A new water-soluble near-neutral ratiometric fluorescent pH indicator

Article abstract of DOI:10.1021/ol7026366

Donor-π-acceptor fluorene derivative 1c is a near-neutral pH indicator whose pK_a of ～7.0 was determined by both absorption and fluorescence methods. 1c satisfies important criteria for a sensitive ratiomeric fluorescent pH indicator with a distinctive isoemissive point, good dispersion in cell cytosol, and low cytotoxicity. Furthermore, its 2PA cross section of 100 GM in its neutral form suggests its potential in two-photon fluorescence imaging applications.

Full text of DOI:10.1021/ol7026366

ORGANIC
LETTERS
2
007
Vol. 9, No. 26
645-5648
A New Water-Soluble Near-Neutral
Ratiometric Fluorescent pH Indicator
5
†
‡
,†
Sheng Yao, Katherine J. Schafer-Hales, and Kevin D. Belfield*
Department of Chemistry and CREOL, College of Optics and Photonics, UniVersity of
Central Florida, P.O. Box 162366, Orlando, Florida 32816-2366, and Winship Cancer
Institute, Emory UniVersity School of Medicine, Atlanta, Georgia 30322
belfield@mail.ucf.edu
Received November 2, 2007
ABSTRACT
Donor−π−acceptor fluorene derivative 1c is a near-neutral pH indicator whose pK
a
of
∼
7.0 was determined by both absorption and fluorescence
methods. 1c satisfies important criteria for a sensitive ratiomeric fluorescent pH indicator with a distinctive isoemissive point, good dispersion
in cell cytosol, and low cytotoxicity. Furthermore, its 2PA cross section of 100 GM in its neutral form suggests its potential in two-photon
fluorescence imaging applications.
The dynamics of intracellular pH is believed to be crucial
for understanding the regulation mechanism of many physi-
ological functions in cells. Of the several methods available
a
to quantitatively measure pH, the pK of the indicator needs
to match the pH of the experimental system. Since the pH
in the cell cytosol is between 6.8 and 7.4, there is tremendous
interesting in developing efficient near-neutral fluorescent
pH indicators. Fluorescent indicators with ratiometric proper-
ties are highly desirable since the ratio of the fluorescence
intensity at peak wavelength vs insensitive isoemissive
wavelength is constant regardless of the change of fluoro-
phore concentration by photobleaching or change of the
external environment such as ion concentration. Here, we
report a sensitive near-neutral ratiometric pH indicator based
on a new class of fluorene derivatives.
1
to determine pH, optical methods have several advantages
including rapid response time, high signal-to-noise ratio,
noninvasiveness, and excellent pH sensitivity. Since the first
use of a trapped intracellular pH probe, 6-carboxyfluorescein,
2,3
was described by Thomas et al., a number of pH indicators
have been reported with a variety of properties.1
,4-11
In order
†
University of Central Florida.
Emory University School of Medicine.
‡
(1) Haugland, R. P.; Spence, M. T. Z.; Johnson, I.; Basey, A. The
Handbook: A Guide to Fluorescent Probes and Labeling Technologies,
Fluorene derivatives are known for exhibiting high fluo-
rescence quantum yields and excellent photostability.1
Recently, fluorene-based conjugated compounds have also
been reported as excellent two-photon fluorescent (2PF)
materials, in which they possess high two-photon absorption
1
2
0th ed.; Molecular Probes, Invitrogen Detection Technologies: Carlsbad,
005; pp 935 and references therein.
(
2,13
2) Thomas, J. A.; Buchsbaum, R. N.; Zimniak, A.; Racker, E.
Biochemistry 1979, 18, 2210-2218.
3) Belt, J. A.; Thomas, J. A.; Buchsbaum, R. N.; Racker, E. Biochemistry
(
1
979, 18, 3506-3511.
(
4) El-Shishtawy, R. M.; Almeida, P. Tetrahedron 2006, 62, 7793-7798.
(5) Bizzarri, R.; Arcangeli, C.; Arosio, D.; Ricci, F.; Faraci, P.; Cardarelli,
F.; Beltram, F. Biophys. J. 2006, 90, 3300-3314.
(10) Dong, S.; Ma, H.; Li, X.; Sun, M.; Duan, X. Anal. Lett. 2004, 37,
2937-2948.
(
6) Zhang, Z.; Achilefu, S. Chem. Commun. 2005, 5887-5889.
(7) Valuk, V. F.; Duportail, G.; Pivovarenko, V. G. J. Photochem.
(11) Diwu, Z.; Chen, C.-S.; Zhang, C.; Klaubert, D. H.; Haugland, R. P.
Chem. Biol. 1999, 6, 411-418.
Photobiol., A 2005, 175, 226-231.
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J. Org. Chem. 2005, 70, 4152-4157.
9) Ertekin, K.; Cinar, S.; Aydemir, T.; Alp, S. Dyes Pigm. 2005, 67,
33-138.
(
(12) Belfield, K. D.; Bondar, M. V.; Przhonska, O. V.; Schafer, K. J. J.
Photochem. Photobiol., A 2004, 162, 489-496.
(13) Belfield, K. D.; Bondar, M. V.; Przhonska, O. V.; Schafer, K. J. J.
Photochem. Photobiol., A 2004, 162, 569-574.
(
1
1
0.1021/ol7026366 CCC: $37.00
© 2007 American Chemical Society
Published on Web 11/22/2007

Scheme 1. Structures of Fluorene Derivatives 1a-c and Synthesis of Fluorescent Probe 1c
2PA) cross sections.1
4-18
As part of our efforts to develop
then introduced in 5 by a Michael reaction. Subsequent
(
high-efficiency two-photon absorbing fluorene derivatives
as novel fluorescent biomarkers, we found D-π-A type
compounds 7-(benzo[d]thiazol-2-yl)-N,N-diphenyl-9H-fluo-
ren-2-amine, such as chromophores 1a and 1b (Scheme 1),
have utility for one-photon and two-photon fluorescence cell
imaging, with near unity fluorescence quantum yields and
reasonable 2PA cross sections, i.e., ∼100 GM (1 GM )
reduction of nitro to amine, diethylation of the primary amine
with triethylphosphate, and hydrolysis of the nitrile groups
afforded the final product 1c in good overall yield. All
compounds were fully characterized by NMR and elemental
analysis (or HRMS in the case of compounds with carboxylic
acid moieties).
As expected, compound 1c exhibits reasonable water
-
50
4
-1 19,20
-4
-3
10
cm s photon ).
However, these two compounds
solubility, i.e., >10 M at pH 1-7 and >10 M at pH
7-12. At pH 4, the absorption and emission maxima of
are rather hydrophobic, thereby limiting their applications
in aqueous biological environments. Thus, compound 1c was
specifically designed to improve the water solubility by intro-
duction of the dipropionic carboxylic acid at the 9-position
of the fluorene ring, and by replacement of the dipheny-
lamino group in 1a and 1b with diethylamino group, resulting
+
protonated 1cH are 341 and 391 nm, respectively (Figure
1A), with a fluorescence quantum yield of 0.21. The 2PA
+
cross section of the 1cH was low in the excitation
wavelength range of 570-750 nm, which may be attributed
to very strong electron-withdrawing nature of the protonated
in a pK
a
close to 7.0. Therefore, the potential of 1c as a near-
nitrogen of the diethylamino group (see the pK measure-
a
neural ratiometric pH fluorescent indicator was investigated
and is reported herein. The 2PF properties of 1c were also
investigated due to its potential in 2PF biological imaging.
The synthetic scheme for probe 1c is shown in Scheme 1.
Converting the iodo group in 2 into cyano afforded precursor
ment). In this A-π-A type structure, the absorption
wavelength is expected to shift to shorter wavelength and
the single-photon excitation allowed S -S transition is not
0
1
2
1
allowed by two-photon excitation. In contrast, at pH 10
buffer, where the neutral form of 1c exists, the absorption
maximum underwent a bathochromic shift of 41 to 382 nm,
and the fluorescence quantum yield increased to 0.56 (Figure
1B). Significantly, a dramatic increase of the 2PA cross
section to ca. 100 GM at the excitation wavelength of 770
nm was observed. The formally 2PA forbidden single-photon
3
that was easily hydrolyzed to carboxylic acid 4. The
carboxylic acid was then transformed to the acid chloride in
situ, and reacted with aminothiophenol in DMSO to form
benzothioazole derivative 5. Two propionitrile groups were
(
14) Belfield, K. D.; Schafer, K. J.; Mourad, W.; Reinhardt, B. A. J.
Org. Chem. 2000, 65, 4475-4481.
15) Belfield, K. D.; Morales, A. R.; Hales, J. M.; Hagan, D. J.; Van
Stryland, E. W.; Chapela, V. M.; Percino, J. Chem. Mater. 2004, 16, 2267-
273.
16) Belfield, K. D.; Morales, A. R.; Kang, B.-S.; Hales, J. M.; Hagan,
D. J.; Van Stryland, E. W.; Chapela, V. M.; Percino, J. Chem. Mater. 2004,
6, 4634-4641.
17) Morales, A. R.; Belfield, K. D.; Hales, J. M.; Van Stryland, E. W.;
allowed S
0
1
-S transition of 1c (382 nm) is accessible by
21
2PA due to the relaxed D-π-A symmetry of the molecule.
(
Even though 2PA was measured at pH 10, as can be seen in
the inset of Figure 2, the deprotonated (high 2PA) form
predominates at pH 7.5 and above, making this probe
especially attractive for 2PF imaging.
2
(
1
(
Hagan, D. J. Chem. Mater. 2006, 18, 4972-4980.
The pH-dependent absorption spectra are presented in
Figure 2 for titration in aqueous buffer. With the increase in
(
18) Yao, S.; Belfield, K. D. J. Org. Chem. 2005, 70, 5126-5132.
(19) Schafer-Hales, K. J.; Belfield, K. D.; Yao, S.; Frederiksen, P. K.;
Hales, J. M.; Kolattukudy, P. E. J. Biomed. Opt. 2005, 10, 051402/1-
51402/8.
20) Schafer, K. J.; Yao, S.; Belfield, K. D.; Hales, J. M.; Hagan, D. J.;
Van Stryland, E. W. Proc. SPIE-Int. Soc. Opt. Eng. 2004, 5329, 201-206.
0
(21) Hales, J. M.; Schafer, K. J.; Morales, A. M.; Belfield, K. D.; Hagan,
D. J.; Van Stryland, E. W. Proc. SPIE-Int. Soc. Opt. Eng. 2003, 5211, 21-
30.
(
5646
Org. Lett., Vol. 9, No. 26, 2007

peak of the neutral form at 382 nm evolved with a well-
defined isosbestic point at 355 nm. It is known that the
22
basicity of the diethylaminobenzene nitrogen is significantly
2
3,24
higher than that of the benzothiazole nitrogen.
Hence,
the structure of the protonated form of 1c is assigned as
protonation at the diethylamino site. The inset in Figure 2
shows the results of the nonlinear regression of the λ at 341
2
5
nm according to a literature method, affording a pK
a
value
of 6.95 ( 0.01. The pK value may also be calculated from
a
the pH dependence of the total integrated fluorescence
emission of 1c excited at 340 and 382 nm, taking the
advantage of the well-separated emission bands of the two
forms. Similar nonlinear regression analysis yielded nearly
same pK value (6.96 ( 0.04).
a
When 1c was excited at the wavelength of the absorption
isosbestic point (355 nm), fluorescence emission from both
protonated and neutral forms was observed, as shown in
Figure 3. The characteristic isoemission point at 493 nm
makes 1c an excellent ratiometric pH indicator.
Figure 1. Absorption (solid line), emission (dashed line), and two-
+
photon absorption (squares) spectra of (A) protonated form 1cH
(in pH 4 buffer) and (B) neutral form 1c (in pH 10 buffer). The
absorption and fluorescence spectra are normalized.
Figure 3. pH-dependence of the fluorescence intensity of 1c in
-
5
buffer (10 M) excited at 355 nm (absorption isobestic point
wavelength in Figure 2) with arrows indicating the change of the
fluorescence intensities with pH increase. (Inset) Ratiometric
calibration curve of I391/I493 (intensity at 391 nm vs intensity at
isoemissive point 493 nm).
pH, the absorption peak at 341 nm, attributed to the
protonated form of 1c, becomes weak, and the absorption
For intracellular applications, one major concern for the
probes is the cell permeativity of the indicator. Therefore,
1
c was incubated with NT2 (NTERA-2 cl.D1 [NT2/D1])
cells, and subjected to two-photon fluorescence imaging
Figure 4). From this cursory study, it was clear the dye had
(
good permeativity and dispersed well in the cytosol. In
addition, this promising result implies that 1c may also serve
as a precursor of 2PF amine reactive probes that can be
readily activated by esterification of the carboxylic acid group
using succinimide hydroxide.
The cytotoxic effect of 1c on proliferating cells is another
parameter of primary interest, particularly for live-cell
(
22) Wiczling, P.; Markuszewski, M. J.; Kaliszan, R. Anal. Chem. 2004,
Figure 2. pH dependence of the absorption spectra of dye 1c in
buffer at concentration of 0.01 mM with arrows indicating the
change of the absorption intensities with pH increase. (Inset)
Nonlinear fitting of the pH-dependent extinction coefficients at 341
nm.
7
6, 3069-3077.
(
(
23) Belfield, K. D.; Schafer, K. J. Chem. Mater. 2002, 14, 3656-3662.
24) Dey, J. K.; Dogra, S. K. Bull. Chem. Soc. Jpn. 1991, 64, 3142-
3
152.
(25) Billo, E. J. Excel for Chemists, A ComprehensiVe Guide; Wiley-
VCH: New York, 1997; p 303.
Org. Lett., Vol. 9, No. 26, 2007
5647

(control) after 48 h, indicating low toxicity of 1c over a
relatively wide concentration range.
In summary, probe 1c was synthesized as a near-neutral
fluorescent pH indicator with pK of 6.96, confirmed by both
a
absorption and fluorescence methods. The distinctive iso-
emissive point observed in the fluorescence spectra at
different pHs, good dispersion in cell cytosol, and low
cytotoxicity indicates that 1c satisfies important criteria for
an excellent one-photon ratiometric fluorescent pH indicator.
Furthermore, its high 2PA cross section in the deprotonated
form at pH > 7 suggests its potential in other 2PF imaging
techniques (a subject of future investigation).
Acknowledgment. We acknowledge the National Science
Foundation (ECS-0524533) and the University of Central
Florida Presidential Initiative for Major Research Equipment
for partial support of this work. We thank Prof. Dr. Kiminobu
Sugaya (Biomolecular Science Center, University of Central
Florida) for his support in cell culturing, Dr. Jie Fu, Prof.
Eric W. Van Stryland, and Prof. David J. Hagan (CREOL,
University of Central Florida) for 2PA measurements.
Figure 4. 2PF image of NT2 cells incubated with 1c excited at
95 nm (164 fs, 76 MHz).
7
fluorescence imaging applications. An Alamar Blue (AB)
2
6
reduction analysis was used to assess cytotoxic effects of
c on proliferating NT2 cells. The cells were treated with
1
different concentrations of compound 1c (0.1-100 µM)
dissolved in buffer, and were also treated with 10% AB
solution. The observed fluorescence intensity of AB reduction
by cells treated with various doses of 1c was similar to that
observed for cells untreated with any fluorene compound
Supporting Information Available: Detailed synthesis
procedures and characterization of the compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(26) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000,
2
67, 5421-5426.
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