A rational approach to tuning the pKa values of rhodamines for living cell fluorescence imaging

Article abstract of DOI:10.1039/c0ob01045f

A novel strategy to systematically tune the pK_a values of rhodamines is described. This strategy was applied to rationally develop compound 1e with a pK_a of 6.5, the highest among rhodamine amide derivatives, and it could be employed

Full text of DOI:10.1039/c0ob01045f

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A rational approach to tuning the pKa values of rhodamines for living cell
fluorescence imaging†
Lin Yuan, Weiying Lin* and Yanming Feng
Received 18th November 2010, Accepted 5th January 2011
DOI: 10.1039/c0ob01045f
A novel strategy to systematically tune the pKa values of rho-
damines is described. This strategy was applied to rationally
develop compound 1e with a pKa of 6.5, the highest among
rhodamine amide derivatives, and it could be employed to
detect acidic pH variations in living cells with a turn-on signal.
Since the seminal work by Czarnik’s group about the first
rhodamine-based fluorescent chemodosimeter for Cu2+,3 rho-
damines have been widely exploited as the scaffold of fluorescence
turn-on probes for various metal ions (e.g. Cu2+, Hg2+, Fe3+, Cr3+,
Pb2+, Zn2+, etc.) and biologically relevant species (e.g. NO, ClO-,
etc.).4–5 When compared to fluoresceins, rhodamines appear to
have better photostability. Another major distinction between
rhodamines and fluoresceins is that they have the opposite trend
of fluorescence response to pH variations. Unlike fluoresceins,
at basic or neutral conditions, rhodamine amide derivatives are
non-fluorescent, as they exist largely in the spirocyclic form (Fig.
1B), whereas rhodamine amide derivatives are fluorescent under
acidic conditions, as the ring-opened form dominates. Thereby,
when the pH values of the environment decrease, intensity-based
rhodamine probes may exhibit a fluorescence enhanced signal.
From the fluorescence response point of view, rhodamines are
superior to fluoresceins in monitoring attenuation of pH values.
It is known that the pKa values of fluoresceins can be tailored
by introducing electron-withdrawing groups nearby the phenolic
alcohol on the xanthene ring.6 However, to our best knowledge,
no strategy has been previously developed to tune the pKa
values of rhodamines. Although scarce examples of rhodamine-
based fluorescent pH probes have been constructed by empirical
experience,7 the rational design of rhodamine-based fluorescent
pH probes is still very challenging due to the lack of a suitable
approach to systematically tune the pKa values of rhodamines.
Thus, it is of interest to develop new molecular strategies for
modulating the pKa values of rhodamines.
Fluoresceins have been extensively employed as fluorescent pH
probes, as their fluorescence properties are sensitive to pH
variations in the environment.1 Under basic or neutral conditions,
fluoresceins are fluorescent, as they exist largely in the ring-opened
form (Fig. 1A). By contrast, under acidic conditions, fluoresceins
are nonfluorescent, as the spirocyclic form dominates. Thus, when
the pH values of the environment decrease (for instance, from
neutral to acidic conditions), intensity-based fluorescein probes
may display a fluorescence quenching signal. Since fluorescent
quenching probes could provide false positive data by other
fluorescent quenchers in real samples and fluorescence quenching
response often leads to a low signal-to-noise ratio,2 it is much more
desirable to detect the diminution of pH values by an enhanced
fluorescence signal.
It is known that rhodamine 6G derivatives are much more
sensitive than rhodamine B derivatives as fluorescent turn-on
probes.8 We reasoned that this behavior may be ascribed to the
steric effect of the methyl group on the xanthene ring of rhodamine
6G derivatives. In other words, the steric methyl group may
facilitate the conversion of the nonfluorescent spirocyclic form
to the fluorescent ring-opened form. Since the pKa of rhodamine
defines the equilibrium between the spirocyclic form and the ring-
opened form, we reasoned that the pKa values of rhodamines
could be systematically modulated by introducing a steric group
on the nitrogen atom of the amide moiety.
In this work, to validate our working hypothesis, we constructed
a series of new rhodamine 6G derivatives 1a–e and rhodamine
B derivatives 2a–e containing different steric bulky substituents
(Scheme 1) to examine their pKa values. Indeed as anticipated,
there is a good correlation between pKa values and the steric bulky
Fig. 1 (A) pH-dependent equilibrium between the spirolactone form
and the ring-opened form of fluoresceins; (B) pH-dependent equilibrium
between the spirolactam form and the ring-opened form of rhodamine
amide derivatives.
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of
Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan
410082, P. R. China. E-mail: weiyinglin@hnu.cn; Fax: (+)81 731 88821464;
Tel: (+)81 731 88821464
† Electronic supplementary information (ESI) available: Experimental
procedures, characterization data and additional spectra. See DOI:
10.1039/c0ob01045f
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Table 1 The pKa values of compounds 1a–e and 2e
a
a
pKa
pKa
1a
1b
1c
2.8
3.2
3.6
1d
1e
2e
3.3
6.5
5.6
a The pKa was calculated according to the Henderson–Hasselbach-type
mass action equation.7a,9
the steric methyl group in rhodamine 6G promotes the formation
of the fluorescent ring-opened form. The analysis of fluorescence
intensity changes at 556 nm as a function of pH by using the
Henderson–Hasselbach-type mass action equation7a,9 gave pKa
values of rhodamine 6G derivatives 1a–d as shown in Table 1.
Indeed, there is a correlation between pKa values and the steric
bulky substituents on the nitrogen atom of the amide moiety, in
good agreement with our working hypothesis.
Although compounds 1a–d have marked fluorescence under
acid conditions, the pKa values of these compounds are too low
(< 4.0) to be useful for biological applications. In order to further
increase pKa, based on our above finding, we decided to introduce
a highly sterically bulky group, 1-adamantanamine, to the nitrogen
atom of the amide moiety in rhodamines 6G and B scaffolds to
give compound 1e and 2e (Scheme 1), respectively.
As shown in Fig. 2 and Table S1, compound 1e is essentially
nonfluorescent at pH 9.0. However, compound 1e showed a
Scheme 1 Synthesis of rhodamine 6G derivatives 1a–e and rhodamine B
derivatives 2a–e.
substituents. Based on this key finding, we were able to rationally
design novel rhodamine 6G derivative 1e with a pKa of 6.5, which
makes it suitable for monitoring acidic pH variations in living cells
with a fluorescence turn-on signal by fluorescence imaging.
The synthesis of rhodamine 6G derivatives 1a–e and rhodamine
B derivatives 2a–e is outlined in Scheme 1. rhodamine 6G acid 3a
was treated with POCl3 to afford the acid chloride intermediate,
which was further reacted with various substituted amines 4a–e
to give the desired products 1a–e. A similar synthetic strategy was
used to prepare rhodamine B derivatives 2a–e from rhodamine B
1
3b. The structures of the products were confirmed by H NMR,
13C NMR, and MS spectroscopy.
With these compounds in hand, we set out to investigate
their spectral properties at different pH values. As expected, the
rhodamine B derivatives 2a–d only showed slight fluorescence
with attenuation of pH values from 7.4 to 2.0 (Figures S1–4A).
Furthermore, when compared to rhodamine B 3b, rhodamine
B derivatives 2a–d have a much weaker fluorescence at pH
2.0 indicating that the non-fluorescent spirocyclic form is still
dominating even at a pH as low as 2.0 (Figures S1–4B). The
same conclusion can be drawn from the absorption profiles
(Figure S5). By sharp contrast, the rhodamine 6G derivatives
1a–d displayed intense fluorescence with decrease of pH values
from 7.4 to 2.0 (Figures S6–9A), and they have the emission
close to rhodamine 6G acid 3a at pH 2.0 (Figures S6–9B). These
findings are further supported by the studies of absorption (Figure
S10). Thus, the comparison of rhodamine 6G derivatives 1a–d
and rhodamine B derivatives 2a–d collaborates the premise that
Fig. 2 (a) pH-dependence of the fluorescence intensity of probe 1e (2 mM)
with the arrow indicating the change of the fluorescence intensities with pH
decrease from 9.0 to 3.0. Spectra were obtained with excitation at 500 nm
in 25 mM PBS aqueous solution (containing 20% DMF as a co-solvent).
(b) Fluorescence responses (fluorescence intensity at 556 nm) of probe 1e
(2 mM) to different pH values (from 9.0 to 3.0). The inset shows the linear
relationship of fluorescence intensity at 556 nm and varying pH values
from 5.5 to 7.5.
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very large fluorescence enhanced signal (70-fold enhancement
of fluorescence intensity at 556 nm) with attenuation of pH
values from 9.0 to 3.0. The pH-dependent absorption spectra
are in good agreement with the pH-dependent emission spectra
(Fig. S11). The fluorescence titration data provides the pKa
of compound 1e as 6.5, which is much higher than those of
compounds 1a–d (< 4.0) and other rhodamine derivatives reported
(4.85).7a This finding confirms the anticipated effect of steric
groups on the pKa values. Similarly, compound 2e also exhibited
a large fluorescence enhanced response with a decrease of pH
values from 8.5 to 3.5 (Fig. S12 and Table S2). The pKa of
compound 2e was calculated to be 5.6, which is smaller than
that of compound 1e, reinforcing the steric effect of methyl
group.
The potential interference of biologically relevant species on the
fluorescence response of probes 1e and 2e was evaluated. As shown
in Figures S14–15, high concentrations of K+, Na+, Ca2+, Mg2+,
Cu2+, Zn2+, Fe3+, Co2+, and other biologically important species
(glucose, Ser, Cys, Arg, Val, GSH, vitamin C, H2O2) caused no
visible effect on the fluorescence signals. In addition, the probes 1e
and 2e are highly stable after irradiation with green light (centered
at 500 nm) for 30 min (Figure S16).
that probe 1e has low cytotoxicity and the cells remained alive
during the imaging process (Fig. S18). The nuclear staining also
revealed that probe 1e associates with the cytoplasm of Hela cells
(Figure S18).
In summary, we have described, for the first time, a general
approach to systematically tune the pKa values of rhodamines
by incorporating a steric group on the nitrogen atom of the
amide moiety. This novel approach was validated by examining
the pKa values of a series of rhodamine 6G derivatives 1a–e and
rhodamine B derivatives 2a–e. On the basis of this approach, new
compound 1e with a pKa of 6.5 was judiciously designed. Notably,
the pKa of compound 1e is higher than that of other rhodamine
amide derivatives known, and it is suitable for detecting acidic pH
variations in living cells with a fluorescence turn-on signal. This
demonstrates the significance of our approach. We anticipate that
the simple and effective strategy introduced herein may be widely
applicable for the rational design of xanthene-based fluorescent
pH probes. Furthermore, the steric effect on the fluorescence
turn-on of rhodamines should have profound implications for
the judicious design of xanthene-based fluorescent probes for
a wide variety of targets including metal ions and biologically
relevant molecules. Although the steric group is incorporated on
the nitrogen atom of the amide moiety in the approach described
herein, we expect that the introduction of steric groups on the
xanthene ring may have the similar effect on pH values and perhaps
on fluorescence turn-on in general. Work along this line is in
progress in our laboratory.
To examine the potential utility of the novel rhodamine-based
probes for fluorescence imaging in living cell◦s, probe 1e (2 mM) was
incubated with Hela cells for 30 min at 37 C, and then the cells
were washed in PBS medium of varying pH values with the addi-
tion of nigericin (1 mg mL-1) to elicit a rapid exchange of K+ for H+
for a fast equilibration of external and internal pH.10 As shown in
Fig. 3b–e–h, the fluorescence in living cells became much brighter
when the pH values were decreased from 7.4 to 5.6, indicating that
probe 1e is cell membrane permeable and could be used to monitor
attenuation of pH values in living cells with a fluorescence turn-on
signal. Furthermore, the MTT assays (Fig. S17 in the Supporting
This research was supported by NSFC (20872032, 20972044),
NCET (08–0175), and the Key Project of Chinese Ministry of
Education (108167).
Notes and references
11
Information) indicate that probe 1e of concentrations below
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Fig. 3 Images of Hela cells treated with probe 1e at different pH values.
(a) Bright field image of Hela cells incubated with probe 1e (2 mM) at pH
7.4; (b) Fluorescence image of (a); (c) The overlay image of (a) and (b);
(d) Brightfield image of Hela cells incubated with probe 1e (2 mM) at pH
6.5. (e) Fluorescence image of (d); (f) The overlay image of (d) and (e); (g)
Brightfield image of Hela cells incubated with probe 1e (2.0 mM) at pH
5.6; (h) Fluorescence image of (g); (i) The overlay image of (g) and (h). The
fluorescence images were acquired with green light excitation.
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