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PRACTICAL SYNTHETIC PROCEDURES
formed when other acids were used (Scheme 1). Figure 3a
shows the aromatic region of the 1H NMR spectrum of the
product 2a formed from the condensation reaction, then
purified via fractional crystallization (see below). Treat-
ment of this with sodium hydroxide, then protonation with
HCl gives a different material (Figure 3b); we propose
this is the cyclic lactone 3a. Conversely, product 2a was
regenerated when 3a was treated with excess methane-
sulfonic acid. Assignment of structures 2a and 2b to the
products of the initial reaction is supported by data from
elemental analyses.
Figure 4 Analytical HPLC traces of: a, compound 2a (98%); and,
b, compound 2b (98%) after two recrystallizations each (reverse pha-
se C-18, Et3N, HOAc, H2O, MeCN).
High field NMR spectra were recorded on a Varian Unity Plus (1H
at 300 MHz, 13C NMR at 75 MHz). Mass spectra were obtained
from the Mass Spectrometry Laboratory at Texas A&M University.
Resorcinol, 1,2,4-benzenetricarboxylic anhydride and methane-
sulfonic acid were purchased from Aldrich and used as received.
Hexanes and MeOH were purchased from EMD Chemicals Inc.
Absolute EtOH-200 proof was purchased from AAPER Alcohol.
All solvents were used as received. Analytical HPLC were run on a
SSI instrument (222C HPLC pump, 232C gradient controller) and a
model 500 variable wavelength detector using a Supelco C-18 col-
umn (SupelcosilTM LC-18-T, 25 × 4.6 mm, 5 mm), gradient elution
was used (A = H2O, B = MeCN, both with 0.1% v/v TEAA) with a
constant flow rate of 0.9 mL/min.
Figure 3 Aromatic 1H NMR (CD3OD) regions of: a, compound 2a;
and, b, compound 3a formed from treatment of 2a with NaOH then
HCl. Differences in the chemical shifts were far less pronounced in
NaOD–D2O.
The reaction shown in Scheme 1 affords compounds 2a
and 2b in approximately a 1:1 ratio. Recrystallization of
5- and 6-Carboxyfluorescein (1a,b)
20 g of that mixture from methanol–hexane at –18 °C 1,2,4-Benzenetricarboxylic anhydride (also called 4-carboxy-
phthalic anhydride, 25.0 g, 0.13 mol) was added to a solution of 1,3-
dihydroxybenzene (also called resorcinol, 28.6 g, 0.26 mol) in
gave a crude sample of compound 2b. A second recrystal-
lization gave 1.0 g of this material in over 98% regioiso-
methanesulfonic acid (1 M). An air condenser was attached to the
meric purity (Figure 4). Combination of the mother
flask and the reaction was heated at 85 °C in an open vessel for 24
liquors, removal of the solvent, then two recrystallizations
h. After cooling to r.t., the reaction mixture was poured into 7 vol-
from a similar solvent system, ethanol–hexane, gave 3.2 g
of the 5-carboxy isomer 2a in over 98% purity. The moth-
er liquors were again combined, the solvents were re-
moved, and two recrystallizations of the residues from the
original solvent system, methanol–hexane afforded an-
other 3.0 g of the 6-isomer 2b. Thus, in this particular ex-
periment, the 5- and 6-isomers were isolated in 3.2 and 4.0
g amounts corresponding to 32 and 40% yields. No at-
tempt was made to crystallize more material from the
mother liquors, but it is likely that this is possible. In other
experiments, compound 2a was isolated first when the re-
crystallizing solvents were used in the reverse order (i.e.
ethanol–hexane, then methanol–hexane).
umes of ice–H2O. An orange–yellow precipitate formed; this was
collected by filtration and dried in an oven at 200 ºC. This residue
was recrystallized from MeOH–hexanes (2 ×) to give 6-carboxy-
fluorescein methanesulfonic acid adduct 2b (1.0 g). The mother li-
quors from this procedure were collected, the solvent was removed
in vacuo, and the residues were recrystallized from EtOH–hexanes
(2 ×) to give 5-carboxyfluorescein methanesulfonic acid adduct 2a
(3.2 g). Finally, the mother liquors from this experiment were com-
bined, evaporated to dryness, and recrystallized from MeOH–hex-
anes (2 ×) to give more of 6-carboxyfluorescein methanesulfonic
acid adduct 2b (another 3.0 g), making a total yield of 4.0 g (40%).
Careful dropwise addition of concd aq HCl to solutions of these
methanesulfonic esters 2a and 2b in aq NaOH (4 M) gave 5- (1a)
and 6-carboxyfluorescein (1b), respectively, in near quantitative
yield.
The methanesulfonic esters 2 are easily converted to the
5- and 6-carboxyfluoresceins 1 by treatment with sodium
hydroxide solution then neutralizing with aq HCl. Conse-
quently, the procedures described here represent extreme-
ly convenient syntheses of the target materials 1a and 1b
as highly enriched regioisomers.
Compound 1a
Mp 385–388 °C (lit8 368–372 °C).
1H NMR (300 MHz, NaOD–D2O): d = 6.56 (d, J = 2.47 Hz, 2 H),
6.66 (dd, J = 2.21, 9.36 Hz, 2 H), 7.20 (d, J = 9.08 Hz, 2 H), 7.33 (d,
J = 7.71 Hz, 1 H), 8.07 (dd, J = 1.65, 7.84 Hz, 1 H), 8.26 (d,
J = 1.93 Hz, 1 H).
Synthesis 2004, No. 15, 2591–2593 © Thieme Stuttgart · New York
Ueno, Yuichiro
