Synthesis and photophysical properties of new fluorinated benzo[c]xanthene dyes as intracellular pH indicators

Article abstract of DOI:10.1016/S0960-894X(01)00595-9

Two new fluorinated benzo[c]xanthene dyes were synthesized by reaction of fluorinated 1,6-dihydroxynaphthalenes with 2,4- (and 2,5)-dicarboxy-3′-dimethylamino-2′-hydroxybenzophenone. The two critical fluorinated 1,6-dihydroxynaphthalene intermediates were prepared via a regioselective route. The fluorinated benzo[c]xanthene dyes exhibit desired lower pK_a values (6.4 and 7.2, respectively) than their parent compound (pK_a = 7.5) while the pH-dependent dual-emission characteristics are well retained. Their cell-permeable esters have been prepared for intracellular applications.

Full text of DOI:10.1016/S0960-894X(01)00595-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2903–2905
Synthesis and Photophysical Properties of New Fluorinated
Benzo[c]xanthene Dyes as Intracellular pH Indicators
Jixiang Liu,a,* Zhenjun Diwub and Wai-Yee Leunga
aMolecular Probes, Inc., 4849 Pitchford Avenue, Eugene, OR 97402, USA
bMolecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089, USA
Received 12 July 2001; accepted 13 August 2001
Abstract—Two new fluorinated benzo[c]xanthene dyes were synthesized by reaction of fluorinated 1,6-dihydroxynaphthalenes with
2,4- (and 2,5)-dicarboxy-30-dimethylamino-20-hydroxybenzophenone. The two critical fluorinated 1,6-dihydroxynaphthalene inter-
mediates were prepared via a regioselective route. The fluorinated benzo[c]xanthene dyes exhibit desired lower pKa values (6.4 and
7.2, respectively) than their parent compound (pKa=7.5) while the pH-dependent dual-emission characteristics are well retained.
Their cell-permeable esters have been prepared for intracellular applications. # 2001 Elsevier Science Ltd. All rights reserved.
Intracellular pH plays an important modulating role in
many cellular events, including cell growth, calcium
regulation, enzymatic activity, receptor-mediated signal
transduction, ion transport, endocytosis, chemotaxis,
cell adhesion and other cellular processes.1ꢀ8 pH-Sensi-
tive fluorescent dyes have been widely applied to moni-
tor changes in intracellular pH in recent years. Imaging
techniques that use fluorescent pH indicators also allow
researchers to investigate these processes with much
greater spatial resolution and sampling density that can
be achieved using other technologies such as microelec-
trode.9 The mostpopular pH-sensiitve probes include
1,4-dihydroxyphthalonitrile (1,4-DHPN),10 8-hydroxy-
pyrene-1,3,6-trisulfonic acid (HPTS),11 5- (and 6)-car-
boxyfluorescein,12 5- (and 6)-carboxy-40,50-dimethyl
fluorescein,13 20,70-bis-(2-carboxyethyl)-5- (and 6)-car-
indicator by fluorinating its benzo[c]xanthene ring sys-
tem. Herein, we report the synthesis and photophysical
properties of the newly synthesized carboxy SNARF-4F
and carboxy SNARF-5F indicators (Fig. 1).
We have tried two approaches to synthesize fluorinated
carboxy SNARF dyes. One is the direct fluorination of
carboxy SNARF-1. The other approach is the con-
densation of 2,4- (and 2,5)-dicarboxy-30-dimethylamino-
20-hydroxybenzophenone with fluorinated 1,6-dihy-
droxynaphthalenes that were prepared by the regiospe-
cific routes, as described below. There are several active
positions on carboxy SNARF-1 aromatic moiety that
are readily available for directelectrophilic fluorination;
however, these routes gave a mixture of fluorinated
products that were difficult to separate. Thus, we deci-
ded to develop the regiospecific synthesis of 5-fluoro-
boxyfluorescein
(BCECF),14
20,70-bis-(2-carboxy-
propyl)-5- (and 6)-carboxyfluorescein (BCPCF),15
carboxyseminaphthofluorescein (carboxy SNAFL) and
carboxyseminaphthorhodafluor-1 (carboxy SNARF-1)
dyes.16 Among these pH sensitive dyes, carboxy
SNARF-1 indicator has both dual emission and dual
excitation with excitability by 488 or 514 nm spectral
lines of the argon-ion laser. These advantages have
made carboxy SNARF-1 exceptionally suitable for both
confocal laser scanning microscopy and flow cytometry.
However, the relatively high pKa (ꢁ7.5) of carboxy
SNARF-1 has limited its cellular applications. This has
motivated us to modify the existing carboxy SNARF-1
*Corresponding author. Fax: +1-541-344-6504; e-mail: jixiangliu@
probes.com
Figure 1. The structures of carboxy SNARF-1, carboxy SNARF-4F,
and carboxy SNARF-5F indicators.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00595-9

2904
J. Liu et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2903–2905
and 4-fluoro-1,6-dihydroxynaphthalenes as key inter-
mediates to achieve an effective synthesis of carboxy
SNARF-4F and carboxy SNARF-5F.
toluene, afforded the desired carboxy SNARF-4F dye.
The carboxy SNARF-4F was purified via the conver-
sion to the corresponding acetate 9 on a silica gel col-
umn, which was then converted back into carboxy
SNARF-4F nearly quantitatively by basic hydrolysis.
Carboxy SNARF-4F was synthesized from commer-
cially available 6-hydroxy-1-naphthoic acid as shown in
Scheme 1. Reaction of 6-hydroxy-1-naphthoic acid with
MeI/K2CO3 in THF followed by fluorination with
SelectFluor [1-chloromethyl-4-fluoro-1,4-diazabicyclo
[2.2.2]-octane bis(tetrafluoroborate)]17 gave a mixture of
compounds 2 and 3 with some unreacted starting mate-
rial. Interestingly, SelectFluor can demethylate com-
pound 2 to give compound 3 under the reported
reaction conditions. Reduction of compound 2 with
Carboxy SNARF-5F was synthesized from commer-
cially available 1,6-dihydroxynaphthalene rather than
1,6-dimethoxynaphthalene. As discussed above, the
demethylation (by SelectFluor) gave a very messy mix-
ture. Fluorination of 1,6-dihydroxynaphthalene with
SelectFluor gave a mixture of the desired product and
other fluorinated by-products, along with some unreac-
ted starting material. The reaction mixture was difficult
to be purified. The mixture was methylated with MeI/
K2CO3, and the resulting ether mixture was purified on
a silica gel column to give pure 4-fluoro-1,6-dimethoxy-
naphthalene 10. Demethylation of compound 10 with
HBr in H2O/AcOH yielded 4-fluoro-1,6-dihydroxy-
naphthalene, which was used to make carboxy SNARF-
5F, utilizing the method analogous to prepare carboxy
SNARF-4F. Finally, both carboxy SNARF-4F and
carboxy SNARF-5F were converted to their corre-
.
BH3 THF gave alcohol 4 in high yield. Oxidation of
alcohol 4 with PCC gave aldehyde 5. Baeyer–Villiger
oxidation of aldehyde 5 with m-CPBA followed by basic
hydrolysis yielded compound 6. Treatment of com-
pound 6 with BBr3 gave the desired intermediate 5-
fluoro-1,6-dihydroxynaphthalene 7. Condensation of 7
with 2,4- (and 2,5)-dicarboxy-30-dimethylamino-20-
hydroxybenzophenone 8, which was prepared by reac-
tion of 3-dimethylaminophenol with trimellitic acid in
Scheme 1. Synthesis of carboxy SNARF-4F dye. Reaction conditions: (a) MeI, K2CO3, THF, reflux, yield: 96%; (b) SelectFluor, CH3CN, 0 ꢂC,
.
yield: for 2: 60%, for 3: 15%; (c) BH3 THF, reflux, yield: 96%; (d) PCC, CH2Cl2, rꢂt, yield: 81%; (e) m-CPBA, CH2Cl2, reflux, yield: 39%; (f) LiOH,
60%; (i) (1) Ac2O, NEt3, THF, reflux; (2) CH3CO2CH2Br, NEt(Pr-i)2, THF, rt; yield 31%.
.
MeOH/H2O, rt, yield: 95%; (g) BBr3, CH2Cl2, rt, yield: 93%; (h) (1) 8, ZnCl2, 160 C; (2) Ac2O, NEt3, THF, reflux; (3) NH3 H2O, MeOH, rt; yield:
Figure 2. Fluorescence emission spectra of (a) carboxy SNARF-4F and (b) carboxy SNARF-5F in buffer solution at various pH values.

J. Liu et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2903–2905
2905
sponding cell-permeable esters. All compounds were
1
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As shown in Figure 2, carboxy SNARF-4F exhibits a
pH-dependent wavelength shift with the emission max-
ima at 587 and 650 nm, and with an isosbestic point at
618 nm. Its pKa was determined to be 6.4 by the fluo-
rescence emission spectra. Similarly, carboxy SNARF-
5F also retains the pH-dependent dual-emssion char-
acteristics, with maxima of 575 and 628 nm. Its pKa was
determined to be 7.2 by the fluorescence emission spec-
tra. As discussed above, the high pKa value of 7.5 of
carboxy SNARF-1 makes itless suitable for measuring
the cytosolic pH of most cell lines (pH ꢁ6.8–7.4). The
new fluorinated carboxy SNARF-4F and carboxy
SNARF-5F apparently have an improved pKa. The low
pKa values of 6.4 for carboxy SNARF-4F and 7.2 for
carboxy SNARF-5F will make these indicators excep-
tionally suitable for pH measurement in the range from
about 6.0 to 7.5 with confocal laser scanning micro-
scopy and flow cytometry.
In summary, carboxy SNARF-4F and carboxy
SNARF-5F were synthesized and characterized as new
dual-emission pH indicators with pKa values of 6.4 and
7.2. The two key intermediates, 5-fluoro-1,6-dihydroxy-
naphthalene and 4-fluoro-1,6-dihydroxynaphthalene
can also be used to prepare other fluorescent probes.
15. Liu, J.; Diwu, Z.; Klaubert, D. H. Bioorg. Med. Chem.
Lett. 1997, 7, 3069.
Acknowledgements
16. (a) Whitaker, J. E.; Haugland, R. P.; Prendergast, F. G.
Anal. Biochem. 1991, 194, 330. (b) Haugland, R. P. Handbook
of Fluorescent Probes and Research Chemicals, 6th ed.; Mole-
cular Probes: Eugene, 1996; p 552.
We thank Ms. Diane Ryan for spectral measurements.
We also thank Dr. Richard P. Haugland for his critical
reading of the manuscript.
17. (a) Banks, R. E.; Mohialdin-Khaffaf, S. N.; Lal, G. S.;
Sharif, I.; Syvret, G. J. Chem. Soc., Chem. Commun. 1992,
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1
18. Carboxy SNARF-4F: H NMR (CD3OD) d 8.82 (s), 8.54
References and Notes
(m), 8.39 (m), 8.00 (s), 7.45 (d), 7.26 (m), 7.00 (m), 6.57 (d),
3.15 (s); 19F NMR f 126.7, 127.1. Carboxy SNARF-5F: 1H
NMR (CD3OD) d 8.90 (s), 8.49 (m), 8.40 (s), 8.02 (s), 7.82 (d),
7.55 (d), 7.44 (m), 7.22 (m), 7.11 (m), 3.40 (s); 19F NMR f
150.4.
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