Synthesis of isomerically pure carboxylate- and sulfonate-substituted xanthene fluorophores

Article abstract of DOI:10.1016/j.tet.2005.01.024

Xanthene-based fluorophores such as fluorescein and rhodamine are typically prepared by acid-catalyzed condensation of the appropriate resorcinol or 3-aminophenol with phthalic anhydride. Condensation of substituted phthalic anhydride species results in functionalized fluorophores that are formed as mixed isomers. Crystallization approaches to isomer separation have been reported elsewhere for symmetric fluorescein carboxylates. We describe crystallization-based separation of protected fluorescein sulfonates and coupling conditions to form sulfonamides, precursors for carboxylate-substituted rhodamines, and precursors for asymmetrically substituted fluoresceins and rhodafluors.

Full text of DOI:10.1016/j.tet.2005.01.024

Tetrahedron 61 (2005) 3097–3105
Synthesis of isomerically pure carboxylate- and
sulfonate-substituted xanthene fluorophores
*
Carolyn C. Woodroofe, Mi Hee Lim, Weiming Bu and Stephen J. Lippard
Department of Chemistry, Massachusetts Institute of Technology, Room 18-498, Cambridge, MA 02139, USA
Received 30 October 2004; revised 5 January 2005; accepted 10 January 2005
Abstract—Xanthene-based fluorophores such as fluorescein and rhodamine are typically prepared by acid-catalyzed condensation of the
appropriate resorcinol or 3-aminophenol with phthalic anhydride. Condensation of substituted phthalic anhydride species results in
functionalized fluorophores that are formed as mixed isomers. Crystallization approaches to isomer separation have been reported elsewhere
for symmetric fluorescein carboxylates. We describe crystallization-based separation of protected fluorescein sulfonates and coupling
conditions to form sulfonamides, precursors for carboxylate-substituted rhodamines, and precursors for asymmetrically substituted
fluoresceins and rhodafluors.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
benzophenones has not been reported. We now describe
methodology for the synthesis of isomerically pure
carboxylic or sulfonic acid-substituted analogues of these
fluorophores.
Xanthene-based dyes such as rhodamines and fluoresceins
are widely used in sensing applications owing to their
brightness, high quantum yields, low-energy excitation and
emission wavelengths, and biocompatibility. Functional
handles are typically incorporated into the benzoic acid ring
of these fluorophores. Bottom-ring-substituted rhodamines
and fluoresceins form by acid-catalyzed condensation of a
3-aminophenol or resorcinol with a substituted phthalic acid
species. This reaction affords the desired product as an
equal mixture of two isomers, substituted at the 5- and
6-positions1 of the benzoic acid ring. Crystallization-based
separations of protected fluorescein carboxylate isomeric
mixtures have been described,1,2 and we now report similar
methods in the separation of fluorescein 5(6) sulfonic acid
isomers. However, there is no general method for separating
rhodamine 5- and 6-carboxylate mixtures by crystallization.
Chromatographic separation of isomers is particularly
tedious with these polar, charged compounds.3,4 Finally,
asymmetrically substituted fluoresceins5–8 and various
hybrid rhodamine-fluorescein compounds,9–13 termed
rhodafluors or rhodols, have been prepared by our group
and others. These compounds share a 20,40-dihydroxybenzo-
phenone-2-carboxylate as a synthon, but the preparation of
isomerically pure dicarboxy-substituted analogues of these
2. Results and discussion
2.1. Fluorescein sulfonamides
In the course of our efforts to produce fluorescent sensors for
Zn2C, the synthesis of fluorescein sulfonamides was of
interest owing to the many reported sulfonamide-based
Zn2C sensors. Sulfonation of the xanthene system was
considered most likely to afford a derivative that would
exhibit a metal-induced change in fluorescence. Direct
sulfonation of unsubstituted fluorescein with fuming
sulfuric acid provided only unreacted starting material,
however. Reaction of sulfonated resorcinol with phthalic
anhydride provided unsubstituted fluorescein as the major
product (Scheme 1). Condensationof resorcinol with 4-sulfo-
phthalic acid was explored next. As expected, this reaction
produced a mixture of two isomers (1a, b, Scheme 2) in
roughly equal amounts, a mixture that is available
Keywords: Fluorescein sulfonic acid; Rhodamine carboxylate; Isomer
resolution; Fractional crystallization; Dibromofluoran; Asymmetric fluor-
escein carboxylate.
* Corresponding author. Tel.: C1 617 253 1892; fax: C1 617 258 8150;
e-mail: lippard@mit.edu
Scheme 1.
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.01.024

3098
C. C. Woodroofe et al. / Tetrahedron 61 (2005) 3097–3105
Scheme 2.
commercially.14 The separation of fluorescein-5(6)-sulfonic
acid isomers, reported previously, claimed isomeric purity
based on infrared spectroscopy and paper chromato-
graphy.15,16 The analytical sensitivity of these techniques
is relatively low. We were able to separate the mixed
isomers by protection as the dipivaloyl esters and sub-
sequent crystallization of the 6-isomer 2a followed by
crystallization of the 5-isomer 2b, each as its diisopropyl-
ethylammonium salt. Isomeric purity of 2a and 2b was
greater than 95% based on NMR spectroscopy. Basic
hydrolysis of the isomerically pure dipivaloates 2a and 2b
yielded the deprotected fluorescein sulfonic acids 1a and 1b.
A crystal structure of fluorescein-6-sulfonic acid (1a) was
obtained (Fig. 1). The crystal formed under strongly acidic
conditions and, consequently, the fluorescein is in the
Table 1. Crystallographic information
1a$3H2O
20H18O11
466.40
P21/c
4
Empirical formula
Molecular weight
Space group
C
S
C26H16N2O5SCl2
539.37
ꢀ
P1
˚
a (A)
10.4324(12)
16.854(2)
11.9615(14)
9.997(4)
11.067(2)
11.401(2)
95.65(3)
100.55(3)
106.81
1171.6(4)
2
1.529
K100
0.409
1.84–28.29
10559
5363
˚
b (A)
˚
c (A)
a, deg
b, deg
g, deg
109.516(2)
3
˚
V, A
1982.3(4)
4
1.563
K85
0.228
2.07–28.29
12393
4568
Z
rcalc, g/cm3
T, 8C
m (Mo Ka), mmK1
q limits, deg
Total number of data
Number of unique
data points
Number of parameters
Ra
316
0.0670
0.1341
0.464, K0.488
352
0.0433
0.1070
0.663, K0.199
wR2b
˚
max, min peaks, e/A
3
Figure 1. ORTEP diagram of fluorescein-6-sulfonic acid (1a) showing 50%
probability thermal ellipsoids. The solvent molecules have been omitted for
clarity.
a RZSjjFojKFcjj/SjFoj.
b wR2Z{S[w(Fo2KFc2)2]/S[w(F2o)2]}1/2
.

C. C. Woodroofe et al. / Tetrahedron 61 (2005) 3097–3105
3099
lactone-opened form, presumably with a positive charge
delocalized over the xanthene system. The charge is
neutralized by the anionic sulfonate group. The C–O
bonds of the phenolic hydroxyls are approximately the
30, 60-dichlorofluoran by X-ray crystallographic analysis of
the 2-picolylamine adduct 4 (Fig. 2; selected bond lengths
and angles listed in Table 3). The dihedral angle between the
two extended aromatic ring systems is 86.68, indicating a
nearly perpendicular orientation. Use of the milder oxalyl
chloride as an activating agent to generate sulfonyl chloride
5 and subsequent reaction with an amine affords the desired
sulfonamide product as a mixture of unprotected (6) and
protected (7) products (Scheme 3).
˚
same length (1.34, 1.33 A) and are each consistent with a
single bond, rather than a ketone tautomer. Three water
molecules were present in the asymmetric unit, with 12 in
the unit cell. Crystallographic parameters are listed in Table
1 and selected bond lengths and angles for 1a, in Table 2.
Table 2. Selected bond lengths and angles for 1a
2.2. Rhodamines and rhodamine carboxylates
˚
(A)
Bond lengths
Bond angles
(Deg)
30,60-Dichlorofluoran can be converted into rhodamines by
direct ZnCl2-catalyzed condensation with excess amine.17,18
This reaction0 suggested that, since 30,60-dihalofluorans
share with 3 ,60-diacetyl- or -dipivaloyl-fluorescein the
fluorescein motif that has been trapped in the lactone
form, it might be possible to synthesize 30,60-dihalofluoran-
5(6)-carboxylates by acid-catalyzed condensation and
subsequently resolve the isomers by selective crystal-
lization. Alternatively, reaction of previously resolved
fluorescein carboxylates with thionyl chloride or thionyl
bromide could provide the desired isomerically pure
dihalofluoran carbonyl halide (vide supra). Subsequent
condensation with amines under appropriate conditions
might afford isomerically pure rhodamine carboxylates.19
S(1)–O(2)
S(1)–O(1)
S(1)–O(3)
S(1)–C(5)
O(8)–C(14)
O(8)–C(16)
O(7)–C(3)
O(6)–C(2)
O(5)–C(8)
O(4)–C(8)
1.442(3)
1.451(3)
1.456(3)
1.780(3)
1.355(4)
1.360(4)
1.342(4)
1.326(4)
1.309(4)
1.219(4)
O(2)–S(1)–O(1)
O(2)–S(1)–O(3)
O(1)–S(1)–O(3)
O(4)–C(8)–O(5)
113.08(15)
112.75(17)
112.10(16)
124.7(3)
Coupling of the sulfonic acids with amines was first
attempted following activation with thionyl chloride
(Scheme 2). Reaction of both protected and unprotected
fluoresceins under these conditions gave a non-fluorescent
material 3a or 3b, however, which was determined to be the
We chose to work with the more reactive dibromofluoran
species, rather than the reported dichlorofluran substrate.
30,60-Dibromofluoran 8 was synthesized by acid-catalyzed
condensation of 3-bromophenol with phthalic anhydride
and used as a model for t0he0 less readily available
dibromofluoran carboxylates. 3 ,6 -Dibromofluoran reacted
smoothly with excess pyrrolidine under literature conditions
to afford the desired rhodamine 9. Palladium-catalyzed
reductive coupling under reaction conditions described in
the literature20 also furnished 9. This chemistry is
summarized in Scheme 4.
30,60-Dibromo-5(6)-carboxylate (10a, b) was synthesized
similarly (Scheme 5). Selective crystallization from pyri-
dine and acetic anhydride afforded the pure 6-isomer
pyridinium salt 10c in 15% yield, and further recrystalliza-
tion of the mother liquor furnished the pure 5-isomer 10b in
11% yield. Unsurprisingly, palladium-catalyzed coupling
conditions were not compatible with a carboxylate-contain-
ing substrate. Nevertheless, heating 10c at 140 8C with
5 equiv of ZnCl2 and 10 equiv of pyrrolidine, followed by
treatment with hydrochloric acid, afforded the desired
rhodamine 11 in O94% yield with no apparent mixing of
isomers. The strongly acidic workup is necessary in order
to hydrolyze any amide byproduct that arises from
carboxylate-amine condensation. The harsh conditions
required for the reaction preclude the use of all but the
most robust amines in the condensation.
Figure 2. ORTEP diagram of fluorescein-6-sulfonamidopicoline (4)
showing 50% probability thermal ellipsoids.
Table 3. Selected bond lengths and angles for 4
˚
(A)
Bond lengths
Bond angles
(Deg)
Cl(1)–C(11)
Cl(2)–C(16)
S(1)–O(5)
S(1)–O(4)
S(1)–N(2)
O(1)–C(13)
O(1)–C(14)
N(2)–C(21)
1.7313(18)
1.7362(19)
1.4263(14)
1.4294(14)
1.5958(16)
1.371(2)
O(5)–S(1)–O(4)
O(5)–S(1)–N(2)
O(4)–S(1)–N(2)
C(21)–N(2)–S(1)
O(3)–C(20)–O(2)
C(10)–C(11)–Cl(1)
C(13)–O(1)–C(14)
121.11(9)
106.95(9)
107.75(9)
122.30(13)
121.94(16)
119.33(14)
118.12(13)
2.3. Isomerically pure synthons for asymmetric carboxy-
substituted xanthene dyes
The syntheses of symmetrically substituted fluorescein-
based zinc(II) biosensors containing a carboxylate or an
amide functionality have been described previously.21,22 To
date, no such methodology exists for preparing similarly
1.374(2)
1.456(2)

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C. C. Woodroofe et al. / Tetrahedron 61 (2005) 3097–3105
Scheme 3.
Scheme 4.
Scheme 5.
functionalized sensors based on asymmetric fluorescein
scaffolds.5,8,6 We, therefore, turned our attention to the
synthesis of dihydroxydicarboxybenzophenones, which
would be the logical starting material for the synthesis of
asymmetric carboxylate-containing fluoresceins. Although
subjecting 1,2,4-benzenetricarboxylic acid to aluminum
chloride-catalyzed condensation with 4-chlororesorcinol13
resulted in quantitative recovery of starting material, the
hydrolysis of previously-resolved fluorescein carboxylates
12a and 12b under harshly basic conditions furnished the
desired benzophenones 13a, 13b as isomerically pure
compounds (Scheme 6). Results from condensation of 13a
with 1,6-dihydroxynaphthalene indicate that seminaphtho-
fluorescein 14 can be obtained in excellent yield with no
apparent scrambling of isomers.
3. Conclusions
We describe here the synthesis and separation of

C. C. Woodroofe et al. / Tetrahedron 61 (2005) 3097–3105
3101
Scheme 6.
fluorescein 5(6)-sulfonic acid. The structural assignment of
fluorescein-6-sulfonic acid was confirmed in an X-ray
crystallographic structure determination. Oxalyl chloride
activation of the separated isomers affords the fluorescein
sulfonyl chloride, whereas thionyl chloride converts the
phenolic hydroxyls to chlorine atoms. Such dihalofluorans
may also be synthesized by acid-catalyzed condensation of
3-halophenol with phthalic anhydride or analogues and can
be converted to rhodamines via Lewis acid- or palladium-
catalyzed reaction with simple amines. Carboxylate-
substituted dihalofluorans are similar to diacetyl- or
dipivaloyl-fluoresceins in that they are formed as a mixture
of isomers, are trapped in lactone form, and may be
separated by fractional crystallization. This methodology
can be applied as a route to isomerically pure carboxylate-
substituted rhodamines. Strongly basic hydrolysis of
previously resolved fluorescein carboxylates affords the
appropriate synthon for isomerically pure asymmetric
fluorescein or rhodafluor carboxylates.
SADABS v 6.2.25 Data were integrated using the SAINT-
PLUS software package,26 and structures were solved and
refined using SHELXTL.27 Procedures for data collection
and structural work have been reported in detail else-
where.28 All non-hydrogen atoms were refined anisotropi-
cally using least-squares methods and Fourier syntheses.
Hydrogen atoms were assigned idealized positions and
given thermal parameters of 1.2 times the thermal parameter
of the carbon or nitrogen atom to which they were attached.
All structure solutions were checked for higher symmetry
with the PLATON program.29
4.2. Synthetic procedures
4.2.1. Condensation of 4-sulforesorcinol with phthalic
anhydride. 4-Sulforesorcinol (424 mg, 2 mmol) was
ground together with phthalic anhydride (148 mg,
1 mmol) and ZnCl2 (41 mg, 0.3 mmol) with a mortar and
pestle, and the resulting mixture was heated in a 190 8C oil
bath for 30 min. Water and MeOH were added and the
resulting solution was extracted with 3!20 mL portions of
CH2Cl2. The combined organic layers were washed with
1!20 mL of brine, dried over MgSO4, and evaporated to
afford a dark orange-brown solid residue. 1H NMR analysis
indicated unsubstituted fluorescein as the major product.
1H NMR (MeOH-d4): d 8.05 (d, 1H, JZ7.5 Hz); 7.77
(td, 1H, JZ7.5, 2.1 Hz); 7.70 (td, 1H, JZ7.5, 2.1 Hz); 7.21
(d, 1H, JZ7.5 Hz); 6.70 (d, 2H); 6.60 (d, 2H, JZ3.2 Hz);
6.61–6.52 (m, 4H).
4. Experimental
4.1. Materials and methods
Reagents were obtained from Aldrich, except for 4-sulfo-
phthalic acid, which was obtained from Lancaster, and
palladium dibenzylideneacetone, sodium tert-butoxide, and
20-dimethylamino-2-dicyclohexyl-phosphinobiphenyl, which
were obtained from Strem. 4-Sulforesorcinol23 and iso-
merically pure 30,6-diacetyl-20,70-dichlorofluorescein-5(6)-
carboxylates22 were prepared as previously described.
The purity of all compounds reported here was judged to
be O95% by NMR spectroscopy. 1H and 13C NMR spectra
were recorded on a 300 or 500 MHz Varian or 400 MHz
Bruker instrument. HRMS data were acquired by personnel
at the MIT DCIF. Low-resolution electrospray mass spectra
were obtained on an Agilent Technologies 1100 Series
LCMS. Single crystals suitable for X-ray crystallography
were covered with Infineum V8512 (formerly called
Paratone N oil) and mounted on a quartz fiber. Data were
collected by using a Bruker CCD X-ray diffractometer with
4.2.2. Fluorescein-5(6)-sulfonic acid (1). A 1.14 mL
portion of a 50% aqueous solution of 4-sulfophthalic acid
(0.736 g, 3 mmol, 80% pure) was neutralized with potas-
sium hydroxide (45% w/v in water) and the solvent was
evaporated to give a purple semi-solid residue, which was
combined with resorcinol (0.661 g, 6 mmol) in 6 mL of
methanesulfonic acid. The reaction mixture was stirred in an
85 8C oil bath for 18 h, then poured into 40 mL of H2O. A
reddish-brown precipitate was filtered and dried under
vacuum to afford 696 mg of a brown powder (71% yield),
which was carried on without further purification. 1H NMR
(MeOH-d4): d 8.81 (s, 1H, JZ1.8 Hz); 8.46 (d, 1H, JZ
8.1 Hz); 8.23–8.30 (m, 2H); 7.84 (d, 1H, JZ1.8 Hz); 7.5 (d,
˚
Mo Ka radiation (lZ0.71073 A) using the SMART soft-
ware package24 and corrected for absorption using

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C. C. Woodroofe et al. / Tetrahedron 61 (2005) 3097–3105
1H, JZ8.1 Hz); 7.5 (s, 2H); 7.46 (s, 2H); 7.36 (t, 4H, JZ
3.0 Hz); 7.2 (m, 4H). MS(M-H): calcd 411.0; Found 411.4.
(0.40 g) and the reaction was heated to reflux overnight. The
ethanol was then removed under reduced pressure, the dark
red solution was acidified, and the resulting orange
precipitate (71 mg, 69%) was collected by filtration and
dried overnight; mp O338 8C (decomp.). 1H NMR (DMSO-
d6): d 7.95 (d, 1H, JZ7.8 Hz); 7.87 (d, 1H, JZ9.3 Hz); 7.25
(s, 1H); 6.70 (s, 2H); 6.55–6.62 (m, 4H). IR: 3400–
2500 cmK1 (br, s), 1707 cmK1 (m), 1638 cmK1 (m),
1603 cmK1 (s), 1457 cmK1 (s), 1314–1122 cmK1 (br, s),
1037 cmK1 (s). 13C NMR (DMSO-d6): d 168.4, 160.0,
154.9, 152.0, 129.3, 127.7, 125.9, 125.0, 120.6, 113.1,
109.5, 102.3. HRMS(MCH): Calcd 413.0331; Found
413.0320. The acidic filtrate obtained was allowed to
stand at RT for 2 weeks, at the end of which time small
bright-orange X-ray quality crystals of 1a had formed.
4.2.3. Diisopropylethylammonium salt of 30,60-dipiva-
loylfluorescein-6-sulfonate (2a). Fluorescein-5(6)-sulfonic
acid (9.08 g, 22 mmol) was dissolved in 35 mL of
trimethylacetic anhydride, 17.5 mL of diisopropylethyl-
amine, and 30 mL of DMF. The solution was heated to
reflux for 4 h and then quenched by addition of ethanol. The
solvents were removed on the rotary evaporator and the
resulting light brown viscous oil was taken up in 200 mL of
CH2Cl2 and 400 mL of ethyl acetate, and washed with 3!
300 mL of 1 M phosphate buffer (pH 7.0). The organic
phase was dried over MgSO4 and evaporated. Diethyl ether
was added, and a heavy fine precipitate formed. The filtered
solid was recrystallized from dichloromethane and diethyl
ether to give 2.10 g (13.5%) of fluorescein 6-sulfonic acid
dipivaloate diisopropylethylammonium salt; mp 227–
4.2.7. 30,60-Dichlorofluoran-6-sulfonyl chloride (3a).
Fluorescein 6-sulfonic acid (412 mg, 1 mmol) was added
to thionyl chloride (2.6 mL, 6 g, 5 mmol) and dimethyl-
formamide (6 mg, 6 mL, 82 mmol) and the reaction was
heated to reflux under Ar for 4 h. The resulting solution was
poured into 150 mL of stirred ice water and stirred for an
additional 10 min. The yellow grainy solid obtained was
lyophilized to give a final mass of 360 mg (87% yield); mp
1
229 8C. H NMR (CDCl3): d 9.00 (br s, 1H); 8.17 (d, 1H,
JZ8.1 Hz); 8.04 (d, 1H, JZ8.1 Hz); 7.64 (s, 1H); 7.04 (d,
2H, JZ2.1 Hz); 6.84 (d, 2H, JZ8.7 Hz); 6.76 (dd, 2H, JZ
8.7, 2.1 Hz); 3.60 (m, 2H); d 3.03 (m, 2H); 1.36 (s, 18H);
1.30–1.34 (m, 15H). 13C NMR (CDCl3): d 176.8, 169.6,
154.1, 152.6, 151.6, 147.0, 131.4, 129.1, 125.1, 124.4,
123.4, 117.8, 116.6, 110.4, 81.4, 39.4, 27.3, 22.3.
HRMS(M-HNiPr2Et): calcd 579.1325; Found 579.1330.
1
O210 8C (decomp.). H NMR (DMSO-d6): d 8.02 (d, 1H,
JZ8.1 Hz); 7.94 (d, 2H, JZ1.2 Hz); 7.92 (d, 1H, JZ
2.4 Hz); 7.58 (s, 2H); 7.39 (s, 1H); 7.22 (dd, 2H, JZ8.6,
1.8 Hz), 6.94 (d, 2H, JZ8.4 Hz). 13C NMR (DMSO-d6): d
168.1, 155.6, 152.6, 150.6, 135.4, 129.9, 128.3, 125.3,
125.0, 124.9, 120.5, 117.4, 117.1, 80.3. FTIR (KBr, cmK1):
3422 (br, m), 1777 (s), 1599 (m), 1566 (w), 1482 (m), 1411
(s), 1266–1060 (br, s), 955 (m). MS(M–ClCOH): Calcd
446.9; Found 447.0.
4.2.4. Diisopropylethylammonium salt of 30,60-dipiva-
loylfluorescein-5-sulfonate (2b). The filtrate from 2a was
allowed to stand at room temperature overnight, and the
resulting light yellow crystals were filtered to give 0.91 g
(5.8% overall) of the diisopropylethylammonium salt of
fluorescein 5-sulfonic acid dipivaloate; mp 154–156 8C. 1H
NMR (CDCl3): d 9.25 (br s, 1H); 8.33 (s, 1H); 8.06 (d, 1H,
JZ6.6 Hz); 6.97 (d, 1H, JZ8.1 Hz); 6.86 (d, 2H, JZ
1.8 Hz); 6.61–6.56 (m, 4H); 3.56 (m, 2H); 2.98 (m, 2H);
1.35 (m, 9H); 1.26 (d, 6H, JZ6.6 Hz); 1.17 (s, 18H). 13C
NMR (DMSO-d6): d 176.0, 168.1, 152.4, 152.2, 150.8,
150.8, 133.4, 129.3, 125.2, 123.9, 121.5, 118.5, 115.9,
110.4, 81.1, 53.6 41.9, 38.7, 27.0, 26.7, 18.1, 16.7, 12.5.
MS(M-HNiPr2Et): Calcd 579.1; Found 579.2. Continued
fractional crystallization brought the final yields to 3.31 g,
21% for the 6-isomer; and 1.59 g, 10.1% for the 5-isomer.
4.2.8. Fluorescein 5-sulfonic acid (1b). 30,60-Dipivaloyl-
fluorescein 5-sulfonic acid diisopropylethyl ammonium salt
(1.42 g, 2 mmol) and potassium hydroxide (3.2 g) were
dissolved in 30 mL of 50:50 v/v ethanol/H2O and heated to
reflux overnight. The ethanol was removed under reduced
pressure, and the aqueous solution was acidified with
concentrated HCl, causing the product to precipitate.
Filtration and drying overnight afforded the desired product
as 696 mg (84%) of a yellow solid; mp O330 8C (decomp.).
1H NMR (DMSO-d6) d 8.06 (s, 1H); 7.98 (d, 1H, JZ
9.3 Hz); 7.22 (d, 1H, JZ8.1 Hz); 6.67 (d, 2H, JZ2.1 Hz);
6.61–6.55 (m, 4H). 13C NMR (DMSO-d6): d 168.3, 160.3,
152.3, 150.2, 133.0, 129.5, 126.2, 124.3, 121.6, 113.2,
109.8, 102.3. FTIR (KBr, cmK1): 3500–2500 (br, s), 1714
(s), 1639–1538 (br, s) 1463 (s), 1383 (m), 1326 (s), 1220–
1126 (s), 1039 (m). HRMS(M-H): Calcd 411.0175; Found
411.0155.
4.2.5. Optimized purification of diisopropylethylammo-
nium salt of 30,60-dipivaloyl-fluorescein-6-sulfonate (2a).
Fluorescein 5(6)-sulfonic acid (6.20 g, 15 mmol) was
combined with trimethylacetic anhydride (35 mL) and
diisopropylethylamine (18 mL) in DMF (20 mL) and heated
to reflux 4 h. The reaction was allowed to cool, then taken
up in 200 mL of CH2Cl2 and 400 mL of ethyl acetate. The
organic solution was washed with 3!300 mL of phosphate
buffer (1 M, pH 7.0), dried over MgSO4, evaporated, and the
resulting residue was crystallized from diethyl ether and
CH2Cl2 at K25 8C overnight. Filtration gave 2.49 g (23.4%)
of the desired product. The mother liquor was placed in the
freezer and a second crop of crystals was obtained (800 mg)
for a total yield of 3.29 g (31% overall).
4.2.9. 30,60-Dichlorofluoran-5-sulfonyl chloride (3b).
Fluorescein 5-sulfonic acid (618 mg, 1.5 mmol) was
combined with thionyl chloride (4 mL) and dimethylforma-
mide (10 mL) and heated to reflux for 4 h. The reaction was
poured into 150 mL of stirred ice water, affording a dark
yellow solid that was filtered and lyophilized to give a final
1
mass of 549 mg (82% yield); mp O232 8C (decomp.). H
4.2.6. Fluorescein 6-sulfonic acid (1a). The diisopropyl-
ethylammonium salt of 30,60-dipivaloylfluorescein 6-sul-
fonic acid (177 mg, 0.25 mmol) was dissolved in 10 mL of
50:50 v/v ethanol/H2O. Potassium hydroxide was added
NMR (DMSO-d6): d 8.10 (s, 1H); 8.00 (d, 1H, JZ9.6 Hz);
7.58 (d, 2H, JZ2.1 Hz); 7.33 (d, 1H, JZ8.7 Hz); 7.20 (dd,
2H, JZ8.6, 2.4 Hz); 6.95 (d, 2H, JZ8.4 Hz). 13C NMR
(DMSO-d6): d 168.1, 152.3, 150.9, 150.7, 135.4, 133.6,

C. C. Woodroofe et al. / Tetrahedron 61 (2005) 3097–3105
3103
130.1, 125.0, 124.0, 121.7, 117.5, 117.1, 80.4. IR:
3091 cmK1 (br, w), 1779 cmK1 (s), 1599 cmK1 (s),
1564 cmK1 (s), 1481 cmK1 (s), 1425–1384 cmK1 (br, s),
1318 cmK1 (m), 1251–1083 cmK1 (br, m), 954 cmK1 (s).
HRMS(M–ClCOH): Calcd 446.9497; Found 446.9503.
Calcd 503.0913; Found 503.0912. The dipivaloyl-protected
sulfonamide product 7 was also isolated from the organic
reaction extract by flash chromatography on silica eluted
with 94:6 CHCl3:MeOH; mp 184–186 8C. 1H NMR
(MeOH-d4): d 8.38 (m, 2H); 8.09 (dd, 1H, JZ8.1,
1.8 Hz); 7.61 (td, 1H, JZ7.8, 2.1 Hz); 7.25 (s, 1H); 7.21–
7.12 (m, 3H), 7.05 (d, 1H, JZ2.1 Hz); 6.84–6.77 (m, 3H);
6.70 (d, 2H, JZ8.4 Hz); 4.40 (s, 2H); 1.36 (s, 18H). 13C
NMR (DMSO-d6): d 176.1, 167.1, 156.5, 155.0, 152.7,
150.9, 148.8, 143.6, 136.8, 133.7, 129.4, 126.1, 125.2,
123.5, 122.6, 122.2, 118.7, 115.3, 110.5, 81.4, 48.0, 38.8,
26.7. HRMS (MCH): Calcd 671.2063; Found 671.2071.
4.2.10. 30, 60-Dichlorofluoran-6-sulfonamido-2-methyl-
pyridine (4). 30,60-Dichlorofluoran-6-sulfonyl chloride (4,
86 mg, 0.2 mmol) was dissolved in 15 mL of CH2Cl2 and
added dropwise to a stirred suspension of 2-aminomethyl-
pyridine (43 mg, 0.4 mmol) and NaHCO3 (84 mg, 1 mmol)
in CH2Cl2. After stirring overnight, the reaction suspension
was filtered and evaporated; the product was purified by
flash chromatography on silica gel (10 mm!17 cm) eluting
with 9:1 CHCl3:MeOH, and then recrystallized from
methanol to give 30 mg (30%) of an off-white powder;
4.2.13. 30,60-Dibromofluoran (8). 3-Bromophenol (1.73 g,
10 mmol) and phthalic anhydride (740 mg, 5 mmol) were
combined in 5 mL of methanesulfonic acid and heated in a
140 8C oil bath for 16 h. The reaction was poured into
120 mL of stirred ice water, stirred for 20 min, and then
filtered. The resulting damp gray solid was taken up in
CHCl3 and filtered through a short plug of silica gel,
evaporated, and recrystallized from CH2Cl2 and MeOH to
afford the desired product as 990 mg of off-white crystals
(43% yield); mp 277–280 8C. 1H NMR (CDCl3): d 8.05 (dd,
1H, JZ7.8, 1.5 Hz); 7.67 (p, 2H, JZ1.2 Hz); 7.50 (d, 2H,
JZ1.8 Hz); 7.20 (dd, 2H, JZ10.5, 2.1 Hz); 7.14 (d, 1H, JZ
7.8 Hz); 6.71 (d, 2H, JZ8.4 Hz). 13C NMR (CDCl3): d
169.4, 153.2, 151.5, 135.9, 130.7, 129.6, 127.9, 126.2,
125.9, 124.6, 124.1, 120.8, 118.3, 81.5. HRMS(MCH):
Calcd 456.9075; Found 456.9084.
1
mp 236–238 8C. H NMR (DMSO-d6): d 8.59 (t, 1H, JZ
5.7 Hz), 8.31 (d, 1H,, JZ4.8 Hz), 8.20 (d, 1H, JZ8.4 Hz),
8.08 (dd, 1H, JZ8.7, 0.6 Hz), 7.76 (s, 1H), 7.66 (td, 1H, JZ
7.8, 2.1 Hz), 7.61 (d, 2H, JZ1.8 Hz), 7.15–7.26 (m, 3H),
6.91 (d, 2H, JZ8.4 Hz), 4.12 (d, 2H, JZ6.0 Hz). 13C NMR
(DMSO-d6): d 168.2, 157.5, 153.6, 151.8, 149.8, 149.3,
137.9, 136.7, 131.1, 130.0, 129.3, 127.6, 126.1, 123.7,
123.2, 122.8, 118.2, 118.0, 81.9, 48.9. MS(M-H): Calcd
537.0; Found 537.1. X-ray quality crystals were obtained by
slow evaporation of a saturated acetonitrile solution of 4 at
RT over 2 days.
4.2.11. 30,60-Dipivaloylfluorescein-6-sulfonyl chloride
(5). 30,60-Dipivaloylfluorescein-6-sulfonate diisopropyl-
ethylammonium salt (708 mg, 1 mmol) was stirred in
10 mL of ethyl acetate (dried over MgSO4) in an ice bath.
Oxalyl chloride (1 mL of 2 M solution in CH2Cl2) was
added, followed by 200 mL of DMF. The ice bath was
removed, and the reaction was stirred for 16 h. The reaction
was then placed on ice and quenched with 10 mL of H2O.
The layers were separated, and the organic layer was
washed with 1!10 mL H2O and 1!10 mL brine, dried,
and evaporated to give a yellow solid residue; mp O160 8C
4.2.014. 30,60-Pyrrolidinorhodamine (9)—method A.
30,6 -Dibromofluoran (46 mg, 0.1 mmol) was combined
with ZnCl2 (68 mg, 0.5 mmol) and pyrrolidine (83 mL,
71 mg, 1 mmol) and heated in a 170 8C oil bath for 4 h. The
reaction was removed from heat, and allowed to cool. Water
and concentrated HCl were added; the suspension was
stirred, filtered, and the solid was washed twice with
dilute HCl to afford a purple solid (42 mg, 95% yield); mp
1
1
O220 8C (decomp.). H NMR (MeOH-d4): d 8.34 (d, 1H,
(decomp.). H NMR (CDCl3): d 8.68 (s, 1H); 8.31 (d, 1H,
JZ7.5 Hz); 7.81 (m, 2H); 7.41 (d, 1H, JZ8.7 Hz); 7.12 (d,
2H, JZ9.3 Hz); 6.90 (dd, 2H, JZ6.9, 1.8 Hz); 6.82 (d, 2H,
JZ2.1 Hz); 3.61 (m, 8H); 2.14 (s, 8H). 13C NMR (DMSO-
d6): d 169.0, 152.7, 152.3, 152.3, 149.2, 135.3, 129.8, 128.9,
128.5, 128.3, 126.7, 125.4, 124.5, 124.0, 108.8, 105.4, 97.4,
85.4, 47.4, 25.0. MS(MCH): Calcd 439.2; Found 439.4.
JZ6.8 Hz); 7.45 (d, 1H, JZ6.5 Hz); 7.18 (s, 2H); 6.84 (s,
2H); 1.37 (s, 18H). HRMS(MCH): Calcd 599.1143; Found
599.1114.
4.2.12. 6-Fluoresceinsulfonamido-2-methylpyridine (6).
The product from 5 was dissolved in 20 mL of CHCl3 and
stirred in an ice bath. 2-Aminomethylpyridine (300 mL) was
added and the reaction was stirred overnight. The reaction
was then extracted with 2!20 mL of H2O, the combined
aqueous layers were washed with 1!20 mL CHCl3,
concentrated on the rotary evaporator to 5 mL, and the
bright red viscous solution was acidified with 2 mL of 1 N
HCl. The resulting yellow precipitate was filtered and the
resulting solid (485 mg) was chromatographed on silica
(20 mm!16 cm), eluting with 89:10:1 CHCl3:MeOH:
AcOH. The desired product (43 mg, 8.5%) was isolated as
a bright yellow–orange solid; mp 78–80 8C. 1H NMR
(CDCl3): d 8.40–8.36 (m, 2H); 8.10 (dd, 1H, JZ8.1,
1.5 Hz); 7.74 (td, 1H, JZ7.8, 1.5 Hz); 7.43 (d, 1H, JZ
7.8 Hz); 7.31–7.23 (m, 2H); 6.69 (t, 2H, JZ0.9 Hz); 6.59 (s,
4H); 4.34 (s, 2H). 13C NMR (DMSO-d6): d 167.4, 156.7,
152.4, 148.8, 142.8, 136.8, 136.5, 129.5, 129.3, 122.5,
122.4, 122.0, 109.1, 102.5, 102.3, 48.0. HRMS(MCH):
4.2.15. 30,60-Pyrrolidinofluoran (9)—method B. 30,60-
Dibromofluoran (229 mg, 0.5 mmol) was combined with
palladium dibenzylideneacetone (11.5 mg, 0.0125 mmol;
0.025 mmol Pd), 0 sodium tert-butoxide (101 mg,
1.05 mmol), and 2 -dimethylamino-2-dicyclohexyl-phos-
phinobiphenyl (10.3 mg, 0.025 mmol) in a thick-walled
tube fitted with a rubber septum. The tube was thrice
evacuated and back-filled with N2, and 1.5 mL of dry
toluene was added, followed by 90 mL (77 mg, 1.08 mmol)
of pyrrolidine. The septum was replaced with a Teflon screw
cap and the reaction was stirred in an 80 8C oil bath for 15 h,
then removed from heat and allowed to cool. Hexanes were
added to the purple slurry, and a purple solid was isolated by
filtration (350 mg, wet) LCMS and 1H NMR analysis of the
solid supported a single product identical to those produced
by method A.

3104
C. C. Woodroofe et al. / Tetrahedron 61 (2005) 3097–3105
4.2.16. Pyridinium salt of 30,60-dibromo-6-carboxy-
fluoran (10c). 3-Bromophenol (3.46 g, 20 mmol) and 1, 2,
4-benzenetricarboxylic acid (2.10 g, 10 mmol) were com-
bined in 10 mL of methanesulfonic acid and heated in a
140 8C oil bath for 3 days. The reaction was poured into
200 mL of stirred ice water, stirred vigorously with
warming for 30 min, and then filtered to yield a greenish
solid which was dried in air to give 3.83 g of 10a and 10b as
a mixture of isomers. Crystallization from 30 mL of acetic
anhydride and 10 mL of pyridine afforded 1.35 g of white
solid, which was recrystallized from 2:1 Ac2O:pyridine to
furnish the desired compound 10c as 1.07 g (18%) of
fine white crystals; mp O327 8C (decomp.). 1H NMR
(DMSO-d6): d 8.58 (m, 2H); 8.25 (d, 1H, JZ6.5 Hz); 8.16
(d, 1H, JZ6.4 Hz); 7.86 (s, 1H); 7.76 (m, 1H); 7.43 (m, 2H);
6.87 (d, 2H, JZ6.4 Hz); 6.67 (d, 2H, JZ6.3 Hz). 13C NMR
(DMSO-d6): d 172.1, 167.6, 166.0, 152.2, 150.8, 149.6,
137.9, 136.2, 131.5, 130.1, 128.6, 127.7, 125.7, 124.8,
124.0, 123.8, 119.9, 117.5, 80.8. HRMS(M-pyH): Calcd
498.8817; Found 498.8804.
4.2.20. 2,4-Dicar0bo0xy-50-chlor0o-20,40-dihydroxybenzo-
phenone (13b). 3 ,6 -Diacetyl-2 ,70-dichlorofluorescein-5-
carboxylic acid (2.12 g, 4 mmol) was suspended in 60 mL
of 50% aqueous NaOH (w/v) and heated at 165 8C for
60 min. The reaction was removed from heat, poured into
400 mL of cold H2O, acidified with conc. HCl, and allowed
to stand at rt overnight. The suspension was filtered, and the
dirty-brown solid was resuspended in 50 mL of H2O,
stirred, and filtered again. The resulting solid was then taken
up in MeOH, filtered to remove residual NaCl, and
evaporated to afford 1.14 g of the desired product (85%
1
yield); mp O265 8C (decomp.). H NMR (MeOH-d4): d
8.72 (s, 1H); 8.34 (d, 1H, JZ5.7 Hz); 7.51 (d, 1H, JZ
4.8 Hz); 6.95 (s, 1H); 6.49 (s, 1H). 13C NMR (CDCl3): d
201.4, 168.1, 167.9, 164.8, 162.0, 145.2, 134.7, 134.6,
133.8, 132.9, 131.2, 129.1, 115.4, 113.6, 105.0. HRMS(M-
H): Calcd 334.9959; Found 334.9944.
4.2.21. 6-Carboxy-20-chloroseminaphthofluorescein (14).
1,6-Dihydroxynaphthofluorescein (24 mg, 0.15 mmol) was
combined with benzophenone 13a (34 mg, 0.1 mmol) in
200 mL of methanesulfonic acid in a 180 8C oil bath. After
14 h, 5 mL of H2O was added and the resulting suspension
was filtered and washed twice with H2O. The resulting
purple solid was dissolved in MeOH, the solution was
filtered, and H2O was added to the filtrate to induce
precipitation. The product was collected by filtration and
dried in air to give 42 mg (91%) of the desired compound;
mp O256 8C (sublimed). 1H NMR (DMSO-d6): d 11.2 (br s,
1H); 10.2 (br s, 1H); 8.40 (d, 1H, JZ8.8 Hz); 8.24 (d, 1H,
JZ1.2 Hz); 8.16 (d, 1H, JZ8.0 Hz); 7.72 (s, 1H); 7.38 (d,
1H, JZ8.8 Hz); 7.27 (d, 1H, JZ9.0 Hz); 7.15 (s, 1H); 7.14
(s, 1H); 6.91 (s, 1H); 6.62 (s, 1H). 13C NMR (DMSO-d6): d
167.9, 166.1, 157.5, 155.2, 152.7, 145.0, 146.4, 137.5,
136.0, 131.2, 129.3, 128.6, 125.6, 124.6, 123.8, 122.5,
119.2, 117.0, 116.6, 110.0, 109.4, 108.7, 103.9, 82.8.
MS(M-H): Calcd 459.0; Found 459.0.
4.2.17. 30,60-Dibromo-5-carboxyfluoran (10b). The filtrate
from the initial crystallization of 10c was concentrated and
recrystallized from pyridine to afford 906 mg of an off-
white solid. Further recrystallization from CHCl3:MeOH
afforded the desired product as 571 mg of white crystals
1
(11.4% yield); mp O324 8C (decomp.). H NMR (DMSO-
d6): d 8.44 (s, 1H); 8.31 (d, 1H, JZ8.0, 1.2 Hz); 7.72 (d, 2H,
JZ2.0 Hz); 7.51 (d, 1H, JZ8.0 Hz); 7.34 (dd, 2H, JZ8.6,
2.0 Hz); 6.90 (d, 2H, JZ8.4 Hz). 13C NMR (DMSO-d6): d
167.5, 165.9, 155.5, 150.6, 136.5, 133.4, 130.8, 127.8,
127.7, 126.0, 125.9, 124.6, 123.9, 119.9, 117.4, 80.6.
MS(MCH): Calcd 500.9; Found 501.0.
4.2.18. 30,60-Dipyrrolidino-6-carboxyrhodamine (11).
30,60-Dibromo-6-carboxyfluoran (116 mg, 0.2 mmol) was
combined with ZnCl2 (136 mg, 1 mmol) and pyrrolidine
(332 mL, 5 mmol) and heated in a 140 8C oil bath for 4 h.
The dark purple residue was dissolved in 15 mL of
concentrated HCl, and the resulting dark red solution was
filtered and then diluted with 30 mL of H2O, allowed to
stand at rt for 2 h, and filtered to yield 98 mg (94%) of the
desired product HCl salt; mp O310 8C (decomp.). 1H NMR
(MeOH-d4): d 8.42 (s, 1H); 8.39 (d, 1H, JZ5.8 Hz); 7.98 (s,
1H); 7.11 (d, 2H, JZ6.4 Hz); 6.94–6.84 (m, 4H); 3.62 (br s,
8H); 2.14 (s, 8H). HRMS(M-H): Calcd 481.1763; Found
481.1744.
Acknowledgements
This work was supported by a grant from NIGMS
(GM65519 to S.J.L.). C.C.W. thanks the Merck/MIT
program for predoctoral support. Instrumentation in the
MIT DCIF is maintained with the aid of grants from
NIH (1S1ORR1388-01) and from NSF (CHE-980861,
DBI-9729592, and CHE-9808061). We are grateful to
M.A. Clark for helpful discussions.
4.2.19. 2,5-Dicarbo0xy-50-chlor0o-20,40-dihydroxybenzo-
phenone (13a). 30,6 -Diacetyl-2 ,70-dichlorofluorescein-6-
carboxylic acid pyridinium salt (2.44 g, 4 mmol) was
suspended in 60 mL of 50% aqueous NaOH (w/v) and
heated at 165 8C for 60 min. The reaction was removed from
the heating bath, poured into 400 mL of cold H2O, acidified
with conc HCl, and allowed to stand at rt for 2 h. The
suspension was filtered, and the pale yellow solid was taken
up in MeOH, filtered to remove residual NaCl, and
evaporated to afford 1.19 g of the desired product (89%
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