission wavelength for a
change in the pH from 7.0 to 3.0 (Fig. S4, ESIw). Thus, we
have clearly demonstrated that Seoul-Fluor-based fluorescent
pH sensors can be tailored through a rational design to display
15 E. Kim, M. Koh, B. J. Lim and S. B. Park, J. Am. Chem. Soc.,
2011, 133, 6642–6649.
16 E. Kim, M. Koh, J. Ryu and S. B. Park, J. Am. Chem. Soc., 2008,
130, 12206–12207.
17 B. B. P. Tice, I. Lee and F. H. Kendall, J. Am. Chem. Soc., 1963,
85, 329–337.
18 J. Han and K. Burgess, Chem. Rev., 2010, 110, 2709–2728.
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