The Pennsylvania Green fluorophore: A hybrid of Oregon Green and Tokyo Green for the construction of hydrophobic and pH-insensitive molecular probes

Article abstract of DOI:10.1021/ol052655g

Fluorescent small molecules are powerful tools for exploring cellular biology. As a more hydrophobic, photostable, and less pH-sensitive alternative to fluorescein, we synthesized Pennsylvania Green, a bright, monoanionic fluorophore related to Oregon Gre

Full text of DOI:10.1021/ol052655g

ORGANIC
LETTERS
2006
Vol. 8, No. 4
581-584
The Pennsylvania Green Fluorophore:
A Hybrid of Oregon Green and Tokyo
Green for the Construction of
Hydrophobic and pH-Insensitive
Molecular Probes
Laurie F. Mottram, Siwarutt Boonyarattanakalin, Rebecca E. Kovel, and
Blake R. Peterson*
Department of Chemistry, The PennsylVania State UniVersity,
UniVersity Park, PennsylVania 16802
brpeters@chem.psu.edu
Received November 1, 2005
ABSTRACT
Fluorescent small molecules are powerful tools for exploring cellular biology. As a more hydrophobic, photostable, and less pH-sensitive
alternative to fluorescein, we synthesized Pennsylvania Green, a bright, monoanionic fluorophore related to Oregon Green and Tokyo Green.
Comparison of membrane probes comprising N-alkyl-3
â
-cholesterylamine linked to 4-carboxy-Tokyo Green (pKa
∼
6.2) and 4-carboxy-
Pennsylvania Green (pKa
living mammalian cells.
∼
4.8) revealed that only Pennsylvania Green was highly fluorescent in acidic early and recycling endosomes within
Molecular probes derived from fluorescein (1, Figure 1) are
widely used as tools for studies of cellular biology. This
green fluorophore is particularly suited for cellular analysis
by confocal laser scanning microscopy and flow cytometry
due to its excitation maximum at 490 nm, closely matching
the 488 nm spectral line of the argon ion laser. In addition,
fluorescein has a high molar absorptivity and excellent
quantum yield (0.92 at pH 9). Under physiological conditions
(pH 7.4), fluorescein is predominantly a highly hydrophilic
dianion. However, the monoanionic form of fluorescein
exhibits the relatively high pKa of 6.5, rendering this dye
much less fluorescent in acidic solutions.1 Fluorescein is also
relatively susceptible to photobleaching, which is thought
to involve reactions with molecular oxygen and proximity-
induced reactions of the dye.2
Figure 1. Structures of known (1-3) and novel (4-6) fluorescent
dyes in their anionic forms.
Oregon Green, a more acidic 2′,7′-difluoro derivative of
fluorescein (2, Figure 1), was developed as a less pH-
sensitive fluorophore.3 The appended fluorine atoms reduce
the pKa of this dye to 4.8, substantially improving fluores-
(1) Sjoback, R.; Nygren, J.; Kubista, M. Spectrochim. Acta A 1995, 51,
L7-L21.
10.1021/ol052655g CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/24/2006

cence at low pH. This compound is also significantly more
photostable than fluorescein. However, the high cost and high
polarity of Oregon Green limits its utility as a building block
for hydrophobic molecular probes.
reported5 convergent eight-step synthesis of this compound.
Cyclization at elevated temperature under precedented condi-
tions afforded the known xanthone 13.5 As shown in Scheme
2, this xanthone (13) and commercially available xanthone
As a more hydrophobic alternative to fluorescein, recent
pioneering work by Urano, Nagano, and co-workers replaced
the carboxylate of fluorescein with a methyl group.4 This
structural modification yielded a highly fluorescent monoan-
ionic fluorophore termed Tokyo Green (3, Figure 1). This
analogue of fluorescein provides a new platform for the
design of fluorescent probes.
Scheme 2. Synthesis of Fluorophores
We report here the synthesis of a novel fluorophore termed
Pennsylvania Green (4). This fluorophore melds the pH-
insensitivity and photostability of Oregon Green with the
hydrophobicity of Tokyo Green. To demonstrate the utility
of the Pennsylvania Green fluorophore, we compared cellular
membrane probes derived from 4-carboxy-Tokyo Green (5)
and 4-carboxy-Pennsylvania Green (6). The lower pKa of
the Pennsylvania Green-derived probe enables visualization
of early/recycling endosomes within living mammalian cells,
and this fluorophore provides a useful tool for analysis of
these and related acidic intracellular compartments.
14 were converted into the novel fluorophores 4-carboxy-
Pennsylvania Green (6) and 4-carboxy-Tokyo Green (5).
Halogen metal exchange with i-PrMgCl7 proved to be an
efficient method for installation of the methyl benzoate
moiety of 17 and 18.
The synthesis of 4-carboxy-Pennsylvania Green (6) was
accomplished in 10 steps from commercially available 1,2,4-
trifluoro-5-nitrobenzene (7). As shown in Scheme 1, 1-amino-
To examine the effects of substitution with fluorine, pKa
values of 4-carboxy-Pennsylvania Green methyl ester (17)
and 4-carboxy-Tokyo Green methyl ester (18) were deter-
mined by absorbance versus pH titrations. As shown in
Figure 2, these experiments revealed that the Pennsylvania
Green derivative is identical in pKa to Oregon Green (pKa
) 4.8) and is 1.4 pKa units more acidic than the correspond-
ing Tokyo Green derivative (pKa ) 6.2).
Scheme 1. An Improved Synthesis of Xanthone 13
Table 1. Comparison of Physicochemical Properties of Known
(1-3)3,4 and Novel (17 and 18) Fluorophores (Q.Y. is Quantum
Yield)
abs/em
(nm)
Q.Y.
(pH)
compound
fluorescein (1)
pKa
2,4-dimethoxy-5-fluorobenzene (9) was prepared via 8 by a
previously reported route.3 Subjection of 9 to Sandmeyer
conditions afforded the novel iodoarene 10. This compound
(10) was converted to the known5 benzophenone 12 in
excellent yield using Larhed’s recently reported6 microwave
and cobalt octacarbonyl-mediated synthesis of diaryl ketones
followed by demethylation with boron tribromide. The five-
step route to 12 shown in Scheme 1 is significantly more
rapid and efficient (40% overall yield) than the previously
490/514
6.5
0.92 (9)
0.37 (5.4)
0.97 (9)
0.85 (13)
0.32 (3.4)
0.91 (9)
Oregon Green (2)
Tokyo Green (3)
490/514
491/510
4.8
6.2
4-carboxy-Pennsylvania Green
methyl ester (17)
496/517
496/517
4.8
6.2
0.68 (5)
0.93 (9)
4-carboxy-Tokyo Green
methyl ester (18)
0.39 (5)
(2) Song, L. L.; Hennink, E. J.; Young, I. T.; Tanke, H. J. Biophys. J.
1995, 68, 2588-2600.
(3) Sun, W. C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J. Org.
Chem. 1997, 62, 6469-6475.
As listed in Table 1, relative fluorescence quantum yields
of fluorophores 17 and 18 were determined at pH 9.0 and
(4) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano,
T. J. Am. Chem. Soc. 2005, 127, 4888-4894.
(5) Chen, C. A.; Yeh, R. H.; Lawrence, D. S. J. Am. Chem. Soc. 2002,
124, 3840-3841.
(6) Enquist, P.-A.; Nilsson, P.; Larhed, M. Org. Lett. 2003, 5, 4875-
4878.
(7) Knochel, P.; Dohle, W.; Gommermann, N.; Kneisel, F. F.; Kopp,
F.; Korn, T.; Sapountzis, I.; Vu, V. A. Angew. Chem., Int. Ed. 2003, 42,
4302-4320.
582
Org. Lett., Vol. 8, No. 4, 2006

amine derivative 19 to afford 20 and 21, followed by removal
of 3â-nosyl protecting groups (Scheme 3). Related com-
Scheme 3. Synthesis of Molecular Probes of Mammalian
Plasma Membranes and Intracellular Endosomes
pounds comprising fluorophores linked to N-alkyl-3â-cho-
lesterylamines have previously been shown to localize on
the exofacial leaflet of plasma membranes and within acidic
intracellular endosomes of living mammalian cells.9 N-Alkyl-
3â-cholesterylamines can rapidly cycle between these two
cellular destinations, similar to many natural cell surface
receptors.10
Living Jurkat lymphocytes treated with molecular probes
22 and 23 were examined by confocal laser scanning
microscopy. This human T-cell line was treated with the
compounds for 1 h, cells were gently centrifuged and washed
to remove unincorporated probes, and cellular fluorescence
was imaged as shown in Figure 3. These experiments
revealed that both compounds could be observed on the
cellular plasma membrane and provide novel markers that
define the cell surface. However, only the Pennsylvania
Green probe (22) exhibited bright fluorescence in intracellular
compartments (compare panels A and B in Figure 3). These
compartments were identified as early and recycling endo-
somes by colocalization with internalized red fluorescent
transferrin protein (data provided in the Supporting Informa-
tion).11 The higher pKa of Tokyo Green results in substantial
fluorescence quenching of 23 in the acidic environment of
endosomes (pH e 6.5).12 This was confirmed by treatment
with 23 and the specific vacuolar H+ ATPase inhibitor
Figure 2. Absorbance versus pH titrations of 17 (panel A) and 18
(panel B) used to determine pKa values (panel C). Overlaid emission
spectra (panels A and B) are in arbitrary intensity units.
5.0 by the method of Williams et al. (data provided in Figure
S1 of the Supporting Information).8 Fluorescein (1) and
5-carboxyfluorescein were used as standards in these experi-
ments. At pH 9.0, both the Pennsylvania Green (17) and
Tokyo Green (18) fluorophores were very similar with
quantum yields of 0.91 and 0.93, respectively. However, as
predicted by the differences in pKa, at pH 5.0, the more acidic
Pennsylvania Green derivative (17) was substantially brighter
with a quantum yield of 0.68 compared with 0.39 for the
Tokyo Green (18) derivative.
Two novel membrane probes (22 and 23) were synthesized
to compare Pennsylvania Green and Tokyo Green in a
cellular environment. These probes were prepared by coup-
ling fluorophores 5 and 6 to the known9 3â-cholesteryl-
(9) Boonyarattanakalin, S.; Martin, S. E.; Dykstra, S. A.; Peterson, B.
R. J. Am. Chem. Soc. 2004, 126, 16379-16386.
(10) Peterson, B. R. Org. Biomol. Chem. 2005, 3, 3607-3612.
(11) Sheff, D.; Pelletier, L.; O’Connell, C. B.; Warren, G.; Mellman, I.
J. Cell. Biol. 2002, 156, 797-804.
(12) Maxfield, F. R.; McGraw, T. E. Nat. ReV. Mol. Cell. Biol. 2004, 5,
121-132.
(8) Williams, A. T.; Winfield, S. A. Analyst 1983, 108, 1067-1071.
Org. Lett., Vol. 8, No. 4, 2006
583

tostability conferred by fluorination.3 To examine whether
Pennsylvania Green is more photostable than Tokyo Green,
Jurkat lymphocytes treated with probes 22 and 23 were
subjected to continuous irradiation with the Ar ion laser (488
nm) of a confocal microscope. As shown in Figure 4, the
Figure 4. Analysis of photobleaching rates of molecular probes
added to living Jurkat lymphocytes. Cells were treated with 22 or
23 (10 µM, 1 h, 37 °C), washed with fresh media, and imaged by
confocal microscopy while subjected to continuous irradiation at
488 nm with a 25 mW argon ion laser at 1% laser power. At specific
time points, the fluorescence intensity of five individual cells was
quantified and averaged. Fluorescence half-lives were calculated
with a one-site exponential decay model.
fluorescence decay of individual cells due to photobleaching
was quantified (examples are provided in the Supporting
Information), and application of a single exponential function
allowed calculation of the half-lives of these fluorescent
probes. This analysis revealed that the Pennsylvania Green
probe 22 is substantially more photostable (t1/2 ) 49 min)
compared to the Tokyo Green probe 23 (t1/2 ) 29 min). The
Pennsylvania Green fluorophore thus has the potential to
provide a valuable new tool for the construction of molecular
and cellular probes.
Figure 3. Confocal laser scanning (left) and differential interference
contrast (DIC, right) micrographs of living Jurkat lymphocytes.
Cells were treated with probes 22 and 23 (10 µM) in RPMI media
for 1 h at 37 °C and washed with fresh media prior to analysis by
microscopy. In panel C, cells were treated with 23 (10 µM) and
the vacuolar H+ ATPase inhibitor Bafilomycin A1 (1 µM) to
prevent acidification of endosomes. White arrows illustrate intra-
cellular endosomal fluorescence. Scale bar ) 10 µm.
Acknowledgment. We thank the National Institutes of
Health (R01-CA83831) for financial support. We thank Prof.
Mary Elizabeth Williams for the use of her spectrometers.
Bafilomycin A1,13 which by blocking acidification of en-
dosomes, increased the intracellular fluorescence of 23
(Figure 3, panel C).
In addition to its lower pKa, a second advantage of Oregon
Green compared with fluorescein relates to enhanced pho-
Supporting Information Available: Supporting figures,
experimental procedures, and characterization data for new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(13) Yoshimori, T.; Yamamoto, A.; Moriyama, Y.; Futai, M.; Tashiro,
Y. J. Biol. Chem. 1991, 266, 17707-17712.
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