Efficient and scalable synthesis of 4-carboxy-pennsylvania green methyl ester: A hydrophobic building block for fluorescent molecular probes

Article abstract of DOI:10.1055/s-0033-1338535

Fluorinated fluorophores are valuable tools for studies of biological systems. However, amine-reactive single-isomer derivatives of these compounds are often very expensive. To provide an inexpensive alternative, we developed a practical synthesis of 4-ca

Full text of DOI:10.1055/s-0033-1338535

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Author Manuscript
Synthesis (Stuttg). Author manuscript; available in PMC 2015 January 01.
Published in final edited form as:
Synthesis (Stuttg). 2014 January 1; 46(2): 158–164. doi:10.1055/s-0033-1338535.
Efficient and Scalable Synthesis of 4-Carboxy-Pennsylvania
Green Methyl Ester: A Hydrophobic Building Block for
Fluorescent Molecular Probes
Zachary R. Woydziak, Liqiang Fu, and Blake R. Peterson
Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045, United
States, Fax: (785) 864-8141
Blake R. Peterson: brpeters@ku.edu
Abstract
Fluorinated fluorophores are valuable tools for studies of biological systems. However, amine-
reactive single-isomer derivatives of these compounds are often very expensive. To provide an
inexpensive alternative, we report a practical synthesis of 4-carboxy-Pennsylvania Green methyl
ester. Derivatives of this hydrophobic fluorinated fluorophore, a hybrid of the dyes Oregon Green
and Tokyo Green, are often cell permeable, enabling labeling of intracellular targets and
components. Moreover, the low pKa of Pennsylvania Green (4.8) confers bright fluorescence in
acidic cellular compartments such as endosomes, enhancing its utility for chemical biology
investigations. To improve access to the key intermediate 2,7-difluoro-3,6-dihydroxyxanthen-9-
one, we subjected bis-(2,4,5-trifluorophenyl)methanone to iterative nucleophilic aromatic
substitution by hydroxide on scales of > 40 g. This intermediate was used to prepare over 15
grams of pure 4-carboxy-Pennsylvania Green methyl ester in 28% overall yield without requiring
chromatography. This compound can be converted into the amine reactive N-hydroxysuccinimidyl
ester in essentially quantitative yield for the synthesis of a wide variety of fluorescent molecular
probes.
Keywords
fluorophore; bioorganic chemistry; chemical biology; molecular probes; fluorine; conjugation
Introduction
Fluorescent molecular probes are powerful tools for visualizing and quantifying biological
1–3
processes. Fluorophores derived from fluorescein (9, Figure 1) have been extensively
used in this way because these compounds can exhibit excellent photophysical properties
under physiological and more basic conditions (fluorescein Abs. λ
= 490 nm, Em. λ
=
max
max
−1
−1
3–8
517 nm, ε = 76,900–87,600 M cm , Φ (aqueous, pH > 8) = 0.92–0.95). However,
© Thieme Stuttgart
Correspondence to: Blake R. Peterson, brpeters@ku.edu.
Supporting Information for this article is available online at http://www.thieme-connect.com/ejournals/toc/synthesis.

Woydziak et al.
Page 2
9
because the phenol of fluorescein exhibits the relatively high pKa of 6.3–6.8, this dye is
much less fluorescent in weakly acidic environments commonly found in biological
systems, such as the lumen of endosomes (pH ≤ 6.5). Fluorescein is also relatively
10
susceptible to photobleaching. These limitations are reduced in certain halogenated
11–13
6
analogues
such as Oregon Green (2′,7′-difluorofluorescein, 10, Figure 1). The fluorine
substituents of 10 decrease the pKa of the phenol to 4.8 and substantially enhance
photostability. However, single isomer 5- and 6-carboxy derivatives of Oregon Green are
costly because condensation of 4-carboxyphthalic anhydride with relatively expensive 4-
fluororesorcinol is used to generate a mixture of regioisomers that need to be further
6
protected as the diacetate for separation. The preparation of the analogous
carboxyfluorescein regioisomers is less expensive as they can be isolated in pure form on a
14
multigram scale by recrystallization of methanesulfonate ester precursors. Moreover,
because the acidic phenol and 3-carboxylic acid functionalities of Oregon Green are both
ionized under physiological conditions, this highly polar dianion does not easily penetrate
biological membranes for studies of targets in the interior of living cells.
To attempt to overcome some of the limitations of Oregon Green, we designed and
15,16
synthesized a hybrid fluorophore termed Pennsylvania Green (12, Figure 1).
Pennsylvania Green incorporates the 2′, 7′-difluoro substituents of Oregon Green (10, Figure
1), but replaces the 3-carboxylic acid with the methyl group found in more hydrophobic
7
Tokyo Green (11, Figure 1). This hybrid fluorophore has been synthesized as the 4-carboxy
methyl ester derivative (8) to install a convenient handle for bioconjugation, structurally
analogous to 5-carboxy-Oregon Green. This ester can be hydrolyzed to the corresponding
carboxylic acid (13), and converted to the highly amine-reactive N-hydroxysuccinimidyl
15,16
ester (14), in nearly quantitative yield.
4-Carboxy-Pennsylvania Green (13) exhibits
= 494 nm, Em. λ = 515 nm, ε = 62,000
excellent photophysical properties (Abs. λ
max
max
−1
−1
M
cm , Φ (aqueous, pH 7.4) = 0.89) that are similar to 5-carboxy Oregon Green.
However, amide derivatives of this hydrophobic analogue can be much more cell permeable,
making this compound a potentially useful probe of intracellular components, targets, and
15
processes in living cells.
To synthesize Pennsylvania Green and derivatives, 2,7-difluoro-3,6-dihydroxy-xanthen-9-
16
one (5, Scheme 1) has been used as a key building block. However, two previously
16,17
reported routes
to this compound are challenging to execute on multigram scales. To
improve access to fluorophores derived from this intermediate, we report here a scalable
synthesis of xanthone 5 from bromoarene 1 and conversion to multigram quantities of
fluorophore 8 (Scheme 1).
17
Xanthone 5 was first prepared in 2002 by Lawrence and co-workers using the nine-step
route shown in Scheme 2 (Panel A). In this report, synthesis of 0.58 grams of 5 in 28%
16
overall yield is described. Based on this precedent, in 2006 we developed a shorter six-
18
step route (Scheme 1, Panel B) to 5 involving cobalt-mediated carbonylation of iodoarene
24 under microwave irradiation to generate benzophenone 25, followed by demethylation to
17
yield the common intermediate 22 previously described by Lawrence. Although
microwave-promoted carbonylation of 24 was effective on small scales of less than one
gram, this reaction was not successful for larger scale preparations, and multiple
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Woydziak et al.
Page 3
chromatographic steps were required for purification of intermediates generated using this
15
route. In 2007, we described a second more concise route for the synthesis of 4-carboxy-
Pennsylvania Green involving condensation of a functionalized benzaldehyde with 4-
fluororesorcinol, but challenging purification steps that hinder production of gram quantities
19
of this fluorophore were also observed. More recently, in 2012, we reported an improved
synthesis of xanthone 5 based on repeated nucleophilic aromatic substitution of
hexafluorobenzophenone 4 by hydroxide (Scheme 1, Panel C). We describe here the
optimization and extension of this methodology to allow synthesis of multigram quantities
of 4-carboxy-Pennsylvania Green methyl ester (8).
Results and Discussion
A multigram synthesis of 2,7-difluoroxanthone (5), based on our iterative nucleophilic
19
aromatic substitution approach, is shown in Scheme 1. The synthesis of key intermediate 5
started with commercially available 1-bromo-2,4,5-trifluorobenzene (1). This compound
was treated with isopropylmagnesium chloride in dry THF at −78 °C to promote metal-
20
halogen exchange, followed by the addition of 2,4,5-trifluorobenzaldehyde 2 to afford
diphenylmethanol 3 in 95% yield. Oxidation with TEMPO and NaOCl provided bis-(2,4,5-
trifluorophenyl)methanone (4) in excellent yield and high purity after recrystallization from
hexanes. During optimization of conversion of 1 to 3, we observed that at least 1.05 equiv of
both i-PrMgCl and 2 are required for addition to 1.0 equiv of 1 to achieve the highest
conversion and minimize impurities and unreacted 2. This slight excess of 0.05 equiv of 2
was oxidized to 2,4,5-trifluorobenzoic acid in the subsequent step, allowing facile removal
by extraction into basic aqueous solution. This optimized process provided benzophenone 4
as a crystalline solid in 90% overall yield from 1, and did not require purification of
intermediate 3.
As summarized in Table 1, we examined the sensitivity of fluorinated benzophenone 4 on a
small scale to base, temperature, and time. The weak base NaOAc was unreactive even after
reflux for 12 h (entry 1). However, reflux of 4 in 1.5 M aqueous KOH afforded bis(4-
hydroxyphenyl)methanone (26) in > 90% yield (entry 2). When the concentration of
aqueous KOH was increased to 10 M, bis(2,4-dihydroxyphenyl)methanone (22) was
obtained in 60% yield, with xanthone 5 isolated in 5% yield (entry 3). Importantly,
conversion to 5 was dramatically accelerated and driven to completion by heating to 200 °C
under these conditions in a sealed tube. Although this approach provided an excellent yield
of 5 (96%) after 4 hours, the ability to safely scale up this method was limited due to the
high pressures involved (entry 4).
Based on the observed product distribution, treatment of 4 with KOH initially substitutes the
reactive fluorines at the 4 and 4′-positions with hydroxyl groups. Subsequent slower
addition of hydroxide at the 2-position, with further substitution at the 2′-position, is
followed by cyclization to yield xanthone 5. To drive this reaction to completion on
multigram scales at ambient pressure, we found that reaction of 4 with 10 M KOH required
refluxing conditions for extended periods of time. However, in glass vessels, silicon-derived
impurities were generated under these conditions that were difficult to separate from 5. To
avoid these impurities, we often used an all-Teflon flask for this reaction. Although this
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Woydziak et al.
Page 4
method could produce 5 in a one-pot procedure, higher yields of 5 (60%) were obtained on
large scale (> 10 g) by a two pot process initially involving heating of 4 to 80 °C for 3 h in a
glass round bottom flask containing DMSO and 10 M aqueous KOH to produce a mixture of
26 and 22. These more water-soluble intermediates were extracted, dried, and subsequently
refluxed for 48 h in an all-Teflon flask charged with only fresh 10 M aqueous KOH. We
used this approach to prepare several ~ 25 g batches of pure 5. Alternatively, simple one-pot
reflux of 4 in a glass round-bottom flask with aqueous 10 M KOH and DMSO for 48 h
could also be used to prepare 5, often in higher yield, presumably due to improved thermal
transfer properties of glass compared with Teflon, but under these conditions silica
impurities contributed to ~10% of the isolated mass of 5. However, crude 5 prepared in this
way could be readily converted without further purification to bis MEM-ether 6, and when
this method was used, any silica impurities were easily removed from this much less polar
derivative.
The phenol of xanthone 5 was protected to afford bis MEM ether 6 (Scheme 1). Initial
studies of reaction of 5 in DMF containing MEM-Cl and NaH (5 equiv) for 16 h at 4–22 °C
provided reasonably high yields of 6 (65%), but a large volume of DMF was necessary to
solubilize 5 under these conditions. To overcome this limitation on multigram scales, 5 was
alkylated at 50 °C with MEM-Cl and the weaker base DIEA (3 equiv) in THF. Under these
conditions, 6 could be isolated in 75% yield after only 3 h. This reaction was quenched with
water, diluted with CH Cl , and the organic layer was isolated. Extraction of the organic
2
2
phase with aqueous KOH (1 M) was used to remove impurities and any unreacted starting
material (5). Crystallization of 6 from hexanes/CH Cl provided pure 6 as a white powder
2
2
(75% yield). This approach allowed preparation of ~ 40 g batches of 6 with standard
laboratory equipment.
To further optimize the synthesis of 4-carboxy-Pennsylvania Green methyl ester (8),
commercially available methyl 4-iodo-3-methylbenzoate (7) in dry THF was subjected to
metal-halogen exchange at −78 °C with i-PrMgCl.LiCl complex in THF (Turbo
21,22
Grignard).
A solution of MEM-protected xanthone 6 in THF at −78 °C was slowly
added, the solution was warmed to room temperature for 12 h, and 6 was completely
consumed. To stop the reaction, and cleave the MEM protecting groups, the reaction was
cooled to 4 °C and 6 M aqueous HCl was slowly added. As previously reported in other
22
systems, if i-PrMgCl in THF (without LiCl) was used for the metal-halogen exchange, this
reagent generated a much less reactive aryl Grignard reactant. Under these conditions, at
least 5 equiv of the aryl Grignard, much higher concentrations, and a reaction time of 24 h
was required to drive the reaction to completion. By contrast, the i-PrMgCl.LiCl complex in
THF (Turbo Grignard) generated a more reactive aryl Grignard reagent, shortened the
reaction time, and required only 1.5 equiv for the conversion of 6 to 8.
Conclusion
To provide access to multigram quantities of 4-carboxy-Pennsylvania Green methyl ester
(8), we developed a new scalable route to 2,7-difluoroxanthone (5). This route involves the
use of iterative nucleophilic aromatic substitution of bis-(2,4,5-trifluorophenyl)methanone
(4) by hydroxide to install four carbon-oxygen bonds in a one-pot process. Optimization of
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Woydziak et al.
Page 5
addition of the Grignard reagent derived from methyl 4-iodo-3-methylbenzoate (7) to bis-
MEM ether 6 allowed preparation of 15 g of pure fluorophore 8 in a single five linear step
process and 28% overall yield without requiring chromatography. Considering only the cost
of materials, the optimized synthetic procedure described here allows access to 4-carboxy-
Pennsylvania Green (13) for ~$5/gram, which is orders of magnitude less expensive than
comparable single-isomer amine-reactive fluorophores available through commercial
sources. The ability to economically produce multi-gram quantities of 4-carboxy-
Pennsylvania Green, especially considering its potential for high compatibility with solid
phase peptide synthesis, should facilitate the preparation of large quantities or libraries of
fluorescent molecular probes that would be inaccessible, much more costly, or difficult to
prepare using other approaches.
Experimental
General Methods. All reagents and solvents were commercially available and were used
without further purification unless otherwise noted. NMR spectra of the intermediates were
recorded on Bruker NMR spectrometers using TMS as an internal standard. EI-MS spectra
were obtained on a Finnigan MAT 95 mass spectrometer and ESI-MS spectra were obtained
on a Kratos MS 80 mass spectrometer. Elemental analysis was obtained using a vario EL
spectrometer. HPLC analysis of 8 shown in the Supporting Information was performed on
an Agilent 1100 liquid chromatograph equipped with a Hamilton reverse-phase PRP-1
column (7 μm, 100 Å, 4.1 × 250 mm); flow rate 0.8 mL/min, detection wavelength: 254 nm,
temperature: 25 °C. Mobile phase gradient: 30:70 to 0:100 water/acetonitrile over 30
minutes (purity > 97%). THF (Sigma-Aldrich, ≥ 99.0%) was rendered anhydrous by use of a
Glass Contour solvent purification system from SG Water USA.
Bis(2,4,5-trifluorophenyl)methanone (4). A one-necked round-bottom flask (1 L) equipped
with a Teflon-coated magnetic stirring bar was removed from a drying oven (150 °C), sealed
with a rubber septum while hot, and purged with dry nitrogen. After cooling to room
temperature (22 °C), 1-bromo-2,4,5-trifluorobenzene (50.0 mL, 90.0 g, 0.427 mol, Oakwood
Products) and 400 mL of anhydrous THF was syringed into the flask. The flask was
submerged in a bath of acetone cooled to −78 °C with dry ice. To this solution was added a
solution of isopropylmagnesium chloride in THF (2.0 M, 224 mL, 0.448 mol, Sigma-
Aldrich) with stirring over 5 min. The resulting mixture was stirred for an additional 10 min,
the dry ice/acetone bath (−78 °C) was removed, and a bath containing ice water was
substituted. This reaction mixture was stirred for an additional 30 min, the ice water bath
was removed, and the flask resubmerged in the dry ice/acetone bath (−78 °C). This reaction
mixture was stirred for 10 min, followed by addition of 2,4,5-trifluorobenzaldehyde (50.4
mL, 71.7 g, 0.448 mol, Oakwood Products) by syringe. The resulting mixture was stirred for
an additional 10 min, the −78 °C bath was removed, the flask was warmed to room
temperature, and the reaction mixture was stirred at room temperature for 3 h. The reaction
was quenched by addition of saturated aqueous NH Cl solution (50 mL). The resulting
4
biphasic mixture was diluted with water (300 mL) and Et O (200 mL), the two phases were
2
separated, and the aqueous fraction was extracted with Et O (2 × 300 mL). The combined
2
organic fractions were dried over anhydrous Na SO and concentrated by rotary evaporation
2
4
to provide a viscous oil. This crude 3 was dissolved in CH Cl (1.0 L) and transferred to an
2
2
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Woydziak et al.
Page 6
Erlenmeyer flask (5 L). To this solution of 3 was added (2, 2, 6, 6-tetramethylpiperidine-1-
yl)oxyl free radical (TEMPO, 3.33 g, 21.0 mmol, 5 mol%, Oakwood Products), KBr (10.1 g,
85.4 mmol, Alfa Aesar), NaHCO (72.0 g, 0.93 mol) and saturated aqueous sodium chloride
3
(500 mL). The resulting mixture was vigorously stirred (1000 rpm), and aqueous NaOCl
(0.72 M, 1.5 L, 1.10 mol, Clorox household-grade bleach) was added. The reaction mixture
was stirred for 3 h, transferred to a separatory funnel, and the phases were separated. The
aqueous layer was extracted with CH Cl (2 × 350 mL), the layers were combined, dried
2
2
over anhydrous Na SO , and concentrated by rotary evaporation to give a crude oil. This oil
2
4
was quantitatively pipetted onto a plug of packed CH Cl -saturated silica gel (50 g, 65 mm
2
2
diameter) and 4 was eluted with CH Cl (400 mL). The filtrate was transferred to a one-
2
2
necked round-bottom flask (500 mL) and the solvent was removed by rotary evaporation to
provide a viscous oil that crystallized upon addition of hexanes (250 mL). The colorless
crystals were collected by vacuum filtration and air-dried for 3 h to give pure benzophenone
1
4 (111.5 g, 90%). Pure 4 has the following properties: mp 80–81 °C; H NMR (400 MHz,
13
CDCl ) δ 7.57 (qd, J = 8.8, 6.4 Hz, 2H), 6.95 (qd, J = 9.2, 6.4 Hz, 2H); C NMR (126
3
MHz, CDCl ) δ 184.7 (s), 156.8 (dd, J = 254.7, 9.8 Hz), 153.3 (ddd, J = 260.0, 14.5, 12.2
3
Hz), 147.1 (ddd, J = 248.1, 12.7, 3.1 Hz), 123.2 (d, J = 14.9 Hz), 118.8 (d, J = 20.1 Hz),
111.6 – 98.1 (m); IR (thin film) ν : 3073, 1667, 1623, 1509, 1437, 1420, 1333, 1293,
max
−1
+
1208, 1138, 889 cm ; HRMS (ESI) m/z 291.0263 (M+H , C H F O requires 291.0245);
13 5 6
Anal. calcd. for C H F O: C, 53.81; H, 1.39; N, 0, found: C, 53.58; H, 1.36; N, <0.05.
13 4 6
2,7-difluoro-3,6-dihydroxy-9H-xanthen-9-one (5). To a glass one-necked round-bottom
flask (500 mL) equipped with a Teflon-coated magnetic stirring bar was added 4 (43.5 g,
150 mmol, 1.0 equiv), aqueous KOH (10 M, 145 mL, 1.45 mol, 9.67 equiv), and DMSO
(145 mL). This mixture was stirred at 80 °C for 3 h. The solution was poured into a beaker
(2 L) containing ice (500 g) and concentrated aqueous HCl (145 mL, 1.74 mol). The
resulting slurry was extracted with Et O (3 × 150 mL). The organic fractions were combined
2
and dried over anhydrous sodium sulfate (45 g). This solution was concentrated by rotary
evaporation (30 °C, 10 mmHg) in a one-necked all-Teflon round-bottom flask (500 mL,
ChemGlass). This Teflon flask was equipped with a Teflon-coated magnetic stirring bar and
an aqueous solution of KOH (10 M, 300 mL, 3.00 mol, 20 equiv) was added. The flask was
vigorously refluxed (bath temp = 150 °C) for 48 h. The resulting slurry was poured, while
hot, into a 2 L beaker containing ice (1 Kg) and concentrated aqueous HCl (300 mL, 3.60
mol). This mixture was briefly stirred with a rod and placed in a refrigerator at 4 °C for 3 h.
A colorless precipitate was collected by vacuum filtration, washed with water (3 × 250 mL),
and dried by vacuum filtration. The colorless solid was transferred to a one-necked round-
bottom flask (500 mL), and exposed to high vacuum (12 h) to remove residual water. The
solid was ground with a mortar and pestle to provide xanthone 5 as a white powder (23.9 g,
1
60%). Pure 5 has the following properties: mp > 300 °C; H NMR (400 MHz, DMSO-d ) δ
6
13
11.56 (apps, 2H), 7.76 (d, J = 10.8, 2H), 7.07 (d, J = 6.8, 2H); C NMR (126 MHz, DMSO-
d ) δ 173.2 (s), 153.1 (s), 152.0 (d, J = 15.0 Hz), 148.8 (d, J = 242.5 Hz), 112.6 (s), 111.0 (d,
6
−1
J = 20.0 Hz), 104.7(s); IR (thin film) ν : 3356, 1627, 1475, 1309, 1188, 1160 cm ;
max
HRMS (ESI) m/z 263.0154 (M-H, C H F O requires 263.0156); Anal. calcd. for
13 5 2
4
C H F O : C, 59.10; H, 2.29; N, 0, found: C, 58.91; H, 2.28; N, <0.05.
13 6 2
4
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Woydziak et al.
Page 7
2,7-difluoro-3,6-bis((2-methoxyethoxy)methoxy)-9H-xanthen-9-one (6). To an oven-dried
one-necked round-bottom flask (500 mL) equipped with a Teflon-coated magnetic stirring
bar was added xanthone 5 (30.0 g, 113.4 mmol, 1.0 equiv). The flask was sealed with a
rubber septum, purged with dry nitrogen, and anhydrous THF (250 mL) and N,N-
diisopropylethylamine (60.2 mL, 44.6 g, 344 mmol, 3.0 equiv) and 2-methoxyethoxymethyl
chloride (MEM-Cl, 39.2 mL, 42.6 g, 344 mmol, 3.0 equiv, AK Scientific) were added
sequentially by syringe. The resulting mixture was heated to 50 °C and stirred for 3 h. The
reaction was allowed to return to ambient temperature and was diluted with CH Cl (250
2
2
mL). The resulting mixture was washed with aqueous KOH (1 M, 3 × 100 mL). The
combined aqueous phases were extracted with CH Cl (200 mL) and the combined organic
2
2
fractions were dried over anhydrous Na SO . Concentration by rotary evaporation yielded a
2
4
pale yellow solid. The solid was further purified by recrystallization by the following
protocol: The solid was dissolved in CH Cl (200 mL) and transferred to an Erlenmeyer
2
2
flask (500 mL). To this flask was also added a boiling stone and hexanes (200 mL). The
resulting solution was heated to a vigorous boil on a hot plate and the total volume of the
solution was reduced to 200 mL. The Erlenmeyer flask containing the solution was removed
from the hot plate, allowed to stand for 12 h, and crystallization was observed. Collection of
the colorless needles by vacuum filtration, washing with hexanes (100 mL), and drying by
exposure to air for 3 h provided pure MEM-protected xanthone 6 (38.0 g, 75%). Pure 6 has
1
the following properties: mp > 300 °C; H NMR (400 MHz, CDCl ) δ 7.97 (d, J = 10.4,
3
2H), 7.28 (d, J = 6.8, 2H), 5.47 (s, 4H), 3.93 (t, J = 4.4, 4H), 3.61 (t, J = 4.3, 4H), 3.41 (s,
13
6H); C NMR (125 MHz, CDCl ) δ 174.6 (s), 153.4 (s), 151.0 (d, J = 25.0 Hz), 149.9 (d, J
3
= 246 Hz), 117.2 (s), 115.4 (d, J = 6.25 Hz), 111. 9 (d, J = 21.0 Hz), 104.8 (s), 94.3 (s), 71.4
−1
(s), 68.6 (s), 59.1 (s); IR (thin film) ν : 2892, 1630, 1503, 1468, 1369, 1272 cm ; HRMS
max
(ESI) m/z 439.1224 (M-H, C H F O requires 439.1205); Anal. calcd. for C H F O :
21 21 2
8
21 22 2 8
C, 52.27; H, 5.04; N, 0, found: C, 57.19; H, 4.92; N, <0.05.
4-Carboxy-Pennsylvania Green methyl ester (8)
An oven-dried one-necked round-bottom flask (500 mL) equipped with a Teflon-coated
magnetic stirring bar was removed from a drying oven, sealed while hot with a rubber
septum, and purged with dry nitrogen. After cooling to room temperature, methyl 4-iodo-3-
methylbenzoate (7, 22.6 g, 81.6 mmol, 1.5 equiv) was added and the flask resealed.
Anhydrous THF (100 mL) was added by syringe, the flask was submerged in a dry ice/
acetone bath (−78 °C), and the solution was stirred for 10 min. Isopropylmagnesium
chloride lithium chloride complex in THF (1.3 M, 62.8 mL, 81.6 mmol, 1.5 equiv, Acros)
was added dropwise over 10 min. The dry ice/acetone bath was removed, replaced with an
ice bath, and the reaction mixture stirred for 1 h. During this 1 h period, 6 (24.0 g, 54.8
mmol, 1.0 equiv) was added to a separate oven-dried one-necked round-bottom flask (250
mL). This flask was sealed with a rubber septum, purged with dry nitrogen, and anhydrous
THF (100 mL) was added to completely dissolve 6. After this 1 h period, the flask
containing the aryl Grignard was resubmerged in the dry ice/acetone bath (−78 °C) and the
reaction mixture stirred for 10 min. The solution of 6 was cannulated into the flask at −78 °C
containing the aryl Grignard over the course of 5 min. The dry ice/acetone bath was
removed, the reaction mixture was warmed to room temperature, and the reaction was
stirred for an additional 12 h. The flask was cooled in an ice-water bath for 5 min. Aqueous
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Woydziak et al.
Page 8
HCl (6 M, 150 mL, 0.9 mol) was added dropwise over 10 min. The resulting slurry was
stirred for 1 h at 4 °C, diluted with 300 mL of H O, and stirred for an additional 30 min. The
2
red-orange precipitate was collected by vacuum filtration, washed with water (3 × 150 mL),
and air-dried thoroughly (1.5 h). The yellow-orange pellet was slurried in CH Cl /hexane
2
2
(3:1, 150 mL). The suspension was stirred at ambient temperature overnight, followed by 4
°C for 1 h, and then was filtered. The filter cake was washed with water (2 × 300 mL) and
air dried to give 8 as an orange-red solid (> 95% pure, 15.0 g, 70%). Pure 8 has the
1
following properties: mp 220–224 °C; H NMR (500 MHz, CD OD/0.15 M NaOCD ) δ
3
3
8.08 (dt, J = 1.6, 0.7 Hz, 1H), 8.03 (ddd, J = 7.8, 1.6, 0.8 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H),
13
6.71 (d, J = 7.4 Hz, 2H), 6.62 (d, J = 11.4 Hz, 2H), 3.39 (s, 3H), 2.12 (s, 3H); C NMR
(126 MHz, CD OD/0.15 M NaOCD ) δ 174.9 (s), 172.5 (d, J = 17.4 Hz), 157.5 – 157.3 (m),
3
3
156.2 (s), 155.5 (s), 140.4 (s), 136.6 (s), 136.0 (s), 132.5 (s), 128.9 (d, J = 177 Hz), 111.7
(s), 111.5 (s), 110.9 (d, J = 8.5 Hz), 106.5 (d, J = 5.5 Hz), 49.9 (s), 19.7 (s); IR (thin film)
−1
+
ν
: 3465, 3000, 1796, 1712, 1213 cm ; HRMS (ESI) m/z 397.0888 (MH , C H O F
22 14 5 2
max
requires 397.0884).
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The authors thank the NIH for financial support (R01 CA83831 and P20-GM103638). Z.W. thanks the NIH for an
IRACDA Postdoctoral Fellowship.
References
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Figure 1.
Structures of fluorophores.
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Scheme 1.
A practical multigram synthesis of 4-carboxy-Pennsylvania Green methyl ester (8).
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Scheme 2.
Previously reported syntheses of xanthone 5.
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Synthesis (Stuttg). Author manuscript; available in PMC 2015 January 01.