Determination of intracellular pH using sensitive, clickable fluorescent probes

Article abstract of DOI:10.1016/j.bmcl.2012.02.021

We synthesized and evaluated a series of acidic fluorescent pH probes exhibiting robust pH dependence, high sensitivity and photostability, and excellent cell membrane permeability. Titration analyses indicated that probe 3 could increase its fluorescence intensity 800-fold between pH 8.0 and 4.1. Additionally, its pK_a value is optimal for intracellular probing of acidic organelles. Fluorescent imaging of HepG2 and Hela cells further revealed that probe 3 demonstrates outstanding capacity for monitoring of intracellular [H⁺] levels. The easily accessible terminal alkyne/azido function groups of these probes offer the possibility of rapidly constructing sensor molecule libraries using 'click' chemistry.

Full text of DOI:10.1016/j.bmcl.2012.02.021

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2440–2443
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Bioorganic & Medicinal Chemistry Letters
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Determination of intracellular pH using sensitive, clickable fluorescent probes
Nazmiye B. Yapici a, Srinivas Rao Mandalapu a, Teng-Leong Chew b, Satya Khuon b, Lanrong Bi a,
⇑
a Department of Chemistry, Michigan Technological University, Houghton, MI 49931, USA
b Northwestern Cell Imaging Facility, Northwestern University Feinberg School of Medicine, 303 E. Chicago Avenue, Chicago, IL 60611, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized and evaluated a series of acidic fluorescent pH probes exhibiting robust pH dependence,
high sensitivity and photostability, and excellent cell membrane permeability. Titration analyses indi-
cated that probe 3 could increase its fluorescence intensity 800-fold between pH 8.0 and 4.1. Addition-
ally, its pKa value is optimal for intracellular probing of acidic organelles. Fluorescent imaging of
HepG2 and Hela cells further revealed that probe 3 demonstrates outstanding capacity for monitoring
of intracellular [H+] levels. The easily accessible terminal alkyne/azido function groups of these probes
offer the possibility of rapidly constructing sensor molecule libraries using ‘click’ chemistry.
Published by Elsevier Ltd.
Received 15 November 2011
Revised 7 February 2012
Accepted 7 February 2012
Available online 16 February 2012
Keywords:
Intracellular pH
Fluorescent probe
Numerous systemic pathologies, including renal tubular acidosis
(RTA), metabolic disorders, diabetes, emphysema, and inflamma-
tion, as well as infection and cancer, manifest acidemia and acidosis
as accompanying pathological features.1 For example, solid tumors
whose intracellular pH ranges from 5.8 to 7.7 are, on average, 0.55
units lower than that of normal tissue associated with a higher level
of anaerobic metabolism via the glycolytic pathway.1b,c Following
excretion from tumor cells, acidic products (primarily lactate) accu-
mulate in extracellular and extravascular spaces due to poor lym-
phatic drainage. Dramatic differences in pH characteristics
between normal and cancerous tissue provides a unique opportu-
nity for optical identification of cancerous cells using a probe with
a robust response to pH. At the same time, monitoring of pH fluctu-
ations in acidic organelles is essential to investigate the normal cel-
lular functions and activities in these compartments.2 A large
number of pH probes with fluorescent capabilities have been widely
used, but most lack sensitivity or are not suitable for characterizing
acidic organelles such as lysosomes (pH 4.5–5.5), endosomes (pH
4.6–5.0) and spermatozoa acrosomes, since their fluorescence is
substantially quenched in acidic environments.3 Ideally, fluorescent
pH probes that are functional under acidic conditions will (i) be
essentially non-fluorescent in the extracellular environment at pH
7.4; (ii) highly fluorescent under acidic conditions (pH <6); (iii) ab-
sorb and fluoresce above 550 nm to avoid auto-fluorescence; (iv)
display a pKa within the typical pH range of acidic organelles; and
(v) be readily synthesized with an additional functional group that
facilitates attachment of other structural moieties to the probe
using mild reactions. Along these lines, rhodamine-based dyes are
superior candidates for the construction of an on/off fluorescent
probe moiety in relation to excellent spectroscopic properties corre-
sponding to large molar extinction coefficients, high fluorescence
quantum yields, and visible wavelength excitation. Accordingly,
we have developed a new series of analogues for the measurement
of intracellular pH, employing the rhodamine scaffold as our precur-
sor molecule. The current report describes our development of these
probes, their spectroscopic characteristics, and their utility, for
intracellular imaging.
First, we undertook our synthetic approach to prepare fluores-
cent pH probes 1–3 using commercially available rhodamine B.
The synthesis of fluorescent probes 1–3 can be readily accom-
plished employing sequential ‘click’ reactions under mild condi-
tions (Scheme 1). Probe 1 is derived via reaction of rhodamine B
with propargylamine (74% yield). It is then readily converted to
probe 2 via formation of the tosylate derivative (96% yield).
]Reaction of probe 2 with tripropargylamine yields probe 3 (28%
yield). All probes are rhodamine lactam-type derivatives and their
spirocyclic structures are confirmed by NMR data. In the lactam
form, the spirocyclic compounds lack significant absorbance and
fluorescence in the visible spectrum, which can be restored when
they are converted to in the amide form. Probes 1–3 have a quater-
nary carbon atom in the alkyl region of their 13C spectra (Fig. S1).
The spiro carbon appears at d 65 ppm, indicative of the spirocyclic
structure for all three probes. Additional evidence confirming the
spirocyclic structure for probes 1–3 was obtained by UV–vis titra-
tion. Absorbance is almost completely lost for all probes as the pH
is adjusted from acidic to basic conditions. Absorption and fluores-
cence emission of probes 1–3 are pH-dependent, with a maximal
absorption peak at 564 4 nm under acidic condition (Fig. 1 and
Figs. S2 and S3).
All probes were non-fluorescent under basic conditions,
whereas a shift to acidic conditions yielded a pink chromophore
⇑
Corresponding author.
E-mail address: lanrong@mtu.edu (L. Bi).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2012.02.021

N. B. Yapici et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2440–2443
2441
Scheme 1. Synthesis of fluorescent pH probes 1–3.
Figure 1. Absorbance and emission spectra of probe 3 at 480 nm excitation wavelength.
with strong fluorescence. These observations indicate that these
probes may serve as ‘naked-eye’ indicators for [H+]. Walter et al.
described amide and lactam forms of rhodamine 101 conjugates
that exist in pH-dependent equilibrium, an observation consistent
with the structural interconversion of probes 1–3 we observed (
Fig. 2). Figure S4 shows the time-dependent emission alternations
Figure 2. Proposed mechanism for fluorescent enhancement of probe 3 upon pH change.

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N. B. Yapici et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2440–2443
of probes 1–3 at 584 4 nm reveal that maintenance of the probes
at 4.10 0.1 for 15 min results in complete interconversion of
lactam-amide. As shown in Figure 1, the fluorescence peak of probe
and lactate production. Reliance of these cells on anaerobic metab-
olism leads to an oxygen-poor environment, offsetting the capacity
of aerobic (normal cells) to thrive and enhancing growth of the
malignant anaerobic tissues. We examined the capacity of probe
3 to detect pH alterations in HepG2 cells (human hepatocellular li-
ver carcinoma cell line) and Hela cells (human cervical cancer
cells). Since cell fixation would induce a myriad of artifacts, our
experiments were carried out in living, non-fixed cells, in order
to prevent alterations of the cellular machinery due to fixation.
Following 1 h incubation with probe 3, intracellular localization
was visible in Hela cells. The fluorescent signal remains strong over
a 24-h period when cells are incubated for 2 h with probe prior to
removal of excess probe by repetitive washing and incubation of
probe-loaded cells dye-free medium (Fig. S5). Additionally, we
examined different concentrations for staining and found no evi-
dence for cell damage in conjunction with dye loading. Bright-field
examination revealed cell viability at the highest concentration
(30 M) of probe 3. To analyze the exact localization of probe 3 in-
side the cells, different cellular localization markers were used.
Double stain of cells (Hela and HepG2 cells) with mitotracker
and Hoechst 33342 revealed that the probe 3 does not co-localize
with mitotracker, even if the staining pattern is very similar (Figs.
S6 and S7). Next, a double staining with lysotracker and Hoechst
33342 indicated that the predominant probe distribution was
likely localizing in acidic vesicles (Fig. 4 and Fig. S8). These differ-
ences in fluorescence intensities within single cells, consistent
with intracellular pH distributions are clearly demonstrated in Fig-
ure 4 and Figure S6–S8. Finally, we performed intracellular micro-
scopic examination of probe 3 in cells to detect changes in regional
pH values. Following treatment with nigericin, an H+/K+ antiporter,
fluorescent images of probe-3 loaded-cells were determined at dif-
ferent pH values. As documented in Figure S9 (representative of all
the cells we examined), the intracellular pH sensitivity of probe 3 is
revealed through the finding of increasing fluorescence as a func-
tion of intracellular pH (the latter in equilibrium with the extracel-
lular pH).
3
was 584 4 nm with minimal intensity beyond pH 6.25;
conversely, fluorescence intensity increased >800-fold as the pH
decreased from 8.0 to 4.1. We subsequently examined the standard
fluorescence pH titration employing 0.07 M buffered solution
(0.2 M K2HPO4–0.1 M citric acid buffer). As described in Figure 3,
the fluorescence emission at 584 4 nm fluctuates with the pH
titration curve of the probes (1 M) in 0.07 M potassium phos-
phate-citric acid buffer solution. Of the three probes, probe 3 was
the most responsive under acidic conditions, which makes this
probe the most applicable for investigation of disorders with al-
tered pH regulation.
The utility of our probes for pH determinations in acidic organ-
elles is underscored by the pKa values of ꢀ4.8 determined by
absorption and fluorescence titration, which closely approximates
the typical pH range of organelles such as lysosomes. Our probes
maintain strong fluorescence at low pH and a constant dye concen-
tration, which makes them superior to other fluorescent pH probes
(e.g., fluoresceine) where fluorescence is heavily quenched under
acidic conditions. In sum, our rhodamine- probes have several un-
ique advantages that can be explored in the development of fluo-
rescent acidotropic probes, including high absorbance and
fluorescence enhancement under acidic conditions, as well as the
ability to form visually discernible chromophores that will enable
‘naked-eye’ detection.
We next assessed the capacity of probe 3 to functionally deter-
mine pH alterations in cancerous cells. Warburg reported that can-
cer cells develop a lower pH associated with increased glycolysis
As shown in this report, several features of our probes (absence
of cytotoxicity, high cell permeability, long-lived intracellular fluo-
rescence) make our pH probes superior candidates for examining
Figure 4. Fluorescence images of probe 3 in Hela cells. Probe 3 (30
was incubated in non-FBS DMEM media for 2 h and counterstained with lysotracker
(1 M, red, B), Hoechst 33342 (1 g/mL, blue, C); and an overlay (D). All images
were acquired with 40Â objective.
lM, green, A)
Figure 3. Absorption and emission titration curves of probes 1–3 as a function of
pH.
l
l

N. B. Yapici et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2440–2443
2443
intracellular pH alterations in intact cells. Unlike other commer-
References and notes
cially available fluorescent pH probes, we do not observe decreased
fluorescent signal once cells are incubated in dye-free medium.
Accordingly, our probes offer novel experimental opportunities
for live-imaging in intact cells over multiple time points (hours),
and will thus add a new tool in understanding intracellular altera-
tions in normal and cancerous cell growth. The easily accessible
terminal alkyne/azido ‘clickable’ function groups of probe 1–3 offer
the possibility of rapidly constructing sensor molecule libraries
using ‘click’ chemistry. Exploitation of this approach to prepare
multiple fluorescent sensors is currently underway in our
laboratories.
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Acknowledgments
L.B. is grateful to NASA (Grant MSGC R85197), Research Excel-
lence Fund (Grant R01121) for support of this project. L.B. is also
grateful to Drs. Nicholas J. Turro, Steffen Jockusch from Columbia
University and Drs. Michael Gibson, Dallas K. Bates, Xiaoqing Tang,
and Jeremy Goldman from Michigan Technological University for
many helpful discussions.
Supplementary data
Supplementary data (experimental details of synthesis, fluores-
cence spectroscopy, NMR data and fluorescence imaging) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2012.02.021.