A newly designed cell-permeable SNARF derivative as an effective intracellular pH indicator

Article abstract of DOI:10.1039/c003167d

We have successfully developed a new cell-permeable SNARF derivative that is activated inside the cell by ester hydrolysis and is notable for having reduced background fluorescence.

Full text of DOI:10.1039/c003167d

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A newly designed cell-permeable SNARF derivative as an effective
intracellular pH indicatorw
Eiji Nakata,*a Yoshihiro Yukimachi,a Yoshijiro Nazumi,a Yoshihiro Uto,a Hiroshi Maezawa,b
Toshihiro Hashimoto,c Yasuko Okamotoc and Hitoshi Hori*a
Received 15th February 2010, Accepted 17th March 2010
First published as an Advance Article on the web 8th April 2010
DOI: 10.1039/c003167d
We have successfully developed a new cell-permeable SNARF
derivative that is activated inside the cell by ester hydrolysis and
is notable for having reduced background fluorescence.
SNARF derivative that is non-fluorescent before hydrolysis
should have advantages for accurate pH monitoring.
The dynamics of intracellular pH (pHi) is crucial for under-
standing the mechanisms of regulation of many physiological
functions of the cell.1 Among the several methods available to
determine pH, optical methods have various advantages
including rapid response time, high signal-to-noise ratio,
non-invasiveness, and excellent pH sensitivity.2 Various types
of fluorescent pHi indicators have been developed,3 and some
have been successfully applied to monitoring changes in pHi.4
Using these indicators, the role of fluctuations in pHi during
the course of endocytic events has been examined.5
We recently reported non-fluorescent SNARF derivatives in
which the phenolic substituents were protected by a benzyl
group, because these compounds exist mainly as SNARF(L)
in aqueous solution.8 These results prompted us to prepare a
newly designed SNARF derivative UTX-40, which emits
fluorescence only after ester hydrolysis (Fig. 1 and Scheme
S1w). UTX-40 is comprised of a p-acetoxybenzyl moiety
directly linked to SNARF through an ether linkage.
SNARF–OAc and SNARF–OAM, the phenolic moieties of
which were masked by acetyl and acetoxymethyl, respectively,
were also synthesized as control compounds (details of
the synthetic procedures for UTX-40, SNARF-OAc and
SNARF-OAM are provided in the ESIw).
Evaluation of the spectroscopic properties of these SNARF
derivatives (Fig. 2a and Table 1) revealed that SNARF–OAc
and SNARF–OAM produced the distinctive absorption and
fluorescent spectra of SNARF(A). On the other hand, 10 times
smaller absorption peaks and very weak fluorescence were
observed when UTX-40 was measured, indicating that
UTX-40 was mainly in the form of SNARF(L). When compared
to the brightness of SNARF(A), UTX-40 was almost 100 times
less bright (1.1% of relative brightness) as shown in Table 1. In
contrast, SNARF–OAM and SNARF–OAc display significant
fluorescence, albeit with somewhat lower brightness (46% and
18%, respectively). These properties were clearly confirmed
under transilluminator observations (Fig. 2b).
SNARF (seminaphthorhodafluor) is one of the most
commonly used pH indicators, because of its unique fluorescent
characteristics. These include to be excited by visible light,
dual-emission properties for ratiometric measurement and a
neutral pKa region suitable for monitoring physiological pH
ranges.6 In aqueous solution, the phenolic substituent of
SNARF is critical for its dual-emission properties in that an
equilibrium exits between the acidic phenol form with a lem of
583 nm (lmax of 544 nm) (written as SNARF(A)) and the basic
phenolate form with a lem of 627 nm(lmax of 573 nm) (written
as SNARF(B)). Thus, the pH is calculated from the ratio of
SNARF(A) and SNARF(B). An additional equilibrium
involves formation of a colorless and non-fluorescent lactone,
which is the dominant form of SNARF in non-polar environ-
ments (written as SNARF(L)).6,7
To increase cell permeability, the phenolic group of SNARF
is converted to esterase substrates such as acetate or acetoxy-
methyl ester derivatives.3a,7 However such cell-permeable
SNARF derivatives retain the fluorescence (lem of 583 nm)
characteristic of SNARF(A) in aqueous solution (Fig. S1,
ESIw). This may result in a low signal-to-noise ratio if the
media have fluorescence or to incorrect pH measurement if the
protected SNARF remains inside the cell. Therefore, a
Next, the behavior of these derivatives as esterase substrates
was evaluated. Though all of these SNARF derivatives could
a Department of Life System, Institute of Technology and Science,
Graduate School, The University of Tokushima,
Minamijosanjimacho 2, Tokushima 770-8506, Japan
b Institute of Health Biosciences, The University of Tokushima,
Tokushima 770-8509, Japan
c Faculty of Pharmaceutical Sciences, Tokushima Bunri University,
Yamashiro-cho, Tokushima 770-8514, Japan.
E-mail: nakata@bio.tokushima-u.ac.jp, hori@bio.tokushima-u.ac.jp;
Fax: +81-88-656-7517; Tel: +81-88-656-7517
w Electronic supplementary information (ESI) available: Further
experimental details and data. See DOI: 10.1039/c003167d
Fig. 1 Schematic illustration of UTX-40 activation by intracellular
ester hydrolysis.
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Fig. 3 Real time absorbance change via esterase-catalyzed ester
hydrolysis at pH 7.5. (a) UTX-40 and (b) SNARF–OAM. (Inset)
Fluorescence spectral change (excited at 534 nm).
Fig. 2 (a) Absorbance (blue) and fluorescence (red) spectra of
SNARF (bold line), UTX-40 (plane line), SNARF–OAM (dash line)
and SNARF–OAc (dotted line) at pH 5.0. (b) Photograph of
SNARF(1), UTX-40(2), SNARF-OAM(3) and SNARF–OAc(4) at
pH 5.0.
which induced the displacement of equilibrium toward
SNARF(L). As UTX-40 is converted into SNARF by enzymatic
hydrolysis, the aggregates become diffused and monomeric
SNARF displays its fluorescent properties.
Table 1 Spectroscopic properties of SNARF derivatives used in
this study
Finally, the properties of the new derivatives as pHi indica-
tors were evaluated.10 The uptake of the SNARF-based
fluorescent intensity inside V79 cells (Chinese hamster lung)
was confirmed by fluorescent microscopic analysis. Immediately
on addition of SNARF–OAM (Fig. 4b) or SNARF–OAc
(Fig. S5w), a strong background signal from medium was
observed. Therefore it was very difficult to distinguish between
the inside and outside of the cell. However in the case of
UTX-40 (Fig. 4a), the medium was scarcely fluorescent under
the same conditions, and only the uptake of the fluorescent
intensity inside the cell was observed after 15 min incubation.11
This result strongly indicates the benefit of the UTX-40 in
reducing the background noise before ester hydrolysis. In
addition, the cellular uptake of the SNARF was quantitatively
evaluated by flow cytometry analysis. As shown in Fig. 4c and
Fig. S6w, the effective cellular uptake of SNARF was confirmed
in the case of UTX-40 as compared to SNARF–OAc or
SNARF–OAM. Furthermore, the actual pHi values of V79
cells in PBS (pH 7.2) were determined using UTX-40. The
fluorescent spectra of SNARF inside cells are shown Fig. 4d.
The ratio values R (= 583 nm/627 nm) were determined to
be 0.81 Æ 0.01. According to a standard curve which was
constructed by conventional methods (Fig. 4e) details are
described in the ESIw,12 an accurate pHi was determined as
7.1, which is consistent with the commonly known range of
pHi.13 After adding Nigericin, serving as an ionophore to
equilibrate pHi and extracellular pH, the value of R became
0.72 Æ 0.03 corresponding to a converted pH of 7.2 which is
the value of extracellular pH. These results establish the
superiority of UTX-40 as a pHi indicator that has notable cell
permeability and that is non-fluorescent before hydrolysis,
properties that will greatly facilitate intracellular applications.
In conclusion, we have developed UTX-40 as a new
SNARF-derived pHi indicator which is notable for its efficient
uptake and lack of fluorescence prior to cellular uptake,
because it formed non-fluorescent aggregates in aqueous
media. Efficient esterase hydrolysis leads to diffusion of the
aggregates and to intracellular-localized fluorescence with low
background fluorescence. The low background noise should
be particularly advantageous for in vivo usage, because for this
application there is difficulty in washing out the redundant
Dye
l
ABS/nm (ea/MÀ1cmÀ1
)
lemb/nm
F
Relc
SNARF(pH 5.0)d 515 (17 700)
544 (21 600)
583
0.030 100%
0.005 1.1%
0.0081 18%
0.019 46%
UTX-40
520 (1344.1)
559 (1383.3)
515 (18 370)
545 (17 388)
515 (15 092)
534 (15 854)
581
581
580
SNARF–OAc
SNARF–OAM
a
b
Measured in pH 5.0 10 mM acetate buffer. Excited at 534 nm.
c
Rel (relative brightness) was calculated using the following equation:
d
Relsample = e F/eSNARF  FSNARF
.
Ref. 6.
be hydrolyzed by esterase, the spectroscopic behavior during
ester hydrolysis was clearly different. When SNARF–OAM
was treated with esterase at pH 7.5, a seesaw type of absorption
spectral change with an isosbestic point (525 nm) was observed
(Fig. 3b). This indicated that the form of SNARF–OAM was
converted from SNARF(A) to SNARF(B) by ester hydrolysis.
A similar spectral change was observed during hydrolysis of
SNARF–OAc with an isosbestic point at 510 nm (Fig. S2 in
the ESIw). However with UTX-40, a simple incremental
absorption change occurred. This indicated that UTX-40
was being converted from SNARF(L) to the quinoid form
(SNARF(A) and SNARF(B)) during ester hydrolysis.
Furthermore, the fluorescent spectral changes during
hydrolysis agreed well with the results obtained from the
absorption measurements (inset of Fig. 3 and Fig. S2w).
Dynamic light scattering (DLS) measurements of UTX-40
and SNARF–OAM before and after esterase treatment
suggested the cause of these different spectroscopic properties
(Fig. S3w).9 DLS intensity showed that the buffer solution of
UTX-40 before esterase treatment consistently contained
aggregates having a diameter of mean size 75.7 Æ 7.7 nm,
whereas negligible DLS intensity was obtained after
treatment with esterase. On the other hand, in the case of
SNARF–OAM, we did not observe DLS intensity either
before or after treatment with esterase. This shows that
SNARF–OAM existed as a monomer throughout the course
of ester hydrolysis. These results suggest that the aggregates of
UTX-40 make a hydrophobic environment by themselves,
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(Tokushima university) for his help with the flow cytometry
measurements, and Ms Maki Nakamura, Ms Emiko Okayama
and the staff at our Faculty for measurement of NMR, and
elemental analysis. We also thank Dr Kenneth L. Kirk (NIH)
for his critical and valuable comments.
Notes and references
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to extracellular solution (EMEM). The scale bars (30 mm) are shown in
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cells analyzed by FACS (n = 4). (d) Intracellular fluorescence spectra
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The standard curve with pH values to determine the intracellular pH.
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This research was financially supported by a Grant-in-Aid
for Young Scientist (B) (No. 21710232) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan. The
authors thank Mr Koji Matsuda (KEYENCE) for his support
with the microscopic measurements, and Dr Atsushi Tabata
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3528 | Chem. Commun., 2010, 46, 3526–3528