Synthesis and photophysical properties of new SNARF derivatives as dual emission pH sensors

Article abstract of DOI:10.1016/j.bmcl.2011.01.105

We report the synthesis and properties of two new seminaphthorhodafluor (SNARF) derivatives, SNARF-F and SNARF-Cl. Both these derivatives exhibit typical red shifts of absorption and fluorescence, and higher cell permeability as compared to traditional SN

Full text of DOI:10.1016/j.bmcl.2011.01.105

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1663–1666
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and photophysical properties of new SNARF derivatives as dual
emission pH sensors
Eiji Nakata a, , Yoshijiro Nazumi , Yoshihiro Yukimachi , Yoshihiro Uto , Hiroshi Maezawa ,
⇑
,
a
a
a
b
c
c
a,
⇑
Toshihiro Hashimoto , Yasuko Okamoto , Hitoshi Hori
Department of Life System, Institute of Technology and Science, Graduate School, The University of Tokushima, Minamijosanjimacho 2, Tokushima 770-8506, Japan
Institute of Health Biosciences, The University of Tokushima, Tokushima 770-8509, Japan
a
b
c
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and properties of two new seminaphthorhodafluor (SNARF) derivatives, SNARF-F
and SNARF-Cl. Both these derivatives exhibit typical red shifts of absorption and fluorescence, and higher
cell permeability as compared to traditional SNARF, while the pH-dependent dual-emission characteris-
Received 19 November 2010
Revised 18 January 2011
Accepted 22 January 2011
Available online 27 January 2011
tics are well retained. In particular, the lower pK
a
(7.38) of SNARF-F makes it more suitable than tradi-
tional SNARF derivatives for intracellular applications.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
SNARF
pH indicator
Ratiometry
Near infrared
Cell permeability
Spatial and temporal observation of intracellular pH changes is
crucial for understanding regulation mechanisms of various cellu-
lar physiologic functions. For such observations, fluorescent spec-
troscopy is particularly advantageous as compared to other
measurement methods, such as the use of microelectrodes, NMR,
and absorbance spectroscopy.1 Accordingly, various fluorescent
pH indicators have been developed and applied to monitor intra-
sized.4 In order to improve cell permeability of SNARF, cell-
permeable SNARF prodrug derivatives have also been synthesized,
wherein the phenolic group is protected by acetate or acetoxy-
methyl ester groups that function as esterase substrates. A compli-
cation with these prodrugs arises if the protected SNARF remains
inside the cell, because SNARF derivatives retain the fluorescence
of the acidic form and may result in incorrect pH measurements.5
As we described previously, one of the solutions to overcome this
drawback is to design a SNARF derivative, that is, nonfluorescent
2
cellular pH changes. Among such indicators, seminaphthorhoda-
fluor (SNARF) is especially useful as a pH indicator because of its
unique fluorescent characteristics.3 First, SNARF can be excited
by visible light, reducing cell damage due to irradiation and cir-
cumventing some disadvantageous effects of intracellular auto-
fluorescence. Second, SNARF has dual-emission properties for
ratiometric measurement, reducing irrelevant parameters such as
optical path length, local probe concentration, photobleaching,
and leakage from cells (Fig. 1). However, it has been pointed out
6
before hydrolysis. Another solution would be the development
that the relatively the high pK
a
of SNARF (ꢀ7.5) has limited its
intracellular applications.2 To overcome this disadvantage, SNARF
,4
derivatives possessing fluorinated xanthene rings and having low-
er pK values because of fluorine substitution have been synthe-
a
⇑
Corresponding authors. Tel.: +81 774 38 3515; fax: +81 774 38 3516 (E.N.) Tel.:
81 88 656 7514; fax: +81 88 656 9164 (H.H.).
+
E-mail addresses: nakata@iae.kyoto-u.ac.jp (E. Nakata), hori@bio.tokushima-
u.ac.jp (H. Hori).
Present address: Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-
0011, Japan.
Figure 1. The equilibria and the structures of SNARF, SNARF-F and SNARF-Cl.
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.105

1
664
E. Nakata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1663–1666
a
OH
N
OH
N
O
F
F
O
F
i
ii
O
O
O
F
F
F
COOH
F
F
F
O
O
F
F
F
b
OH
N
OH
N
O
Cl
Cl
O
Cl
Cl
iii
iv
O
O
O
Cl
Cl
COOH
Cl
Cl
Cl
O
O
Cl
Cl
Cl
3
Scheme 1. Synthesis of (a) SNARF-F and (b) SNARF-Cl. Reaction conditions: (i) 3-dimethylaminophenol, toluene, reflux, yield: 55%; (ii) 1,6-dihydroxynaphtalene, MeSO H,
reflux, yield: 33%; (iii) 3-dimethylaminophenol, toluene, reflux, yield: 36%; (iv) 1,6-dihydroxynaphtalene, MeSO H, reflux, yield: 48%.
3
Table 1
Photophysical properties of SNARF derivatives described in this study
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
(⁄10ꢁ2
c log P9
pK a
a
SNARF derivatives
SNARF
pH
5.0
k
Abs, max
(
e
M
cm
515 nm (17,700)
44 nm (21,600)
)
k
Abs, iso
(e
M
cm
)
k
em, max (nm)
U
)
534 nm (25,750)
546 nm (23,100)
532 nm (26,700)
583
3.0
4.99
7.62
7.38
7.90
5
1
1
1
0.0
5.0
573 nm (44,100)
533 nm (25,600)
71 nm (28,900)
603 nm (54,400)
539 nm (27,200)
74 nm (31,000)
627
603
9.0
1.0
SNARF-F
5.69
7.29
5
0.0
5.0
650
603
2.0
1.0
SNARF-Cl
5
0.0
605 nm (47,500)
651
3.0
a
a
pK value were determined by UV spectrum change (see Supplementary data).
Figure 2. (a–c) Absorbance (plane line) and fluorescence (dash line) spectra of 10
were excited at the isosbestic point, respectively.) (d–e) Photos of (d) color and (e) fluorescence of SNARF (10
0.0.
l
M of (a) SNARF, (b) SNARF-F and (c) SNARF-Cl at pH 5.0 (red) and pH10.0 (blue). (They
l
M), SNARF-F (10 M) and SNARF-Cl (10 M) at pH 5.0 and pH
l
l
1

E. Nakata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1663–1666
1665
of unprotected SNARF derivatives with higher inherent cell
permeability.
These considerations motivated us to derivatize existing SNARF
reagents by another strategy, that is, substitution of a hydrogen
atom of the benzene moiety with a halogen atom, such as fluorine
or chlorine, in order to increase hydrophobicity as well as to mod-
ulate photophysical properties. So far, there are only limited re-
2
,7
ports on the derivatization of benzo[c]xanthene fluorophores.
Herein, we report the synthesis and photophysical properties of
the newly synthesized SNARF-F and SNARF-Cl indicators.
SNARF-F was synthesized from commercially available tetraflu-
orophthalic anhydride as shown in Scheme 1a. Reaction of
3
-dimethylaminophenol with tetrafluorophthalic anhydride in
0
0
toluene produced 2-carboxy-3,4,5,6-fluoro-3 -dimethylamino-2 -
hydroxybenzophenone. Condensation of this product with
1
,6-dihydroxynaphthalene afforded the desired SNARF-F dye.
SNARF-Cl was synthesized from tetrachlorophthalic anhydride in
a manner similar to the preparation of SNARF-F (Scheme 1b).
1
Structures of all compounds were confirmed with H NMR, high-
MS, and elemental analysis (see Supplementary data).
a
The absorbance and fluorescence properties, pK , and calculated
log P (c log P) of the SNARF derivatives are summarized in Table 1.
In aqueous solution, SNARF produced maximal absorption and
strong fluorescence at 544 and 583 nm, respectively, at pH 5.0,
and 573 and 627 nm, respectively, at pH 10.0 (Fig. 2a). Notably,
as compared to SNARF, the absorption and fluorescence of
SNARF-F exhibited a typical red shift, that is, SNARF-F produced
maximal absorption and strong fluorescence at 571 and 603 nm
at pH 5.0, respectively, and maximal absorption strong fluores-
cence at 603 and 650 nm, respectively, at pH 10.0 (Fig. 2b). In
the case of SNARF-Cl, a similar red shift was observed (Fig. 2c).
The altered properties of absorption and fluorescence were clearly
confirmed under bright-field and transilluminator observations
Figure 4. Microscopic images of V79 cells fluorescently labeled with SNARF-F, Mito
Tracker Green FM and Hoechst 33258. Cells were imaged simultaneously for (a)
SNARF-F, (b) Mito Tracker Green FM and (c) Hoechst 33258. The overlay image of
(
a), (b) and (c) is shown in (d). (e) is the transmission image. The scale bars (20
lm)
are shown in the photograph.
(
Fig. 2d and e). As described above, it should be noted that a pH
indicator with longer wavelength emission is beneficial for intra-
cellular applications. The pK values of SNARF-F and SNARF-Cl
were determined to be 7.38 and 7.90, respectively (Figs. S1 and
S2). It has been pointed out that the relatively high pK value of
SNARF (pK = 7.62) is problematic for measuring the cytosolic pH
(Fig. 4 and S3). Along with SNARF, SNARF-F and SNARF-Cl were
localized mainly in the mitochondria and cytosol.
2
,6a
a
In conclusion, we designed and synthesized two new SNARF
derivatives, SNARF-F and SNARF-Cl. Their photophysical and char-
acteristic properties indicated both to be useful as dual-emission
pH indicators. SNARF-F, in particular, appears to be more promis-
ing for intracellular application as compared to traditional SNARF,
because SNARF-F combines the typical red shift of absorption and
a
a
2,4,7,8
of most cell lines (pH ꢀ 6.8–7.4).
In this regard, SNARF-F is
more suitable as a pH indicator for intracellular pH measurement.
The c log P values of SNARF, SNARF-F, and SNARF-Cl were calcu-
lated to be 4.99, 5.69, and 7.29, respectively. These findings indi-
fluorescence with
permeability.
a
lower pK
a
together with higher cell
9
cate that SNARF-F and SNARF-Cl are more hydrophobic than
SNARF, and thus, should be more cell permeable than SNARF. In or-
der to verify this, the cellular uptake of the two SNARF derivatives
was quantitatively evaluated by flow cytometry. As shown in Fig-
ure 3, we confirmed more effective cellular uptake of SNARF-F
and SNARF-Cl than of SNARF. Further, the localization of SNARF-F
and SNARF-Cl were confirmed by a multistaining procedure
Acknowledgments
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
Dr. Atsushi Tabata (Tokushima University) for his help with the
flow cytometry measurements, and Ms. Maki Nakamura, Ms. Emi-
ko 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.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.105.
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Figure 3. Flow cytometry analysis of the cellular uptake of the fluorescence of
SNARF into V79 cells. The cells were incubated with SNARF-F (red), SNARF-Cl (blue)
or SNARF (green) or without SNARF derivatives (gray).
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