A concise synthesis of the pennsylvania green fluorophore and labeling of intracellular targets with O6-benzylguanine derivatives

Article abstract of DOI:10.1021/ol7015093

We report improved syntheses of the Pennsylvania Green and 4-carboxy-Pennsylvania Green fluorophores; the latter compound was prepared from methyl 4-iodo-3-methylbenzoate in a three-pot process (32% overall yield). Chinese hamster ovary cells expressing O

Full text of DOI:10.1021/ol7015093

ORGANIC
LETTERS
2007
Vol. 9, No. 19
3741-3744
A Concise Synthesis of the
Pennsylvania Green Fluorophore and
Labeling of Intracellular Targets with
O6-Benzylguanine Derivatives
Laurie F. Mottram,† Ewa Maddox,† Markus Schwab,‡ Florent Beaufils,‡ and
Blake R. Peterson*,†
Department of Chemistry, The PennsylVania State UniVersity,
UniVersity Park, PennsylVania 16802, and CoValys Biosciences AG,
CH 4108 Witterswil, Switzerland
brpeters@chem.psu.edu
Received June 25, 2007
ABSTRACT
We report improved syntheses of the Pennsylvania Green and 4-carboxy-Pennsylvania Green fluorophores; the latter compound was prepared
from methyl 4-iodo-3-methylbenzoate in a three-pot process (32% overall yield). Chinese hamster ovary cells expressing O6-alkylguanine-DNA
alkyltransferase fusion proteins were treated with Pennsylvania Green and Oregon Green linked to O6-benzylguanine (SNAP-Tag substrates).
Analysis of living cells by confocal microscopy revealed that Pennsylvania Green derivatives exhibit substantially higher cell permeability
than analogous Oregon Green-derived molecular probes.
Fluorescent molecular probes are powerful tools for studies
of biological systems. Structurally diverse fluorophores,
including small molecules, intrinsically fluorescent proteins,
and semiconductor nanoparticles, have been particularly
useful for investigating the localization and function of
proteins in cells.1 Among the small molecules, derivatives
of fluorescein (1, Figure 1), a classic green fluorophore,2
have been especially important. Some fluorescein derivatives
such as 5- and 6-carboxyfluorescein can be readily synthe-
sized3 and exhibit high quantum yields in neutral and basic
aqueous solutions (quantum yield of fluorescein ) 0.925 in
0.1 N aqueous NaOH4). However, the high pKa (∼6.4)5 of
fluorescein and its 5- and 6-carboxy derivatives renders the
quantum yield of these dyes quite sensitive to pH fluctuations
observed under physiological conditions such as the environ-
ment in acidic endosomes of mammalian cells (pH ∼5-
6.5). Other limitations of many fluorescein derivatives
include a relatively high susceptibility to photobleaching6
and limited cell permeability7 due to the predominant
(3) Ueno, Y.; Jiao, G.-S.; Burgess, K. Synthesis 2004, 15, 2591-2593.
(4) Magde, D.; Wong, R.; Seybold, P. G. Photochem. Photobiol. 2002,
75, 327-334.
† The Pennsylvania State University.
‡ Covalys Biosciences AG.
(1) Giepmans, B. N.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science
2006, 312, 217-224.
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1280.
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10.1021/ol7015093 CCC: $37.00
© 2007 American Chemical Society
Published on Web 08/17/2007

strates) are useful probes of cellular biology because they
selectively alkylate the DNA repair protein O6-alkylguanine-
DNA alkyltransferase (AGT), enabling covalent labeling of
AGT fusion proteins (SNAP-Tag fusion proteins).11 The
native human AGT protein is a monomeric protein of 207
amino acids that reacts with aberrant O6-alkyl guanine
residues to repair this mutagenic lesion in DNA. As shown
in Figure 2 (panel A), X-ray crystallography has illuminated
Figure 1. Structures of fluorophores and molecular probes in
ionization states observed at physiological pH.
dianionic charge state of 1 at physiological pH (7.4).8 The
cell permeability of fluorescein can be improved by protec-
tion as a diester, but this modification adds synthetic
complexity, eliminates fluorescence, and requires that intra-
cellular esterases unmask the fluorophore.7
We recently reported the synthesis of a more hydrophobic,
less pH-sensitive, and more photostable analogue of fluo-
rescein termed Pennsylvania Green (2, Figure 1).9 This new
fluorophore and its 4-carboxy derivative (3) incorporate
structural elements from the previously reported fluorophores
Oregon Green5 and Tokyo Green.10 Oregon Green is a more
acidic and photostable 2′,7′- difluoro derivative of fluorescein
first described by Haugland and co-workers. By contrast,
Tokyo Green is a more hydrophobic analogue of fluorescein,
more recently reported by Urano, Nagano, and co-workers,
that replaces the carboxylate with a methyl group. By
combining these structural features, Pennsylvania Green and
derivatives represent potentially valuable tools for the
construction of molecular and cellular probes. However, our
previously reported 10-step synthesis of 4-carboxy-Pennsyl-
vania Green (3), from 1,2,4-trifluoro-5-nitrobenzene in 16%
overall yield, limited the availability of this material. To help
overcome this limitation, we report here a three-pot synthesis
of 4-carboxy-Pennsylvania Green (3) that affords a 32%
overall yield, employs less expensive methyl 4-iodo-3-
methylbenzoate as the starting material, and allows prepara-
tion of gram quantities of the fluorophore.
Figure 2. X-ray structure of human AGT bound to O6-methylgua-
nine on DNA (panel A, pdb code 1T38) and mechanism of covalent
labeling of AGT fusion proteins with O6-benzylguanine-fluoro-
phores (panel B).
the molecular details of interactions of AGT with modified
guanine residues on the natural DNA substrate.12 Nucleo-
philic attack of cysteine-145 of human AGT on alkylguanine
derivatives repairs these modified guanines by transferring
the alkyl group to cysteine and liberating free guanine (Figure
2, panel B).13 In the absence of AGT, O6-benzylguanine
derivatives are stable under physiological conditions, and this
approach can be used to selectively label AGT fusion proteins
with fluorophores and other probes linked to O6-benzylgua-
nine. This technology has been improved and generalized
by construction of mutants of AGT that increase the
specificity of AGT for O6-benzylguanines and that eliminate
To further characterize the biological properties of deriva-
tives of Pennsylvania Green, fluorescent molecular probes
4 and 5 derived from O6-benzylguanine (Figure 1) were
investigated. O6-Benzylguanine derivatives (SNAP-Tag sub-
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3742
Org. Lett., Vol. 9, No. 19, 2007

the DNA binding activity of this protein to facilitate studies
of AGT fusion proteins localized outside the nucleus.14 This
method has been used to fluorescently label subcellular
compartments in mammalian cells,15 detect AGT fusion
proteins in SDS-PAGE gels,16 and immobilize proteins on
solid support.17 Other methods for site-specific labeling of
proteins with fluorophores have also been reported.18-24
5- and 6-carboxyfluorescein and related compounds are
typically synthesized by condensation of 1,3-dihydroxyben-
zene (resorcinol) with 1,2,4-benzenetricarboxylic anhydride
(4-carboxyphthalic anhydride) under acidic conditions at
relatively high temperatures.25 To prepare pure isomers of
5- and 6-carboxyfluorescein on large scale, Burgess and co-
workers reported the synthesis of isomeric methanesulfonic
esters that can be readily separated by recrystallization.3
Alternatively, Van Vranken and co-workers demonstrated
that the fluorophore scaffold commonly used in metal-
sensitive fluorescein derivatives (fluo indicators) can be
constructed by condensation of resorcinol with an aldehyde
rather than an anhydride in the presence of dilute methane-
sulfonic acid, followed by oxidative cyclization with DDQ.26
These milder reaction conditions avoid the generation of
isomers and have the potential to allow access to a broader
range of fluorophores.
Scheme 1. Synthesis of Pennsylvania Green (2, Panel a),
4-Carboxy-Pennsylvania Green (3, Panel B), and Related
Molecular Probes Derived from O6-Benzylguanine (4, 5, Panel
B)
Based on these precedents, we investigated the condensa-
tion of o-methylbenzaldehyde (6) with 4-fluororesorcinol (7)
as a concise method to synthesize Pennsylvania Green (2).
4-Fluororesorcinol is commercially available, but we typi-
cally prepare it on a 5 gram scale using the two-step method
of Vij and Shreeve27 starting with 1,3-dimethoxybenzene and
Selectfluor. As shown in Scheme 1, by condensing 6 with 7
in the presence of 9% methanesulfonic acid, triarylmethane
8 could be isolated in 83% yield. Initial attempts to prepare
Pennsylvania Green (2) by oxidative cyclization with DDQ
in AcOH/benzene afforded 2 in 28% yield. Further optimiza-
tion revealed that treatment of 8 with excess p-TsOH in
refluxing toluene, a method previously reported by Thong-
panchang for synthesis of oxa-helicenes,28 was more effec-
tive, providing 2 in 37% yield. To prepare 4-carboxy-
Pennsylvania Green (3), commercially available methyl
4-iodo-3-methylbenzoate (9) was employed (Scheme 1, panel
B). This aryl iodide (9) was subjected to halogen-metal
exchange using i-PrMgCl and subsequently formylated with
DMF in 95% yield.29 The resulting aldehyde (10) was
condensed with 4-fluororesorcinol (7) in 9% methanesulfonic
acid to afford triarylmethane 11 in 88% yield. Treatment of
11 with DDQ or chloranil did not afford the cyclic product,
presumably due to the influence of the electron-withdrawing
ester substituent. However, heating to reflux in toluene with
excess p-TsOH sucessfully promoted cyclization; subsequent
base-promoted hydrolysis of the methyl ester in the same
pot yielded the desired product (3) in 38% yield. Attempts
to further improve the yield by addition of Lewis acids
analogous to the approach reported by Suzuki30 decreased
the yield or impeded purification of the final product.
(14) Keppler, A.; Pick, H.; Arrivoli, C.; Vogel, H.; Johnsson, K. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 9955-9959.
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2006, 41, 167-175.
(16) Sielaff, I.; Arnold, A.; Godin, G.; Tugulu, S.; Klok, H. A.; Johnsson,
K. ChemBioChem 2006, 7, 194-202.
Molecular probes 4 and 5 were prepared by acylation of
commercially available O6-[4-(aminomethyl)benzyl]guanine
(14)11 with succinimidyl ester derivatives of 4-carboxy-
Pennsylvania Green (12) and 5-carboxy-Oregon Green (13)
(Scheme 1). These structurally similar AGT substrates were
prepared to examine differences in cellular permeability
resulting from substitution of the carboxylate of 5 with the
methyl substituent of 4. Previous studies of AGT fusion
proteins expressed within mammalian cells have reported that
the efficiency of labeling is critically dependent on the cell
permeability of the substrate.15 To confirm that differences
in cellular labeling do not result from changes in physico-
(17) Kindermann, M.; George, N.; Johnsson, N.; Johnsson, K. J. Am.
Chem. Soc. 2003, 125, 7810-7811.
(18) Chen, I.; Ting, A. Y. Curr. Opin. Biotechnol. 2005, 16, 35-40.
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Angew. Chem., Int. Ed. 2004, 43, 1672-1675.
(21) George, N.; Pick, H.; Vogel, H.; Johnsson, N.; Johnsson, K. J. Am.
Chem. Soc. 2004, 126, 8896-8897.
(22) Los, G. V.; Wood, K. Methods Mol. Biol. 2007, 356, 195-208.
(23) Yin, J.; Liu, F.; Li, X.; Walsh, C. T. J. Am. Chem. Soc. 2004, 126,
7754-7755.
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99-104.
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2001, 5, 45-49.
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Shreeve, J. M. Heteroatom Chem. 1998, 9, 229-239.
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Org. Lett., Vol. 9, No. 19, 2007
3743

chemical properties, the maximal absorbance (abs) and
emission (em) wavelengths, molar extinction coefficients (ꢀ),
and quantum yields (Φ) of compounds 1-5 were deter-
mined31 (see the Supporting Information for details), and
these values are listed in Table 1. We next examined the
Table 1. Physicochemical Properties of Fluorophores and
Fluorescent Probesa
abs/em
(nm)
ꢀ
compound
fluorescein (1)
Pennsylvania Green (2)
4-carboxy
(M-1 cm-1
)
Φ (pH)
490/514
494/514
494/515
77 000
82 000
62 000
0.925 (9)
0.91 (7.4)
0.89 (7.4)
Pennsylvania Green (3)
O6-benzylguanine-
Pennsylvania Green (4)
O6-benzylguanine-
Oregon Green (5)
494/515
494/522
58 000
73 000
0.92 (7.4)
0.97 (7.4)
aValues for fluorescein (1) were previously reported.4,8 Values for
compounds 2-5 were determined in phosphate-buffered saline (PBS) at
pH 7.4.
ability of probes 4 and 5 to efficiently label intracellular
targets in Chinese hamster ovary-K1 (CHO) cells. These cells
were transiently transfected with commerically available
plasmid vectors (Covalys Biosciences) encoding AGT fusion
proteins that become targeted to distinct subcellular locations.
To image the plasma membrane, AGT was expressed fused
to a C-terminal CAAX peptide sequence from a human Ras
protein, which is associated with the inner leaflet of the
cellular plasma membrane.14 Conversely, AGT was expressed
fused to the C-terminus of the human histone 2B (H2B)
protein to promote localization in the cell nucleus.15
CHO cells transiently expressing AGT fusion proteins
were treated with molecular probes and analyzed by confocal
laser scanning microscopy. As shown in Figure 3, only the
Pennsylvania Green derivative 4 (5 µM, 1 h treatment) was
capable of clearly labeling the cellular plasma membrane
upon expression of the AGT-CAAX fusion protein. Under
identical conditions, the Oregon Green probe 5 showed only
punctate fluorescence throughout the cytoplasm of the subset
of transfected cells (compare panels A and C of Figure 3).
Transient transfection of CHO cells with the nuclear localized
AGT-H2B fusion protein revealed strong nuclear fluores-
cence upon treatment with 4 (5 µM, 1 h) but only weak
nuclear fluorescence upon treatment with 5 under these
conditions (compare panels B and D of Figure 3).
Figure 3. Confocal laser scanning and differential interference
contrast (DIC) micrographs of CHO cells expressing AGT fusion
proteins and treated with probes 4 and 5. CAAX: C-terminal
peptide derived from the Ras protein that associates with the inner
leaflet of the plasma membrane. H2B: nuclear-localized histone
2B protein. Molecular probes were added at 5 µM for 1 h at 37
°C. Scale bar ) 10 µM.
sylvania Green and derivatives reported here has the potential
to facilitate studies of cellular biology requiring cell perme-
able and pH-insensitive green fluorescent molecular probes.
Acknowledgment. We thank the National Institutes of
Health (R01-CA83831) for financial support. We thank Mr.
Kyle Schmid (Penn State University) for technical assistance.
We thank Prof. Mary Elizabeth Williams for the use of her
spectrometers.
Our results demonstrate that the subtle molecular substitu-
tion of the carboxylate of Oregon Green for the methyl group
of Pennsylvania Green can profoundly enhance the cellular
permeability and consequent biological activity of O6-
benzylguanine derivatives. The concise synthesis of Penn-
Supporting Information Available: Experimental pro-
cedures and characterization data for new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(31) Williams, A. T.; Winfield, S. A. Analyst 1983, 108, 1067-1071.
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